THE UBIQUITIN PROTEASOME SYSTEM IN THE CENTRAL NERVOUS SYSTEM: FROM PHYSIOLOGY TO PATHOLOGY
THE UBIQUITIN PROTEASOME SYSTEM IN THE CENTRAL NERVOUS SYSTEM: FROM PHYSIOLOGY TO PATHOLOGY
MARIO DI NAPOLI AND
CEZARY WÓJCIK EDITORS
Nova Biomedical Books New York
Copyright © 2007 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA The ubiquitin proteasome system in the central nervous system : from physiology to pathology / Mario Di Napoli and Cezary Wojcik, (editors). p. ; cm. Includes bibliographical references and index. ISBN-13: 978-1-60692-524-9 1. Ubiquitin. 2. Central nervous system--Physiology. 3. Central nervous system--Pathophysiology. I. Napoli, Mario Di. II. Wójcik, Cezary, 1968[DNLM: 1. Central Nervous System--metabolism. 2. Central Nervous System Diseases--metabolism. 3. Proteasome Endopeptidase Complex--physiology. 4. Proteins--metabolism. 5. Ubiquitin-Protein Ligase Complexes--physiology. WL 300 U15 2007] QP552.U24U2577 2007 612.8'2--dc22 2007016546
Published by Nova Science Publishers, Inc.
New York
CONTENTS Preface
ix
Chapter 1
Focusing on the Ubiquitin Proteasome System in Nervous System Mario Di Napoli and Cezary Wójcik
Chapter 2
Intracellular Protein Degradation: from a Vague Idea thru the Lysosome and the Ubiquitin-Proteasome System and Onto Human Diseases and Drug Targeting Aaron Ciechanover
Chapter 3
Ubiquitin and Ubiquitination: An Overview of the UbiquitinProteasome System for Protein Degradation Yulia Matiuhin and Michael H. Glickman
Chapter 4
Diversity and Cellular Functions of Deubiquitinating Enzymes Kyuhee Oh, Ok Sun Bang and Chin Ha Chung
Chapter 5
Ubiquitin Domain Proteins Functional Variations of a Common Structure Andrea Schulze, Michael Seeger and Rasmus Hartmann-Petersen
1
15
41 71
93
Chapter 6
Structure and Function of the 20S Proteasomes Manila Amici and Anna Maria Eleuteri
117
Chapter 7
Structure and Function of the 26S Proteasomes Cezary Wójcik and George N. DeMartino
137
Chapter 8
Assays with Natural Substrates: Novel Tools to Address the Complexity of Protein Degradation by 26S Proteasomes K. Matthew Scaglione and Dorota Skowyra
159
Chapter 9
Proteasome Activators, Inhibitors and Associated Proteins Geoffrey M. Goellner and Carlos Gorbea
169
Chapter 10
The Molecular Chaperones in the Ubiquitin-Proteasome System Mihiro Yano and Hiroshi Kido
207
vi
Mario Di Napoli and Cezary Wójcik
Chapter 11
Intercellular Localization of Proteasomes Cezary Wójcik and George N. DeMartino
Chapter 12
Aggresome Formation: A Failure of the Ubiquitin-ProteasomeSystem Patrick H. Thibodeau, Michael J. Corboy and W. Christian Wigley
241
Endoplasmic Reticulum (ER) Stress, ER-Associated Degradation and Unfolded Protein Response Cezary Wójcik and Mario Di Napoli
271
Chapter 13
Chapter 14
Chapter 15
Chapter 16
Chapter 17
Chapter 18
Chapter 19
Chapter 20
Chapter 21
Chapter 22
Chapter 23
Theoretical Models for Proteasome Function: Predictive Methods to Understand Ubiquitin Protein System in Neurodegenerative Diseases Fabio Luciani, Morten Nielsen, Can Kesmir and Mario Di Napoli
227
307
The Ubiquitin-Proteasome System and the Development of the Nervous System Douglas S. Campbell
343
General Aspects of Ubiquitin Proteasome System in the Mature Central Nervous System Lukasz P. Bialy and Izabela Mlynarczuk-Bialy
373
Ubiquitin-Proteasome System in the Peripheral Nervous System: Functional and Morphological Aspects Lucia Notterpek
393
Dynamic Regulation of Synaptic Plasticity by the Ubiquitin Proteasome System Jennifer A. Johnston
411
The Role of the Ubiquitin Proteasome System in Modifying Cellular Stress Responses Mario Di Napoli and Francesca Papa
443
The Homeostatic Control of Glucocorticoid Receptor in the Central Nervous System by the Ubiquitin-Proteasome System Theo Rein and Donald B. DeFranco
495
Role of the Ubiquitin- and Proteasome System in Neuronal Apoptosis Cezary Wójcik
513
Molecular Chaperones and the Ubiquitin Proteasome System in Aging Stuart K. Calderwood
537
Impairment of the Ubiquitin-Proteasome System: A Common Pathogenic Mechanism in Neurodegenerative Disorders Lian Li and Lih-Shen Chin
553
Contents Chapter 24
Chapter 25
Neuroprotective and Pro-Apoptotic Responses of Ubiquitin Proteasome System Meng Shyan Choy and Nam Sang Cheung
579
Ubiquitin Proteasome System Pathway in Dopaminergic Neurodegeneration Hideto Miwa
599
Chapter 26
The Ubiquitin-Proteasome System in Axon Degeneration Michael P. Coleman
Chapter 27
The Ubiquitin Proteasome System in Pain Transmission and Neuropathic Pain Clare W. J. Proudfoot, Darren C Robertson, Emer M Garry and Susan M Fleetwood-Walker
Chapter 28
Chapter 29
Chapter 30
Mechanisms Involved in the Aggregation of Ubiquitinated Proteins in Neurodegenerative Disorders Kenyon D. Ogburn and Maria E. Figueiredo-Pereira
717
The Regulation of TAU Protein Proteolysis by the Ubiquitin Proteasome System in Neurodegenerative Foldopathies Patrice Delobel and Isabelle Lavenir
737
Chapter 32
The Ubiquitin-Proteasome System in Huntington’s Disease and other Polyglutamine Diseases Louise Kelly and Elsdon Storey
Chapter 35
Chapter 36
651
671
Ubiquitin-Dependent Proteolysis in Parkinson’s Disease Jian Feng,, Thomas Schmidt-Glenewinkel and Mario Di Napoli
Chapter 34
627
The Ubiquitin Proteasome System in Neurological Disorders: From Conformational Diseases to Proteasomepathies Mario Di Napoli, and Francesca Papa
Chapter 31
Chapter 33
vii
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases Birkir Thor Bragason and Astridur Palsdottir Role of the Ubiquitin Proteasome System in Antigen Presentation, Autoimmune Disorders and Inflammation in the Central Nervous System Jakub Golab and Dominika Nowis
763
795
815
835
The Functional Role of Proteasome Antibodies in Neurological Disorders Anette Storstein and Christian Vedeler
851
The Ubiquitin Proteasome System in the Pathobiology of Human Gliomas Marco Piccinini, Maria Teresa Rinaudo and Davide Schiffer
865
viii Chapter 37
Chapter 38
Mario Di Napoli and Cezary Wójcik The Ubiquitin Proteasome System in the Pathobiology of Human Pituitary Tumors Mădălina Muşat, Márta Korbonits and Ashley B. Grossman
877
The Role of the Ubiquitin-Proteasome System in Epilepsy and Seizure Susceptibility Edward Glasscock
899
Chapter 39
The Ubiquitin Proteasome System in Cerebral Ischemia Mario Di Napoli, and Francesca Papa
Chapter 40
Pharmacology of the Ubiquitin Proteasome System: Proteasome Inhibitors and Modulators Halina Ostrowska, and Marek Z. Wojtukiewicz
Chapter 41
Chapter 42
Chapter 43
Preclinical Data on the Use of Proteasome Inhibitors: A New Approach to Treatment of Nervous System Diseases Anthony J. Williams,, Jitendra R. Dave, Peter Elliott and Frank C. Tortella
923
963
991
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases Imtiaz M. Shah and Mario Di Napoli
1013
Clinical Application of Proteasome Inhibitor Bortezomib: Characterization of Neurotoxicity Jens Voortman and Giuseppe Giaccone
1039
Book Glossary
1057
Index
1105
PREFACE The book focused on the role of ubiquitin protesome system (UPS) in central nervous system. Proteasomes are large multicatalytic proteinase complexes that are found in the cytosol and in the nucleus of eukaryotic cells with a central role in cellular protein turnover. The UPS has a central role in the selective degradation of intracellular proteins. In addition to serving as a means to rapidly eliminate short-lived regulatory proteins involved in cell cycle, cell growth, and differentiation, in periods of stress rapid elimination of denatured, misfolded and damaged proteins by the proteasome becomes a critical determinant of cell fate. These aspects are analyzed in central nervous system physiology and pathology. Chapter 1 - Multiple critical cellular processes are regulated by maintaining the appropriate levels of proteins. Whereas de novo protein synthesis is a comparatively slow process, proteins are rapidly degraded at a rate compatible with the control of cell cycle transitions, signaling events and induction of cell death. The ubiquitin-proteasome system (UPS) plays a pivotal role in the degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen presentation. On the other hand the UPS also displays an important quality control function, removing abnormal proteins from the cytosol, the nucleus and the endoplasmic reticulum. The pathway involves an enzymatic cascade through which multiple 76–amino acid ubiquitin monomers are covalently attached via a three-step process to the protein substrate, which is then degraded by the 26S proteasome complex, a cylindrical organelle that recognizes ubiquitinated proteins, degrades the proteins, and recycles ubiquitin. It is now clear that regulated protein degradation by the UPS affects practically every cellular process. In the nervous system, ubiquitination plays a role, among others, in neuronal signaling, synapse formation and function, as well as, in various diseases. It is becoming increasingly evident that altered activities of the UPS are crucially involved in the pathophysiology of Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, prion diseases and in spinocerebellar ataxia, just to name a few. Protein degradation pathways are also targets for therapy as shown by the successful results obtained with the inhibitors of the 26S proteasome. Further work in this area holds great promise toward our understanding and treatment of a wide range of neurological disorders. Chapter 2 - Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded
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has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin–proteasome system (UPS) resolved the enigma. The authors now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism based drugs. Chapter 3 - Cells contain many different kinds of proteins, each fulfilling structural, functional, or regulatory roles. The presence of either damaged or mutated proteins, or of altered levels of normal proteins could cause pathological conditions and even cell death. Therefore, monitoring the state of all these proteins, as well as continuously adjusting their levels to suit demands, is paramount to survival. To exercise such quality control, cells are continuously spending energy both to synthesize new proteins, and to simultaneously degrade them, even though many may still be functional. An important characteristic of regulatory degradation is that it is specific; only the correct proteins are removed in a time-coordinated manner. Such extraordinary specificity is achieved by a modular system that identifies proteins that are to be degraded, marks them by covalently attaching ubiquitin to an amino residue, and finally proteolyses them into amino acids. This sequence of events is executed by the following components. Recognition of target proteins is carried out by a specific ubiquitin-protein ligase, called an E3. This protein recognizes the substrate and usually directs a ubiquitin-conjugating enzyme, an E2, to attach ubiquitin, a small 76 amino acid protein, onto an amino group on the substrate. Ubiquitin molecules are often added one to another, resulting in chains of ubiquitin extending from the protein targeted for degradation. These polyubiquitin conjugates are then shuttled to the 26S proteasome, a large ATPdependent proteolytic complex, where they are degraded. Interestingly, ubiquitination is a reversible process, with deubiquitinating enzymes able to remove ubiquitin from the target before it can be recognized by the proteasome. Hence, transfer of the polyubiquitinated conjugate to the proteasome must happen swiftly or be shielded from these enzymes. The cumulative balance of these processes allows the ubiquitin-proteasome system to control the cellular levels and half lives of thousands of proteins making it a key player in basic biological pathways such as cell division, differentiation, signal transduction, trafficking, and quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases, certain malignancies, neurodegenerative disorders, inflammation and immune response. Understanding of the underlying mechanisms involved is important for the development of novel, mechanism-based drugs. Chapter 4 - Covalent modification of proteins by ubiquitin is a key mechanism for the control of cellular processes as diverse as cell proliferation, differentiation, apoptosis. Deubiquitination, reversal of this modification, is catalyzed by deubiquitinating enzymes that belong to the superfamily of proteases. Deubiquitinating enzymes occupy the second largest family of enzymes in the ubiquitin system, implying their functions in the control of diverse cellular processes by regulating the fate, function of ubiquitinated proteins. Cellular functions
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of deubiquitinating enzymes include the regulation of proteasome activity, protein stability, signal transduction, DNA repair, chromatin dynamics, transcription, endocytosis. Deubiquitinating enzymes also play important roles in the processing of inactive ubiquitin precursors for the generation of matured ubiquitin monomers, the removal of ubiquitin from ‘distal’ end of poly-ubiquitination chains conjugated to proteins for controlling the fidelity of the ubiquitination process, the cleavage of ubiquitin adducts for the release of free ubiquitin. Deubiquitinating enzymes consist of five families that have distinct catalytic domain structures: the ubiquitin-specific protease (USP) family, the ubiquitin C-terminal hydrolase (UCH) family, the ovarian tumor protease (OTU) family, the Machado-Joseph disease protein (MJD) family, the Jab1/MPN/Mov34-domain protease (JAMM) family. While the JAMM family members are metalloproteases, the other family members are cysteine proteases. As the names of certain families imply, deubiquitinating enzymes play critical regulatory roles in a multitude of processes from cancer to neurodegenerative diseases. In this chapter, the authors summarize the catalytic properties of deubiquitinating enzymes so far been identified, the recent findings on their functions as cellular regulators. The authors also describe the specific features of deubiquitinating enzymes that are related with neuronal diseases. Chapter 5 - Ubiquitin-like (UBL) domain proteins (UDPs) constitute a family of proteins with a modular architecture, which is characterized by an integral UBL-domain. Although members of the UDP family display a variety of different functions, many of them are on some level connected with the ubiquitin-proteasome system, a central pathway, which accommodates intracellular protein degradation in eukaryotic cells. While some UDPs are involved in substrate recruitment for the 26S proteasome, also a ubiquitin-specific hydrolase, an ER-membrane resident protein, a co-chaperone, and a ubiquitin ligase belong to this family. Several of these proteins have been implicated in the development of neurodegenerative diseases. Of the initially studied UDPs, most bound the proteasome in a UBL-dependent manner. Therefore it appeared that proteasome binding was a general feature of this protein family. However, evidence is accumulating that a number of UDPs also bind to other components of the ubiquitin pathway, while some appear not to bind the proteasome at all. Hence UDPs appear functionally more diverse than one would expect from their structural appearance. Here the authors provide insight into the UDP family and attempt to summarize what is known about their physiological role, especially with respect to neurodegenerative diseases. The authors come to the conclusion that, despite their striking structural similarity, UDPs display rather diverse binding features, and appear to be part of a sophisticated protein network within the ubiquitin system. Chapter 6 - The 20S proteasome, a 700 kDa multicatalytic proteinase complex, is responsible for the extralysosomal protein degradation that occurs in the cytosol and nucleus of eukaryotic cells. It represents the proteolytic core of the 26S proteasome, a 2000 KDa elongated structure formed by the 20S capped, at each side, by the 19S regulatory complex (also called PA700). The 26S complex is involved in the ATP, ubiquitin-dependent and ubiquitin-independent proteolytic pathways. The proteasome constitutes up to 1% of protein in the cells and the free 20S proteasomes are the major portion of the total amount of proteasomes. Its molecular architecture is extremely conserved from archaebacteria to higher eukaryotes and is organized in four stacked 7-membered rings of α and β subunits, in a cylinder-like shape. The two inner rings are composed of β subunits, harbouring the active
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sites, flanked by the two outer rings made up of non-catalytic α subunits which regulate the substrate access through the opening of the outer ring and the binding of regulators. The 20S proteasome is a member of the N-terminal nucleophile (Ntn)-hydrolases family. Its Nterminal threonine residues are exposed as the nucleophile in peptide bond hydrolysis. The three β subunits, β1, β5 and β2 (also called Y/delta, X and Z, respectively) express the three catalytic activities, designated peptidyl-glutamyl peptide hydrolyzing, chymotrypsin-like and trypsin-like, based on the ability to cleave peptide bonds on the carboxyl side of hydrophobic, basic and acidic aminoacids, respectively. Furthemore, two additional activities cleaving bonds after branched chain and small neutral amino acids have been described and called branched chain amino acid preferring and small neutral amino acid preferring. They enable the 20S proteasome to degrade alone a wide variety of protein substrates: poorly folded or unfolded proteins and oxidized proteins characterized by an increased surface hydrophobicity. Under the influence of γ-interferon, a major immunomodulatory cytokine, vertebrate proteasomes assemble catalytically-active subunits, named β5i, β1i and β2i (also called LMP7, LMP2 and MECL1, respectively) which replace their constitutive homologues, β5, β1 and β2, respectively, and associate to a regulatory particle, PA28, (or 11S regulatory complex) also induced by γ-interferon. Such a complex has been demonstrated to be specialized in generating MHC class I antigenic peptides. This review focuses on recent progress concerning the structure, including the assembly pathway, and the enzymatic activities that are involved in physiological/pathological functions exerted by the eukaryotic 20S proteasomes in the cell. Chapter 7 - 26S proteasomes are ~2.4 MDa supramolecular assemblies that function as protein degrading complexes in neuronal as well as other cell types. They constitute the final, common destination of the proteins degraded by the ubiquitin-proteasome pathway, and perhaps by some non-ubiquitin-dependent pathways as well. 26S proteasomes are formed by association of the core 20S proteasomes with one or two PA700 activators (19S caps). While the core 20S proteasomes harbor the proteolytic activities, the remaining features of 26S proteasomes are conferred by components of the PA700. Mammalian PA700 is composed of 18 subunits, including 6 AAA ATPases (Rpt1-6) and several non-ATPase subunits (Rpn1-3, Rpn5-12 and Uch37). PA700 is physically divided into the lid and base subcomplexes. PA700 allows the recognition of polyubiquitinated proteins, their attachment, unfolding, opening of the closed proteasomal ‘gates’ and translocation of the unfolded polypeptide chain of the substrate towards the central catalytic cavity of the proteasome. At the same time PA700 allows the release of free ubiquitin through at least two different deubiquitinating activities. All of these functions are coupled to the ATP-ase activity of the complex, making them highly susceptible to ATP depletion such as during episodes of limited hypoxia or ischemia. Moreover, under those conditions 26S proteasomes tend to separate into free 20S proteasomes and PA700 complexes. Besides the canonical 26S proteasome subunits, several proteins associate loosely with the 26S proteasome, including additional deubiquitinating enzymes, ubiquitin ligases and polyubiquitin binding and delivery factors. Chapter 8 - The 26S proteasome has long been viewed as a major therapeutic target. However, in the past 20 years only inhibitors of the proteolytic sites have been developed. Such a focus was primarily the result of the limited availability of assays for monitoring activity of the 26S proteasome. Due to the difficulties in preparation of naturally
Preface
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polyubiquitinated proteins, these assays were based on artificial model substrates, typically monomeric proteins that could be either polyubiquitinated in vitro without a specific E3 ubiquitin ligase (lysozyme, DHFR, Ub-Pro-β-gal) or degraded without polyubiquitination (fluorogenic peptides, loosely structured caseine, denatured ovalbumin). Although these reagents proved invaluable in uncovering the basic principles of proteasomal function, it becomes increasingly clear that they did not allow one to address the puzzling complexity of the 19S cap composition, indispensable in the highly controlled and rapid (T1/2 50 glutamines) in ataxin-3 is thought to confer gain-of-function toxicity by inducing protein misfolding and aggregation, resulting in formation of nuclear and cytoplasmic inclusions [87]. Interestingly, homozygous SCA-3 patients with two mutant alleles exhibit earlier disease onset and more severe phenotypes than heterozygous individuals with one mutant allele [87], suggesting a role for the normal function of ataxin-3 in modulation of SCA-3 disease pathogenesis and progression. Recent bioinformatic analysis reveals that ataxin-3 contains two ubiquitin-interacting motifs (UIMs) and a Josephin domain that shares homology with the catalytic sites of UCH
Impairment of UPS: A Common Pathogenic Mechanism
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(ubiquitin carboxy-terminal hydrolase) and UBP (ubiquitin processing protease) classes of DUB enzymes [88]. The solution structure of ataxin-3 Josephin domain has been solved by NMR, which confirms that this domain indeed assumes the papain-like cysteine protease fold characteristic of other DUBs [89]. Moreover, biochemical studies have demonstrated that ataxin-3 binds polyubiquitin chains through its UIMs and exhibits deubiquitinating activity [89,90]. Further analysis of ataxin-3 enzymatic activity suggests that ataxin-3 functions as a polyubiquitin chain-editing enzyme that shortens K48-linked polyubiquitin chains [89,91]. Ataxin-3 also associates with the proteasome and has been implicated in regulation of UPSmediated protein degradation [90,92]. A very recent study has shown that, while polyglutamine expanded mutant ataxin-3 protein induces neurodegeneration in Drosophila, wild-type human ataxin-3 suppresses neurotoxicity induced by polyglutamine disease proteins, including mutant ataxin-3 protein itself as well as mutant huntingtin protein [93]. These data suggest that the normal function of ataxin-3 is neuroprotective.
IMPAIRMENT OF UPS COMPONENTS BY OXIDATIVE STRESS IN SPORADIC NEURODEGENERATIVE DISEASES Despite recent progress in identification of the genetic defects responsible for rare monogenic familial forms of neurodegenerative diseases, the causes of other forms of neurodegenerative diseases, particularly sporadic cases, remain largely unknown. Oxidative stress has been strongly implicated in the pathogenesis of many age-related neurodegenerative diseases, including AD, PD, and ALS [4,94,95]. For example, these diseases have been associated with increased production of reactive oxygen species (ROS) and/or impaired antioxidant defense systems, which could result from aging, genetic predisposition, and environmental factors [4]. Epidemiological studies suggest that exposure to pesticides, herbicides, and other environmental toxins that inhibit mitochondrial complex I, can lead to excess production of ROS and increased incidence of sporadic PD [96]. In addition to the mitochondria, the ER is also a major source of ROS [97,98]. ER stress caused by the accumulation of misfolded proteins, such as Pael-R in PD, leads to increased production of ROS which is damaging to neurons [99]. Dopaminergic neurons of the substantia nigra are thought to be particularly vulnerable to increased oxidative stress because of the intrinsic ability of dopamine to promote oxidative damage [100]. Consistent with this notion, oxidative stress induced by rotenone, paraquat, and 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), has been shown to produce PD-like phenotypes in rodents [101]. In spite of the overwhelming evidence linking oxidative stress to the pathogenesis of PD and other neurodegenerative diseases, relatively little is presently known of the biochemical pathways by which increased oxidative stress leads to neuronal dysfunction and, ultimately, neuronal cell death. Although it was initially thought that targets of oxidative damage by reactive oxygen species were random and indiscriminate, it has become increasingly clear that the susceptibility of proteins to oxidative damage is highly dependent on specific properties of individual proteins, such as unique sequence motifs, surface accessibility, and subcellular localization [102,103]. Emerging evidence indicates that oxidative stress can directly damage UPS components (Table 2). The oxidative damage to the UPS may
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contribute to neurodegeneration in sporadic neurodegenerative diseases in a manner similar to the genetic mutations of UPS components in causing familial neurodegenerative diseases (Figure 2).
Oxidative Damage to the Ubiquitination Machinery As mentioned earlier, a majority of E3 ubiquitin-protein ligases in cells are RING fingercontaining E3s. The RING finger motif is a cysteine/histidine-rich (C3HC4), Zn2+-binding domain that serves as the E3 catalytic core with the binding site for the E2 enzyme [7,14]. The Zn2+-bound cysteine thiolate anion (Cys-S-) is more reactive than the sulfhydryl group (Cys-SH), and can be readily modified by a variety of ROS and reactive nitrogen species (RNS) [104]. For example, the cysteine residues in the RING finger of APC11, a component of the multi-subunit E3 called anaphase-promoting complex, are oxidized in response to oxidative stress induced by H2O2. The cysteine oxidation induces dissociation of Zn2+ from the RING finger and disrupts the E2-binding site, leading to the loss of E3 activity [105]. Such oxidative stress-induced inactivation mechanism may also apply to other RING-type E3 ligases, including parkin. In fact, parkin has been reported to undergo misfolding and aggregation in response to H2O2 [106]. Furthermore, the cysteine residues in the RING fingers of parkin have been shown to be S-nitrosylated by nitric oxide (NO), resulting in a dramatic reduction in parkin’s E3 activity and neuroprotective function [107,108]. A very recent study has demonstrated that dopamine quinone, a reactive metabolite of dopamine oxidation, can covalently modify the cysteine residues in parkin RING fingers and functionally inactivate parkin, providing a mechanism linking the loss of parkin function with selective degeneration of dopaminergic neurons [109]. The dopamine-derived parkin adducts as well as S-nitrosylated parkin have been detected in brain samples from patients with sporadic PD [107-109], suggesting the involvement of oxidative and nitrosative stressinduced damage to parkin in the pathogenesis of sporadic PD. In addition to E3s, E1 and E2 enzymes also contain reactive cysteine residues that have the potential to serve as the targets for oxidative and nitrosative stress-induced modifications. In support of this possibility, the E2 enzyme UbcH7 is robustly modified by dopamine quinone in vitro [109]. Since these cysteine residues participate in the formation of highenergy thioester intermediates that are crucial for the catalysis of the ubiquitination reaction [7], oxidative and nitrosative stress-induced modifications of these residues would be expected to inactivate these enzymes, leading to a general inhibition of the UPS function which ultimately results in neuronal cell death in sporadic neurodegenerative diseases.
Oxidative Damage to DUB Enzymes The most widely used marker for oxidative damage to proteins is the presence of carbonyl groups, which can be introduced into proteins by direct oxidation of Pro, Arg, Lys, and Thr side chains, or by Michael addition reactions with products of lipid peroxidation or glycooxidation [94,102,103]. Postmortem analyses reveal that the total levels of protein
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carbonyls are elevated in brains from patients with AD, PD, or other neurodegenerative diseases [28,110]. However, the identities of the oxidized proteins that have been altered by carbonylation or other types of oxidation remain largely unknown. As a first step towards a molecular understanding of the pathogenic mechanism of oxidative stress in neurodegenerative diseases, we performed a search for specific protein targets of oxidative damage in sporadic AD and PD brains by using a proteomic approach that combined twodimensional gel electrophoresis, immunological detection of protein carbonylation, and mass spectrometry [111]. Interestingly, a major target of oxidative damage in AD and PD that we identified is UCH-L1. As described earlier, UCH-L1 is a DUB/E3 dual function enzyme whose mutations have been linked to early-onset familial PD in human and to gracile axonal dystrophy in mouse. Table 2. UPS components damaged by oxidative stress in sporadic neurodegenerative diseases Protein Function Modification Ubiquitination/deubiquitination machinery Parkin E3 S-nitrosylation Dopamine adduct APC11 E3 subunit Cys oxidation UbcH7 E2 Dopamine adduct UCH-L1 DUB/E3 Carbonylation Met oxidation Cys oxidation HNE adduct 26S proteasome S6 ATPase 19S cap subunit Carbonylation 20S core subunit HNE adduct α6 20S core HNE adduct α2, α6, α7 subunits 20S catalytic Acrolein adduct β subunits subunits
In vitro
Diseases
References
Yes Yes Yes Yes n.d. n.d. n.d. Yes
PD PD n.d. n.d. PD, AD PD, AD PD, AD n.d.
[107,108] [109] [105] [109] [111] [111] [111] [112]
Yes Yes Yes
n.d. n.d. IRI
[115] [116] [117]
Yes
n.d.
[118]
HNE, 4-hydroxy-2-nonenal; IRI, ischemia/reperfusion injury; n.d., not determined.
In addition to carbonylation, we found that UCH-L1 is also oxidatively modified by methionine oxidation and cysteine oxidation in sporadic AD and PD brains [111]. Oxidative damage to UCH-L1 by the identified modifications may result in irreversible alteration in the conformation and/or DUB/E3 enzymatic activities of UCH-L1, and thus has deleterious effects on neuronal function and survival similar to the pathogenic effects caused by the UCH-L1 genetic mutations as described earlier. Consistent with this notion, a recent in vitro study showed that the DUB activity of recombinant UCH-L1 was decreased upon oxidation of UCH-L1 by 4-hydroxy-2-nonenal, a lipid peroxidation product that generates carbonyl groups in proteins via Michael addition reactions [112]. Oxidative modifications may also render UCH-L1 itself more resistant to proteolysis and promote its aggregation into hallmark lesions of AD and PD brains. In support of this possibility, we and other groups have found
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the presence of abundant UCH-L1 protein in neurofibrillary tangles in AD and in Lewy bodies in PD brains [111,113]. Although UCH-L1 is the only identified DUB that is oxidatively damaged in sporadic neurodegenerative diseases, it is possible that other DUBs might also be the targets for oxidative and nitrosative stress-induced modifications. Out of the five known classes of DUB enzymes, four classes are cysteine proteases [17]. The active site cysteine residues usually have high propensity for being modified by a variety of ROS and RNS [104]. Modifications of the active site cysteine residues would inactivate DUB enzymes and result in abnormal protein ubiquitination and degradation, thereby contributing to the pathogenesis of sporadic neurodegenerative diseases (Figure 2).
Oxidative Damage to the Proteasome Accumulating evidence indicates that oxidative stress not only impairs the ubiquitination/deubiquitination machinery, but also causes direct damage to the 26S proteasome (Table 2). The endogenous product of inflammation 15-deoxy-delta (12, 14)prostaglandin J2 (PGJ2) is a potent inducer of intracellular oxidative stress implicated in the pathogenesis of a number of neurodegenerative diseases, including AD, PD, and ALS [114]. A recent proteomic study has shown that in human neuroblastoma SH-SY5Y cells, one of the subunits in the 19S regulatory complex of the 26S proteasome, S6 ATPase, is oxidatively damaged by carbonylation in response to oxidative stress induced by PGJ2 or H2O2 [115]. The oxidative damage to S6 ATPase is accompanied by a significant reduction in the S6 ATPase activity and in the ability of the 26S proteasome to degrade substrate proteins. In addition, the lipid peroxidation product 4-hydroxy-2-nonenal, a putative endogenous mediator of oxidative stress, has been shown to modify several α subunits (α2, α6, α7) of the 20S proteasome and inhibit the proteasome activity in vitro [116] as well as in a rat model of ischemia/reperfusion injury [117]. Furthermore, in SH-SY5Y cells, PD-associated environmental toxin rotenone has been shown to inhibit the proteasomal proteolytic activity by inducing oxidative modification of the catalytic β subunits of the 20S proteasome with the lipid peroxidation product acrolein [118]. The susceptibility of the proteasome components to oxidative stress-induced modifications as described above raises the possibility that the 26S proteasome is oxidatively damaged in brains of patients with sporadic neurodegenerative diseases. However, it remains to be determined whether this indeed is the case. Recently, the levels of 20S proteasome α (but not β) subunits and 20S proteasomal enzymatic activities have been reported to be reduced selectively in the substantia nigra of sporadic PD patients compared to age-matched controls [119,120]. Furthermore, an animal model study has shown that systemic exposure to proteasome inhibitors causes rats to develop PD-like phenotypes, including dopaminergic neurodegeneration, motor behavioral deficits, and accumulation of Lewy body-like protein aggregates [46]. These findings suggest that proteasome impairment plays a crucial role in the pathogenesis of sporadic PD. In addition to being damaged via direct oxidation of its subunits, the proteasome may be blocked or inhibited by oxidative stress-induced misfolded proteins and aggregates
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[47,48,121]. However, as pointed out earlier, the evidence supporting this view has come from studies using purified proteasome or cultured cells. It remains to be resolved whether oxidized proteins or aggregates can directly inhibit the proteasome in vivo.
CONCLUSIONS The UPS is an elaborate system that not only controls protein degradation via proteasome-mediated proteolysis, but also regulates protein function via multiple types of ubiquitination. Recent genetic studies of familial forms of neurodegenerative diseases have provided direct evidence linking dysregulation of ubiquitination to neurodegeneration. The list of disease-causing mutations in E3 ubiquitin-protein ligases and deubiquitinating enzymes is growing. Identification of the physiological substrates and the cellular processes that are regulated by each of these enzymes is crucial for understanding the role of the UPS in neuronal function and survival. It is important to investigate the proteasome-dependent as well as the proteasome-independent mechanisms of aberrant ubiquitination in the pathogenesis of neurodegenerative diseases. Moreover, future studies are needed to better understand the interplay between UPS dysfunction, oxidative stress, and protein aggregation. A mechanistic understanding of the UPS and its malfunction in various neurodegenerative diseases will undoubtedly facilitate the development of novel rational therapies for treating these devastating disorders.
ACKNOWLEDGEMENTS This work was supported in part by US National Institutes of Health Grants (AG021489, NS047199, NS050650, and NS047575) and Emory Center for Neurodegenerative DiseaseMerck Scholar Award.
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[117] Bulteau AL, Lundberg KC, Humphries KM, Sadek HA, Szweda PA, Friguet B, Szweda LI: Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion. J Biol Chem 2001, 276:30057-30063. [118] Shamoto-Nagai M, Maruyama W, Kato Y, Isobe K, Tanaka M, Naoi M, Osawa T: An inhibitor of mitochondrial complex I, rotenone, inactivates proteasome by oxidative modification and induces aggregation of oxidized proteins in SH-SY5Y cells. J Neurosci Res 2003, 74:589-597. [119] McNaught KS, Jenner P: Proteasomal function is impaired in substantia nigra in Parkinson's disease. Neurosci Lett 2001, 297:191-194. [120] McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW: Altered proteasomal function in sporadic Parkinson's disease. Exp Neurol 2003, 179:38-46. [121] Le Pecheur M, Bourdon E, Paly E, Farout L, Friguet B, London J: Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice. FEBS Lett 2005, 579:3613-3618.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 579-598 © 2007 Nova Science Publishers, Inc.
Chapter 24
NEUROPROTECTIVE AND PRO-APOPTOTIC RESPONSES OF UBIQUITIN PROTEASOME SYSTEM Meng Shyan Choy and Nam Sang Cheung∗ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597.
ABSTRACT The accumulation of unfolded, misfolded or damaged proteins in cells is a threat to cell survival. The ubiquitin-proteasome system (UPS) is responsible for the degradation of these abnormal proteins. UPS dysfunction has been postulated to play a key role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Both normal and misfolded proteins can undergo highly specific degradation by the UPS. Selective degradation of correctly folded proteins underlies many cellular regulations. Examples include degradation of cyclins or their inhibitors in the regulation of the cell cycle and the degradation of IκB in the activation of immunity responses. The endoplasmic reticulum (ER) is the site of synthesis of membrane proteins and secretory proteins. In the ER, defective or unfolded proteins are degraded [a process known as ERassociated proteins degradation (ERAD)], whereas correctly folded proteins are spared. In the familial form of Alzheimer’s disease, transcriptional misreading of the stressinduced polyubiquitin gene produces ubiquitin with aberrant C-terminal extensions that competitively inhibit proteasomal function. This inhibition of UPS may impair ERAD, thereby causing the accumulation of misfolded proteins in the ER, resulting in ER stress and induction of cell death through the activation of calpain and caspase-3. Proteasome inhibitors such as lactacystin have been reported to activate the pro-apoptotic transcription factor C/EBP-homologous protein (CHOP) and to cause cell death in ∗
Correspondence concerning this article should be addressed to Dr. Nam Sang Cheung, PhD; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597. Phone: 65-65164780; Fax: 65-67791453 ; E-mail:
[email protected].
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Meng Shyan Choy and Nam Sang Cheung cultured cortical neurons. Although the inhibition of proteasomes has been linked to cell death, recent studies have shown that, below a threshold level, proteasome inhibition can activate neuroprotective responses — proteasome inhibition has been shown to induce various molecular chaperones such as heat shock proteins (HSPs) that increase cell tolerance to the accumulation of unfolded and damaged proteins, stimulate the expression of UPS components through a feedback mechanism, and suppress inflammatory responses by inhibiting IκB degradation. Although the inhibition of proteasomes may stimulate neuroprotective responses, prolonged ER stress ultimately leads to apoptosis. Further studies to elucidate the impact of proteasomal inhibition on other cellular signaling pathways may provide insights on the interplay between the UPS and cell physiology. A better understanding of the function and activation of the neuroprotective or pro-apoptotic responses would provide a means to manipulate this pathway in order to cure diseases associated with unfolded proteins. Much remains to be discovered about the inducibility and functioning of chaperones and neuroprotective ubiquitin-proteasome pathways in neurons. Such studies would be useful, since genetic polymorphism in these protective systems and changes in their expression with ageing may play critical roles in the accumulation of unfolded or damaged proteins, and in the pathogenesis of disease. Moreover, pharmacological induction or activation of these protein repair-anddegradative systems could in future be developed into innovative therapies for neurodegenerative diseases.
Keywords: Neuroprotective, pro-apoptotic, neuron, microarray, lactacystin.
ABBREVIATIONS ARJP, autosomal recessive juvenile parkinsonism; Atf4, activating transcription factor 4; ATF6, activating transcription factor 6; C/EBP, CCAAT/enhancer binding protein; Cebpb, C/EBP beta; CHOP, C/EBP-homologous protein; COX-2, cyclooxygenase-2; Ddit3, DNAdamaged inducible transcripts 3; EGCG, (-)-epigallocatechin-3-gallate; ER, endoplasmic reticulum; ERAD, ER associated proteins degradation; EST, expressed sequence tag; GSH, reduced glutathione; HSP, heat shock protein; ROS, reactive oxygen species; SREBP, sterol regulatory element binding proteins; UBB, ubiquitin B; UPS, ubiquitin-proteasome system.
INTRODUCTION Aggregation of Misfolded Proteins and Neurodegeneration The abnormal accumulation of misfolded proteins as protein aggregates in neurons is a hallmark of many neurodegenerative diseases [1] and can lead to stress and cell death [2]. Misfolded proteins tend to aggregate because the normally buried hydrophobic domains of these proteins associate with one another. Eukaryotic cells have two main strategies to counteract the formation of such protein aggregates. First, the induction of heat shock proteins (HSPs) and molecular chaperones that are involved in the protein refolding systems and second, targeted degradation of damaged and misfolded proteins by the UPS [3,4,5]. In
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conditions where UPS activity is affected, the homeostasis between protein synthesis and protein degradation is disrupted, and protein aggregation occurs [6]. The dysfunction of UPS has been proposed as one of the main causes of neurodegeneration [1,7,8]. The identification of a number of genes responsible for rare familial forms of neurodegenerative diseases has provided insights into the underlying mechanisms of these diseases [9,10]. For example, lossof-function mutations in the gene encoding ubiquitin protein ligase (E3), Parkin, are linked to autosomal recessive juvenile parkinsonism (ARJP) [9,11,12], while over-expression of Parkin could counter misfolded protein stress-induced cell death [2]. Recent studies also revealed a frame-shift mutation of the ubiquitin B gene (UBB) that produces a variant form of ubiquitin, UBB+1, a major component in intracellular protein inclusions in Alzheimer’s disease and progressive supranuclear palsy. This mutation results in the absence of the C-terminal G76 of ubiquitin, preventing the ligation of UBB+1 to target protein substrates or poly-ubiquitin chains. Instead, UBB+1 is itself readily poly-ubiquitinated, so that it acts as a potent competitive inhibitor of 26S proteasome [13,14,15]. Familial types of neurodegenerative diseases, such as certain forms of Parkinson’s disease, are linked to loss-of-function mutations of ubiquitin ligase. The sporadic forms of neurodegeneration are, however, often associated with ageing. In ageing cells, the capacity to handle the accumulation of misfolded or damaged proteins is insufficient to prevent their accumulation and resultant toxicity [16]. Furthermore, recent studies reported that expression of neuroprotective antioxidants and molecular chaperones decreases in ageing cells [16; and Chapter 22]. The resultant accumulation of misfolded protein aggregates leads to inhibition of the UPS. The expression, functions and regulation of heat shock proteins (HSPs), molecular chaperones and the UPS components in mammalian cells are largely not well understood (see Chapter 19). Investigations in these areas could therefore provide valuable insights into the molecular mechanisms behind the pathogenesis of neurodegenerative diseases. Pharmacological induction or activation of these protein repair-and-degradative systems could lead to the development of innovative therapies for neurodegenerative diseases [1]. Proteasome inhibitors have been used widely in the study of the UPS system in mammalian cells [17,18]. In addition to being useful research tools for dissecting the roles of the proteasome, these inhibitors have potential applications in biotechnology and medicine. Proteasome inhibitors are known to induce cell death [19]. On the other hand, they can also protect cells against other insults. For example, the proteasome inhibitor MLN-519 has been shown to be very effective in suppressing the production of inflammatory mediators in stroke models [20]. Furthermore, proteasome inhibition can also induce the synthesis of various HSPs, which increase tolerance of cells to stressful conditions [21]. It appears that proteasome inhibition induces both protective and apoptotic effects in mammalian cells, depending on the presence or absence of a number of factors (see Chapter 21 and [19,22]). The factors that seem to be involved in these contradictory outcomes of proteasome inhibition are (i) the duration of exposure, (ii) the concentration of proteasome inhibitor used, (iii) the type of cells used and (iv) the type of proteasome inhibitor used [19,23,24]. The fundamental objectives in any neurodegeneration and neuroprotection research are to determine the factors constituting the primary event, the sequence in which these events occur, and whether they act in concurrence in the pathogenic process [25]. Our study of genes
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differentially expressed during lactacystin-induced neuronal apoptosis using microarray technique has shown the induction of potentially neuroprotective and pro-apoptotic transcriptional responses [22]. However, the sequence of events leading to the process of neuronal apoptosis is not clear. This paper focuses on our research findings from the time course microarray study of the genes differentially expressed during lactacystin-induced neuronal apoptosis, and on other recent findings in this area of research. Our microarray study revealed that treatment of cultured cortical neurons with 1 μM lactacystin resulted in apoptosis and a large number of genes being differentially expressed: out of a total of 12488 genes and expressed sequence tags (ESTs) in the murine genome GeneChip® U74Av2, the expressions of 1168 genes were enhanced more than two-fold, according to the one-way ANOVA, p99% pure tubulin preparation [101]. Consistent with this tight interaction, parkin exhibits punctuate subcellular localization along microtubules [90]. The ability of parkin to ubiquitinate α- and β-tubulin and to facilitate their degradation may be very important for cell survival. The synthesis of α- and β-tubulin polypeptides are very tightly regulated at the transcriptional and translational levels to ensure the equimolar production of both tubulins [104], as overexpression of either tubulin gene is toxic to the cell [105,106]. The formation of polymerization-competent α/β heterodimers requires a series of folding reactions that are perhaps the most complicated for all known proteins (reviewed in [109]. First, α or β polypeptide is folded into quasi-native conformers with the help of cytosolic chaperonins, which are ribosome-sized multisubunit complexes that facilitate protein folding in an ATP-dependent manner. Second, α and β monomers are captured by tubulin-specific folding cofactors. In a sequential, coordinated and reversible folding process catalyzed by cofactors A through E, α/β heterodimers are formed with the hydrolysis of GTP. Misfolded tubulins produced during this complicated process are quickly degraded through an unknown mechanism. As an E3 ligase for tubulins, parkin may well ubiquitinate misfolded tubulins to facilitate their degradation. Because the tubulin folding process is dependent on ATP and GTP hydrolysis, mitochondrial complex I-inhibiting PD toxins that can reduce ATP production (e.g. rotenone and MPP+) may adversely affect the folding reaction and lead to increased production of misfolded tubulin. In addition, the ability of these PD toxins to depolymerize microtubules [113-116] would further increase the amount of tubulin that needs to be degraded. It has been known for a long time that microtubule depolymerization leads to rapid degradation of tubulin, a protein that normally has a very long half-life [117]. By ubiquitinating and degrading tubulin, parkin could prevent the same kind of toxicity caused by overexpression of tubulin [105,106]. Because nigral DA neurons have very long axons projecting to striatum, a very high percentage of total cell volume is in the axon, which contains large quantities of microtubules. Thus, exposure of these PD toxins may result in much higher demand to ubiquitinate and degrade tubulin in nigral DA neurons than that in other types of cells with smaller volume and shorter processes. Our studies have shown the TH+ neurons in midbrain neuronal cultures are much more vulnerable than TH-
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neurons to microtubule depolymerizing agents such as rotenone, colchicine or nocodazole [118]. Many parkin substrates, such as Pael-R [83], DAT [119], synaptotagmin XI [120], CDCrel-1 [82], are transmembrane proteins or membrane-associated proteins. At least some of these proteins are prone to misfold in the endoplasmic reticulum (ER) [83,119], which causes UPR if left unchecked [121] (see Chapter 13). Previous studies have demonstrated that misfolded membrane proteins are retroversely translocated from the ER to the cytosol, where they must be immediately ubiquitinated to avoid aggregation due to the abundance of hydrophobic residues left exposed by the disordered polypeptide chain [122]. Under normal situations, the ER is attached to microtubules to maintain its morphology and stability [123125]. The proximity of the ER to parkin, which is anchored on microtubules [90,126], gives parkin ideal accssess to misfolded substrates as they are retrotranslocated from the ER. Many misfolded proteins are transported along microtubules to the proteolytic center of the cell, a perinuclear area around the centrosome, where under conditions of impaired proteolysis area to form a single large inclusion is formed [127], termed the ‘aggresome’ [128] (see Chapter 12). The localization of parkin along microtubules [90] would greatly facilitate the ubiquitination of its substrates and their subsequent transport on microtubules to the proteolytic centeraggresome. Previous studies have shown that parkin and its substrates, such as CDCrel-1, Pael-R and DAT, are accumulated in the perinuclear aggresomesarea when protein degradation is compromised by proteasome inhibitors or an excess of misfolded proteins themselves [119,129,130]. Accumulation of these proteins around the centrosome is a microtubule-dependent process, since disruption or overt stabilization of microtubules abolishes this phenomenon [130]. It appears that parkin anchored on microtubules may serve as sentinels to efficiently ubiquitinate misfolded proteins for their destruction by the 26S proteasome or transportation along microtubules to the aggresome, proteasomes should be overwhelmed by misfolded proteins. Thus, a unique combination of several features – parkin as an E3 ligase anchored on microtubules, the attachment of ER to microtubules, and the need to retrotranslocate misfolded transmembrane proteins from the ER for ubiquitination and degradation in the cytosol – makes parkin ideally suited to ubiquitinate transmembrane or membrane-associated substrates. The ubiquitination of DAT by parkin appears to be a novel mechanism to regulate the functions of DAT, which are critical to dopaminergic transmission. In addition to these functions that are dependent on the E3 ligase activity of parkin, our recent studies have shown that parkin suppresses the transcription of monoamine oxidases through an E3-independent mechanism [131]. Monoamine oxidase has two isoforms, MAO-A and MAO-B, which are encoded by two distinct genes [132]. Both MAOs are located on the cytoplasmic side of the mitochondrial outer membrane through a Cterminal tail anchored in the membrane [133,134]. MAO-catalyzed oxidative deamination of monoamines produces H2O2 that has been shown to damage mitochondrial DNA [135]. Thus, ROS produced during dopamine oxidation can affect mitochondrial functions and may lead to reduced ATP production and increased generation of ROS, if the electron transfer chain is disrupted. Several lines of evidence have indicated a strong involvement of MAO in PD. It has been found that PD patients have elevated MAO-B activity in the substantia nigra [136]. Consistent with these, MAO inhibitors such as deprenyl have been widely used to delay the progression of PD symptoms [137]. On the other hand, MAO-B knockout mice are resistant
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to the PD toxin MPTP [138], because MAO-B is responsible for the conversion of MPTP to MPP+ [139], the active toxin that is selectively taken up by DA neurons through the dopamine transporter [140]. Thus, results from both basic research and clinical studies point to MAO as an important factor in PD pathogenesis. Our studies have shown that overexpression of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y or mouse fibroblast NIH3T3 cells significantly reduces the mRNA levels of both MAO-A and MAO-B. In B-lymphocyte cells obtained from a PD family with deletion of parkin exon 4, levels of both MAOs are significantly increased in the homozygous, but not heterozygous, carrier of this mutation [131]. This study complements several studies that have indicated the involvement of parkin in mitochondrial functions. In parkin-deficient Drosophila, the flight and climbing problems are caused by apoptotic degeneration of indirect flight muscles, which is accompanied by abnormal mitochondrial morphology [38]. Mitochondrial defects are also found in spermatids of male mutant flies, which are sterile due to the lack of mature sperm [38]. Consistent with this, overexpression of parkin in PC12 cells delays ceramide-induced mitochondrial swelling and subsequent release of cytochrome C and apoptosis [141]. Parkin is shown to be associated with mitochondria and may interact with an unknown target that is critical for mitochondrial functions [141]. A recent report shows that parkin enhances mitochondrial biogenesis in proliferating cells by regulating the expression of many mitochondrial proteins [142]. How does parkin, a cytosolic protein, regulate the gene expression of mitochondrial proteins such as MAOs? Parkin has a RING-IBR-RING motif in the C-terminus [143]. RING (Really Interesting New Gene) finger is a variation of zinc finger, while IBR (In-between RING fingers) domain is a zinc finger-like, putative metal-binding motif found between two RING fingers in a group of proteins implicated in transcription regulation. One member in the family, RBCK1 binds to DNA and activates transcription in in vitro assays [144,145]. More recently, parc (p53-associated, parkin-like cytoplasmic protein) has been found to contain the RING-IBR-RING motif in the C-terminus. Although parc also has E3 ligase activity and can ubiquitinate itself, it does not ubiquitinate p53, to which it binds strongly. Parc suppresses p53-dependent gene transcription and apoptosis by tethering it in the cytosol [146]. Previous studies have also shown that p53 is anchored in the cytoplasm through interaction with microtubules [147]. It seems likely that parc and microtubules may act in a concerted manner to sequester a portion of p53 in the cytoplasmic pool, away from its transcriptional activities in the nucleus [148]. Our previous studies have found that parkin binds to microtubules strongly and appears to be absent in the nucleus [90]. In a similar manner to parc, parkin might tether a transcription factor for mitochondrial proteins in the cytosol to regulate their transcription. Thus, a general picture about cellular functions of parkin appears to center on its unusually strong interaction with tubulin and microtubules. Three different but connected functions of parkin need to be considered within the context of its interactions with tubulin and microtubules. First, the strong binding between parkin and microtubules stabilizes the microtubule network against depolymerizing agents [126], which exert much higher toxicity on dopaminergic neurons than non-dopaminergic neurons [118]. The unique combination of cell morphology (long axons enriched with microtubules) and neurochemistry [production of
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reactive oxygen species (ROS) during dopamine metabolism] of dopaminergic neurons renders these cells particularly vulnerable to PD toxins such as rotenone and MPP+, which depolymerize microtubules and inhibit mitochondrial respiratory chain. Since microtubule depolymerization induces rapid degradation of tubulin [117], the ability of parkin to ubiquitinate and degrade tubulin would also be critical for the survival of the cell in the presence of these PD toxins. Second, the effectiveness of parkin to handle misfolded transmembrane proteins such as DAT appears to be related to several important facts: (i) misfolded transmembrane proteins need to be retrotranslocated from the ER to the cytosol to be ubiquitinated and degraded; (ii) ER is attached to microtubules; (iii) parkin binds strongly to microtubules and exhibits punctated localization along microtubules. It seems plausible that parkin, which is anchored on microtubules, may act as sentinels to efficiently ubiquitinate misfolded transmembrane proteins as they are reverse translocated from the ER to the cytosol. If this process goes awry (e.g. when parkin is mutated and loses its E3 ligase activity), these misfolded transmembrane substrates of parkin would easily aggregate and trigger UPR. In the case of dopamine transporter, parkin increases its surface expression by ubiquitinating and degrading misfolded DAT molecules [119]. This allows native DAT conformers to oligomerize with each other into functional transporters destined for the plasma membrane. This function of parkin increases dopamine uptake and thereby enhances the temporal and spatial precision of dopaminergic transmission. Third, the ability of parkin to regulate expression of mitochondrial proteins such as MAO may be mediated by sequestration of key transcription factor(s) in the cytosol. It is quite clear at least in the case for MAO that, parkin affects gene expression through a mechanism independent of its E3 ligase activity [131]. Instead, parkin may serve as a cytosolic anchor to tether transcription factors away from DNA and transcriptional machinery in the nucleus. By suppressing the expression level of MAO, parkin may limit the production of reactive oxygen species generated during dopamine oxidation by MAO.
UCH-L1 UCH-L1 (ubiquitin carboxy-terminal hydrolase L1) was originally identified in neurons and testis of mammals [149] but is known now to occur as widespread as Aplysia [150] and Drosophila [151]. The enzyme is a member of a family of ubiquitin hydrolases which in mammals consists of UCH-L1, UCH-L3, UCH-5 and BAP1. UCH-L1 is one of the most abundant proteins in the mammalian brain (1-2% of total brain protein content) [149,152]. The primary function of the enzyme is the deubiquitination of C-terminal esters and amides of ubiquitin resulting in monomeric ubiquitin, an essential step prior to degradation of polyubiquitinated proteins by the proteasome. UCH-L1 has been implicated in the pathogenesis of PD because of a misense mutation (I93M) in the fourth exons of the uch-l1 gene which was identified in a German family, leading to an increased susceptibility for PD. This mutation reduces enzyme activity by about 50% [153]. It has also been identified as a constituent of LB [154]. Curiously enough, another polymorphism in the uch-l1 gene (S18Y) in humans results in a decreased
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susceptibility for PD [32,155-157]. The S18Y mutation in the enzyme increases the hydrolase activity when compared with the wild-type enzyme [158]. However, it has been observed that UCH-L1 in its dimeric form has ubiquityl ligase activity with a potential for polyubiquitinating K63 of α-synuclein resulting in an elevated cytoplasmatic concentration of this protein [32]. The effect of the S18Y mutation is a reduction in dimerization while maintaining almost wild-type or higher hydrolase activity which would explain the reduced risk for PD. The issue of involvement of UCH-L1 in PD is further complicated by the fact that complete lack of UCH-L1 activity as found in the gad mouse does not result in a Parkinson phenotype in the mouse. The gad mouse carries a spontaneous, autosomal recessive mutation in the Uchl1 gene because of an intragenic deletion of exons 7 and 8 resulting in an Uchl1 null mutant [159]. Analysis of the clinical phenotype demonstrates severe sensory ataxia at the early stage of the disease followed by motor paresis [160]. The underlying defect in the gad mouse is axonal degeneration in the gracile tract, the formation of spheroid bodies in nerve terminals and progressive accumulation of ubiquitinated proteins [161]. The loss of UCH-L1 function might lead to a decreased pool of available ubiquitin which in turn would interfere with protein degradation via the UPS pathway.
DJ-1 A possible role for the gene DJ-1 in PD was established when mutations in DJ-1 were identified in the PARK7 locus, causing an early onset form of PD [162]. The recessive mutation is quite rare, being responsible for an estimated 1-2% of early onset cases [163]. The DJ-1 gene has a size of about 24kb containing 8 exons and giving rise to a major mRNA of about 1kb with an open reading frame of 570bp encoding a protein of 189 amino acids [162]. The protein is widely expressed in the brain and peripheral tissues but a clear physiological role for the protein has yet to be established [164,165]. Based on its structure, the protein is related to ThiJ/PfpI/DJ1 superfamily [162]. The wild-type protein can form homodimers and some of the known mutations disrupt dimer formation –e.g. L166P but not M26I and E64D which have also been identified in patients with early onset parkinsonism. It has been suggested that the protein modulates gene expression under conditions of cellular stress, so providing a protective effect against oxidative stress [113,162]. The possible stress sensor might be the sulfhydryl group of cysteine C106 which can be oxidized to sulfonic acid, resulting in a shift of the pI of the DJ-1 protein [166,167]. There is some experimental evidence indicating that some of the oxidized form of DJ-1 are localized to mitochondria but the physiological significance of this translocation is unclear. There is currently no evidence that this step interferes with mitochondrial function and especially ATP generation which is important for proteasome assembly and function (for a review of these issues see [39]). In summary – there is currently no evidence that DJ-1 and its known mutations are linked to UPS malfunction.
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PINK 1 The Pink1 (PTEN-induced kinase 1) gene was identified as part of the PARK 6 locus [168]. Homozygous mutations in this gene are causing PD of the early onset type. Although disease transmission is recessive, it should be noted that about 5% of the sporadic early-onset type of PD have single heterozygous PINK1 mutations. The gene spans 18 kb, containing 8 exons and encodes a 581 amino acids protein [169]. Based on a highly conserved protein kinase domain (amino acids 156 to 509) it has been suggested that the protein encodes a putative serine/threonine kinase of the Ca2+ -calmodulin type with a mitochondrial targeting sequence. Since the identified mutations are in and around the kinase domain it is assumed that loss of the kinase activity is responsible for the disease but this as well as the physiological function of the enzyme remains to be clarified. Recent studies in Drosophila have shown that PINK 1 mutants show apoptotic muscle degeneration, defects in mitochondrial morphology, degeneration of dopaminergic neurons and increased sensitivity to oxidative stress [170-172]. Expression of human PINK1 in the Drosophila mutants rescues the defects observed in the mutants, indicating evolutionary conservation of PINK1 function. It was further noted that loss of Drosophila parkin showed phenotypes similar to loss of pink1 function. Overexpression of parkin rescues the mitochondrial morphological defects in pink1 mutants. These and other observations indicate that parkin and pink1 appear to function in the same pathway with pink1 functioning upstream of parkin. More recently it was shown that inactivation of PINK1 in Drosophila results in progressive loss of dopaminergic neurons and in degeneration of the ommatidia in the compound eye. The degeneration was significantly inhibited by expression of human SOD1 or by Vitamin E indicating that PINK1 contributes to neuronal survival by preventing oxidative stress [173]. A recent report indicates that PINK1 and other mitochondrial proteins can localize to the aggresome (see below) upon proteasome inhibition with MG132 as part of a process which also localizes mitochondria to the aggresome [170]. This appears to happen only under proteasome stress when either proteasome function is diminished or when there is an excessive build-up of damaged or misfolded proteins. It has been suggested that the recruitment of mitochondria to the aggresome might facilitate protein degradation by providing additional ATP.
LRRK2 Leucine-rich repeat kinase 2 (LRRK2, dardarin) recently identified as part of the PARK8 locus, is associated with late-onset PD [174]. The protein is expressed in the brain, predominantly in the basal ganglia and hippocampus but is also found in other tissues [175,176]. The LRRK2 gene is very complex, containing 51 exons and encodes a very large protein of 2527 amino acids. Sequence analysis reveals 6 independent major domains – an ankyrin repeat region, a leucine-rich repeat domain, a ROC GTPase domain, a COR (C terminal of ROC)-domain, a tyrosine kinase-like domain and a C-terminal WD40 domain
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[177,178]. The region between amino acids 180 to 660 is predicted to fold as armadillo repeats. In addition to the unusual feature of containing potentially two enzymatic centers – the predicted protein kinase and the GTPase activities - LRRK2 contains four protein interaction domains, making it potentially the scaffold of a multiprotein signaling complex. This idea is supported by the fact that the 22 identified mutations in the gene, known to cause PD, are distributed across all the catalytic and protein-protein interaction domains of LRRK2. Relevant to the discussion here is the observation that LRRK2 can interact with the E3ligase parkin through the COR domain although there is currently no evidence that this interaction results in polyubiquitination or degradation of the protein [179]. Clearly, this issue requires more experimental clarification.
AUTOPHAGY An alternative to the UPS for the degradation of intracellular proteins is autophagy which is carried out in the lysosomal compartment. It has been observed that perinuclear aggregates (later named ‘aggresomes’) of misfolded proteins formed upon proteasome inhibition are removed from the cells by formation of autophagosomes [127], a finding which was recently extended [180,181]. Autophagy is responsible for removal of bulk cytoplasmatic constituents including cell organells. This might represent a functional survival strategy of eukaryotic cells in response to sublethal damage. Based on the mechanism of autophagic vacuole formation and the delivery of material to the autophagic vacuole, three types of autophagy can be distinguished - macroautophagy, microautophagy and chaperone- mediated autophagy. Macroautophagy involves the sequestration of parts of the cytosol, including not only soluble proteins but also cell organelles into a double membrane vesicle called the autophagosome which is subsequently fused with the lysosome to provide the proteolytic and acidic pH environment required for degradation of the sequestered proteins. Microautophagy involves the wrapping of and degradation of cytosolic regions as well as cell organelles by engulfing directly into the lysosome. In chaperone- mediated autophagy the substrate proteins degraded thru this pathway share the pentapeptide motiv KFERQ. This motive is recognized in the cytosol by the chaperone hsc73, a heatshock protein of 73 kDa, leading ultimately to the binding of this complex to the LAMP2a receptor (lysome-associated membrane protein 2a) and translocation into the lumen of the lysosome. (for reviews see.[182-185]). Autophagy is mediated by the Atg family of proteins, which consist of 29 different members in yeast (see Table 1) [186]. So far, 18 of these genes have also been shown to occur in other eukaryotic systems. Two of the Atg proteins are conjugated to acceptor protein and phospholidpid in a manner similar to ubiquitin [187-189]). A distinct and important role of autophagy in the nervous system has been known for a long time in cases of injury and regeration of peripheral axons [190]. Only in the last few years experimental evidence also clearly revealed an important role of autophagy in neurodegenerative diseases (for a review see [185] and Chapter 28). Specifically, in PD the occurence of autophagic vacuoles engulfing mitochondria in the substantia nigra point to an activation of macroautophagy [191]. In cellular models of of PD – e.g. the overexpression of mutant A53T α-synuclein in PC12 cells produced a marked increase in autophagy and cell
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death different from apoptosis [192]. A marked decrease in chaperone-mediated autophagy was noticed in PC12 cells under these conditions because the mutant α-synuclein blocks the LAMP2a receptor, leading to an activation of macroautophagy [193,194]. It has further been observed that in neuronal cell lines inhibition of the UPS also causes activation of macroautophagy presumably as compensatory response. The molecular linkage between the UPS system, macroautopagy and chaperone-mediated autophagy is currently unknown. Because α-synuclein constitutes a major portion of LB a large number of studies have focused on its degradation. The protein carries the KFERQ motif which would suggest chaperone-mediated autophagy as an important degradation pathway [185,193]. There exist conflicting data if α-synuclein is mainly degraded by the UPS or by autophagy in vivo [195]. Interestingly, addition of rapamycin which inhibits the nutrient sensor kinase mTor, leads to an increased formation of autopaghosomes resulting in an increased clearance of α-synuclein [195]. Macroautophagy might be an compensatory response if chaperone-mediated autophagy and/or the UPS is blocked or at least reduced in activity. Table 1. Autophagy-Related Genes Gene Atg1 Atg2 Atg3 Atg4 Atg5 Atg6 Atg7 Atg8 Atg9 Atg10 Atg11 Atg12 Atg13 Atg14 Atg15 Atg16 Atg17 Atg18 Atg19 Atg20 ATG21 ATG22 Atg23 Atg24 Atg25 Atg26 Atg27 ATG28 ATG29
Protein Function Protein Kinase Peripheral membrane interacts with Atg9 E2-like enzyme, conjugates Atg8 to phosphatidylethanoleamine (PE) Cysteine protease Conjugated to Atg12 via internal lysine Component of Ptdlns 3-kinase complexes I and II E1-like enzyme, activates Atg8 and Atg12 Ubiquitine-like protein, conjugated to PE via C-terminal glycine Integral membrane protein E2-like enzyme, conjugates Atg12 to Atg5 Involved in cargo recognition Ubiquitine-like protein, conjugated to Atg5 via C-terminal glycine Modifier of Atg1 activity by phosphorylation Component of Ptdlns 3-kinase complex1 Lipase required for breakdown of intravacuolar vesicles Part of Atg12-Atg5 complex Modifier of Atg1 activity Peripheral membrane protein required for localization of Atg2 Cargo receptor for Cvt pathway PX domain protein needed for the Cvt pathway Specific to Cvt pathway Integral membrane protein involved in autophagic breakdown Needed for Cvt vesicle completion Sorting of nexin Coiled-coil protein involved in macropexphagy UDP-glucose:sterol glucosyltransferase-containing GRAM domain Ptdlns(3)P binding protein required for Cvt pathway Degradation of peroxisomes (pexophagy) Autophagosome formation
References [3-7] [12-16] [20-23] [15,27-29] [33-36] [7,33,41,42] [7,15,47,48,48,49] [6,7,27,28,35,35,57-59] [6,16,63-65] [68,69] [6,47] [34-36,73,74] [33,41] [80,81] [6,84,85] [87] [7,91,92] [94,95] [98] [99] [100] [102] [98] [103] [6,107,108] [110] [111] [112]
Summary of the currently known ‘autophagy-related’ (Atg) genes and their functions. Genes which have been demonstrated so far to occur only in yeast are italicized. Adapted from Klionsky et al., 2003 [186].
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The essential role of autophagy in neurodegeneration was recently demonstrated using conditional mutations of Atg5 and Atg7 in mice [196,197]. Atg5 flox/flox and Atg7 flox/flox mice were crossed with mice expressing Cre- recombinase under the nestin promoter which is expressed in neuronal precursor cells after embryonic day 10.5. Both mice developed progressive deficits in motor function which was accompanied by the accumulation of polyubiquitinated cytoplasmatic inclusion bodies. These results suggest that autophagy is essential for survival of neuronal cells. Finally, it should be emphasized that an emerging body of data indicates that autophagy plays also an important role in a variety of other neurodegenerativer diseases as Alzheimer’s disease and Polyglutamine disease (for a review see [185]) presumably because of the occurence of protein deposits are a main histopathological finding of these neurodegenrative diseases.
CONCLUSIONS For our current understanding of PD two observations appear to be important: first, αsynuclein is the major component of filamentous LB [131] and secondly, certain point mutations in α-synuclein are pathogenic resulting in various forms of PD. However, while the case for an involvment of α-synuclein in PD is very strong, it remains somehow mysterious why wild-type α-synuclein in the majority of PD patients accumulates in LB. In addition, although PD is a major neurodegenerative illness affecting 5% of individuals older than 85 years [198,199], it is unknown why the remaining 95% of individuals are disease-free. Although α-synuclein has a propensity for misfolding and fibrillization into filamentous LB due to its conformation in an aqueous environment, other factors appear to be neccessary to trigger PD. Genetic screens to search for the causative factor(s) of PD have resulted in the identification of 11 genetic loci which cause a disease which variably resembles PD (see Chapter 25 of this volume). At least five of these genes (PARK1, PARK2, PARK4, PARK5 and PARK7) are linked directly or indirectly to the UPS. Recent studies of the proteasome indicate that its activity declines with age (for a review, see [200]. Because of its propensity for misfolding and denaturation, the maintenance of functional levels of α-synuclein might be under the best of circumstances a race against time to eliminate misfolded forms of this protein by either refolding by molecular chaperones or by degradation via the protein degradation machinery of the cell. It has been suggested that the failure of the UPS is the main cause of PD [201], but the causes for the decline are currently unknown. Systemic administration of a lipid-soluble proteasome inhibitor able to penetrate the blood-brain bareer induces parkinsonism in rats [202], raising the question of a possible induction fo parkinsonism in patients receiving Velcade and other proteasome inhibitors. One critical factor for proteasome function is the assembly of the 26S proteasome from the 19S and 20S precursor complexes which is ATP dependent. In Drosophila, proteasome activity is reduced by half in old vs. young flies with a concomitant decrease in ATP levels by 50% [203]. While in rats no or only a very mild decline in ATP concentration is found with increasing age a pronounced reduction in ATP turnover has been observed with age in the parietotemporal
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cortex and hippocampus of rats which also performed poorly in a memory test [204]. This effect might be attributable to a decline in ATP/ADP translocase which would diminish the release of ATP from the mitochondria into the cytoplasma [205]. It is however well established that mitochondrial function in the nervous system declines with age [206,207]. A reduction in ATP levels might also impair the ability of the cell to collect protein aggregates into perinuclear the aggrosomes. It has been reported that in Drosophila reduced ATP levels selectively affect molecular motors [208] which might reduce the ability of the cell to form aggresomes and so to diminish the cellular burden of protein aggregates. In the sporadic form of PD a reduction in mitochondrial complex I activity has been observed in the substantia nigra [209,210] indicating a reduction in ATP which would not only affect proteasome activity but also other steps of the UPS system. It is further known that treatment with the pesticide rotenone, a known inhibitor of mitochondria, results in degeneration of the nigrostriatal dopaminergic system [11]. While no detailed studies of the ATP requirements of autophagy have been carried out it is clear that this complex system requires ATP in a variety of steps. It is also known that chaperone-mediated autophagy declines with age. So, a unifying theme – the decline in the ability of cells to maintain protein structure and to degrade proteins possibly because of a decrease in ATP, might be linking unfolded protein stress, aggresome formation, UPS and autophagy. Obviously, the mechanistic details for the decline in protein degradation need to be investigated. If indeed mitochondrial malfunction is at the core of the problem appropriate scientific and clinical studies should be initiated to investigate if this will lead to new drug discoveries for the treatment of PD.
ACKNOWLEDGEMENTS We wish to apologize to the many colleagues whose work for reasons of space could not be included in this chapter. The work of J.F. is supported by NIH grant NS41722. Work in the laboratory of T.S-G is supported by National Institute of Health Grants NIGMS GM60654 (T.S-G. and M.F.-P.), NIGMS/RCMI RR03037 core facility grant to Hunter College and a PSC-CUNY Grant 68753. Dr.T.S-G. wishes to thank Dr. Maria FigueiredoPereira and Dr.Yong Zhang for critical comments on the manuscript.
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In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 795-814 © 2007 Nova Science Publishers, Inc.
Chapter 32
THE UBIQUITIN-PROTEASOME SYSTEM IN HUNTINGTON’S DISEASE AND OTHER POLYGLUTAMINE DISEASES Louise Kelly∗ and Elsdon Storey¥ Department of Neurosciences, Monash University, Alfred Hospital, Melbourne, Victoria 3001, Australia.
ABSTRACT Huntington’s disease (HD) belongs to a group of nine polyglutamine (polyQ) tract disorders, which also includes spinocerebellar ataxias (SCA's) types -1, 2, 3, 6, 7, and 17, spinobulbar muscular atrophy (SMBA) and dentatorubral-pallidoluysian atrophy (DRPLA). The proteins involved in each of these disorders show no homology to one another except for an expanded polyQ tract. Although each protein is ubiquitously expressed throughout the central nervous system (and most non-neural tissues), only a distinct subset of neurons is affected in each disease, with only partial overlap between each. A common feature of these diseases is the formation of polyQ-containing intraneuronal inclusions, which are typically also immunoreactive for ubiquitin. However, the pathogenesis of these diseases is unknown, and there is much debate as to whether it is the inclusions themselves that are pathogenic or whether they are merely markers of disease. One suggestion, on the basis of numerous studies showing colocalisation of various other proteins with the inclusions, has been that the inclusions contribute to pathogenesis, interfering with normal cellular functioning by trapping components such as transcription factors, molecular chaperones, and components of the ∗
¥
Correspondence concerning this article should be addressed to Dr. Louise Kelly, Monash University, Department of Neurosciences, 4th Floor Centre Block, Alfred Hospital, Commercial Road, Melbourne, Victoria 3001, Australia. Phone: + 61 3 9276 2697; Fax:+ 61 3 9207 1043; Email:
[email protected]. Correspondence concerning this article should be addressed to Prof. Elsdon Storey, Monash University, Department of Neurosciences, 4th Floor Centre Block, Alfred Hospital, Commercial Road, Melbourne, Victoria 3001, Australia. Phone:+ 61 3 9276 2552; Fax:+ 61 3 9276 2458; Email:
[email protected].
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Louise Kelly and Elsdon Storey ubiquitin-proteasome system (UPS), and thus preventing them from carrying out their normal functions. However, this theory is disputed, with other studies suggesting that the inclusions may in fact be a form of cellular defense. Other evidence against a toxic role for aggregates includes the short-stop HD animal model, where increased inclusion formation accompanies decreased neuronal death, and SCA-1 models in which the protein is mutated so as not to form aggregates but toxicity is still seen. As the inclusions in these polyQ diseases are ubiquitinated, the role of the UPS in their pathogenesis has come under scrutiny. While some studies show that the function of the UPS is impaired in these disorders, other studies report no loss of function. Any impairment of the UPS may relate to difficulty with degradation of expanded or perhaps just aggregated polyQ proteins, although the evidence for such difficulty is also conflicting. It has been reported that UPS components are sequestered irreversibly into aggregates of polyQ-containing huntingtin (Htt) fragments. These Htt fragments were incompletely degraded, and a stable interaction between the polyQ-bearing proteins and the proteasome was seen. Such a stable interaction with non-degradable, aggregated polyQ proteins might result in depletion of proteasomal activity. In support of this suggestion, other studies have reported that UPS impairment is seen in the presence of aggregated polyQ-bearing proteins, and that this is evident in both the cytoplasmic and the nuclear compartments, even when the aggregated protein sequences are targeted to either the nucleus or cytoplasm alone. However, this impairment is also seen in cells where there are no detectable aggregates or toxicity, suggesting that UPS overload may not be a factor in neurotoxicity. Furthermore, an animal model of SCA-7 has shown that while there is neuronal damage in susceptible cells, the UPS remains functional in these neurons. Here we review the conflicting evidence from previous studies of the UPS in polyQ disorders, and discuss both the possible roles of the UPS in the pathogenesis of these diseases and the effect of inclusion formation on the UPS.
Keywords: Huntington’s disease, Polyglutamine, Spinocerebellar ataxia, ubiquitin, pathogenesis, proteasome, nuclear inclusion.
ABBREVIATIONS AD, Alzheimer’s disease; CBP, CREB-binding protein; DRPLA, dentatorubralpallidoluysian atrophy; ER, endoplasmic reticulum; Htt, huntingtin; HD, Huntington’s disease; HRPT, hypoxanthine phosphoribosyltransferase; polyQ, polyglutamine; SMBA, spinobulbar muscular atrophy; SCA, spinocerebellar ataxia; SCA-1, spinocerebellar ataxia type 1; SCA-2, spinocerebellar ataxia type 2; SCA-3, spinocerebellar ataxia type 3; SCA-7, spinocerebellar ataxia type 7, VCP, valosin-containing protein.
INTRODUCTION The dominantly inherited polyglutamine diseases include Huntington’s disease (HD), spinocerebellar ataxias (SCA's) types -1, 2, 3, 6, 7, and 17, spinobulbar muscular atrophy (SMBA) and dentatorubral-pallidoluysian atrophy (DRPLA). The various proteins involved in each of these diseases all contain a polyglutamine tract (polyQ) that is expanded beyond a
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specific threshold, resulting in neuronal cell toxicity [1]. The expanded polyQ tract is the only homology shared by these proteins. Despite their ubiquitous expression within (and beyond) the CNS, only certain subsets of neurons are affected, with incomplete overlap between the diseases. These expanded polyQ proteins tend to misfold and aggregate, giving rise to one of the characteristic features of these disorders: intranuclear inclusions (Figure 1). These inclusions consist of the full-length expanded polyQ protein, and/or a fragment of the mutant protein bearing the polyQ tract. Other proteins associated with these nuclear inclusions include components of the ubiquitin-proteasome system (UPS), various chaperones, and transcription factors [2, 3]. Although intranuclear inclusions are the characteristic feature of these diseases cytoplasmic inclusions have been reported in SMBA [4], HD [5], and SCA1 [5]. These cytoplasmic inclusions (aggresomes, see Chapter 12) are similar in composition to the nuclear inclusions but do not bind transcription factors, they are removed from the cell via autophagy and appear to be less toxic to the cell [5]. Whether these inclusions are pathogenic or merely markers of disease is subject to debate, with a number of studies supporting each premise.
Figure 1. Intranuclear inclusions in a PC12 cell. The nucleus (red) of a PC12 cell (rat pheochromocytoma cell line) showing ataxin-1 intranuclear inclusions (green).
There is only limited information on the mechanisms of pathogenesis of the polyQ diseases, but much discussion revolves around whether it is the inclusions that are themselves toxic, or whether they are merely markers of protein misaggregation and reflect a protective mechanism of the cell trying to rid itself of misfolded proteins via the UPS. The polyQ proteins have been shown to have a propensity to misfold and aggregate [7]. This was confirmed in a mouse model with an expanded polyQ tract out of context, [inserted into the hypoxanthine phosphoribosyltransferase (HRPT) protein], which led to both inclusion formation and neurological abnormalities [8]. Further to this, an in vitro study showed inclusion formation and cell death following transfection of primary rat neurons with an expanded polyQ tract devoid of host proteins [9]. While these findings suggest that the polyQ tract is important in the formation of the inclusions, they do not address the question of
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whether it is the inclusions themselves that are pathogenic. Some studies have noted that neurotoxicity can occur without the formation of visible inclusions: for example in SCA-1 transgenic mice [10], and in a transfected cell model of HD [11]. In both studies it was shown that nuclear localization of the mutant protein rather than inclusion formation was critical for the development of neurotoxicity. A SCA-7 mouse model also showed dysfunction in neurons weeks before inclusions became visible [12], and in SCA-2 patients the most vulnerable neurons — Purkinje cells — do not appear to develop inclusions, which supports the hypothesis that inclusions may be protective [13]. Moreover, numerous different neurons are seen to have inclusions, including both those that are susceptible and those that are nonsusceptible to the disease in SCA-7 [14]. A dissociation between inclusion formation (increased) and neurotoxicity (decreased) is also seen in the short-stop mouse model of HD [15]. Conversely, and despite the evidence from human SCA-2, there is evidence that neurotoxicity does not occur in cells that do not develop nuclear inclusions in a SCA-1 transgenic mouse model [10] (Figure 2).
Figure 2. Possible outcomes of proteolysis of expanded polyQ proteins. Whether proteolysis of expanded polyQ proteins is unaffected, enhanced, reduced or the result of novel proteolysis there are several possible toxic outcomes for the cell. Proteolysis can lead to the formation of truncated expanded polyQ proteins which may have deleterious effects on the cell, including; (A) formation of cationic channels in the cell membrane, (B) formation of intranuclear inclusions which recruits components of the UPS, (C) resistance to proteolysis by the proteasome, (D) disruption of the nuclear matrix, (E) alteration of normal protein binding partner interactions. (Adapted from Tarlac & Storey (2003) [6])
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It is apparent from these studies that the expanded polyQ proteins tend to form aggregates, but it is possible that toxicity precedes or is independent of aggregate formation. Needless to say, extensive research efforts to elucidate the mechanism(s) of polyQ disease pathogenesis continue. Given that proteins from the UPS and chaperones have been shown to interact with these inclusions, it is hardly surprising that the role of the UPS in the pathology of these diseases has come under increasing scrutiny over recent years. Much of the resulting evidence is contradictory, and consensus on the role of the UPS in these disorders is yet to be reached. The following two sections summarize the evidence for (Table 1) and against (Table 2) UPS involvement in the pathogenesis of the polyQ diseases, respectively. Table 1. Summary of evidence FOR UPS involvement/impairment in the polyQ diseases 1.
2.
3. 4.
5. 6. 7.
UPS components found within inclusions and re-distributed in inclusion-bearing cells - ubiquitin - chaperones (HDJ2/HSDJ, HSP90 , HSP70, HSP90) - proteosome components (26S, 20S, 19S, 11S, LMP2,VCP) Degradation of polyQ-containing proteins by the UPS is impaired - polyQ cannot be degraded by the UPS, or - polyQ degradation by the UPS is is polyQ tract length dependent , or - poly Q cannot be degraded by the UPS once the polyQ protein has formed inclusions Inhibition of UPS leads to increased polyQ aggregate formation Mutations in UPS and over-expression of chaperones have an opposite effect on polyQ neurotoxicity - loss-of-function mutations in UPS lead to neurological disease - loss-of-function mutations in UPS lead to increased aggregate formation - over expression of chaperones leads to decreased inclusion formation - over-expression of chaperones leads to improved motor function in polyQ animals UPS is inhibited in cells transfect with polyQ proteins ER stress is seen cells transfected with polyQ proteins UPS involvement has been reported in other neurological diseases AD, PD, Prion diseases
EVIDENCE FOR THE INVOLVEMENT OF THE UPS IN THE POLYQ DUSEASE Association of Ubiquitin Proteasome System Proteins and Chaperones with PolyQ Inclusions The inclusions seen in the polyQ diseases not only contain the polyQ proteins, but also numerous other proteins including those of the UPS, chaperones and transcription factors. Ubiquitinated polyQ inclusions have been seen in SCA-1 transgenic mice, ataxin-1 and ataxin-3 transfected cells, and affected brain regions of SCA-1 patients [16], SCA-3 patients
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[17, 18], and HD patients [19], although not all inclusions seen in the brains of HD patients were ubiquitinated [20]. Interestingly, while SCA-7 patients have numerous inclusions in neurons unaffected by disease, only those inclusions in susceptible cells are ubiquitinated [21]. This finding is strengthened by the fact that ataxin-7-containing inclusions in COS cells are not ubiquitinated, suggesting that there is some cell specificity to ubiquitination, which may be necessary for — or at least a marker of — neurotoxicity. The 26S proteasome is comprised of a number of subunits which include the barrel-shaped 20S catalytic core, and the 19S and 11S regulatory caps (see Chapters 5, 6 and 7). Various components of the proteasome are also reported to be associated with polyQ inclusions, including the immunoproteasome subunit (LMP2) (a subunit that is induced in antigen-presenting cells and results in generation of peptides that are optimal for MHC-I antigen presentation) in a transgenic HD mouse [22], the 20S proteasome subunit in HD [23], and the 26S proteasome in SCA-3 [24]. SCA-1 inclusions in human and transgenic mice stain immunopositive for the 20S proteasome subunit and the heat shock protein, HDJ2/HSDJ, and these findings have been confirmed in transfected cell models [25]. Another molecule associated with the polyQ proteins and inclusions is VCP [26], a poly-ubiquitin binding protein with a function in the UPS (see Chapter 13). VCP has been shown to bind ataxin-3 in a manner dependent on the length of the polyQ tract [27, 28]. A loss-of-function in the ter94 gene (a homologue of VCP) in a Drosophilia model of polyQ disease led to the suppression of polyQ- mediated neurodegeneration, though not inclusion formation, and co-expression of ter94 and polyQ protein led to severely enhanced degeneration [29]. Over-expression of ter94 also led to degeneration, suggesting a role as a cell death effector molecule. While these findings demonstrate that there is interaction between the polyQ proteins and those of the UPS, and hint at impaired UPS function as a possible mechanism of neurotoxicity, they still do not confirm this suggestion. However, from these initial findings it would at least seem reasonable to hypothesise that the UPS has a role to play in the development of, or perhaps the defence against, neurotoxicity. Various experiments have therefore examined whether there is direct inhibition or overload of the UPS, preventing it from carrying out its other cellular functions. Table 2. Summary of evidence AGAINST UPS involvement/impairment in the polyQ diseases 1.
2. 3.
4. 5.
UPS components are found within inclusions, BUT This is expected as it is the job of the UPS to remove misfolded proteins inclusions are dynamic and UPS components are not irreversibly bound to inclusions Degradation of polyQ-containing proteins by the UPS is not impaired Inhibition of UPS leads to increased aggregate formation, BUT This is expected as its the job of the UPS to remove misfolded proteins there is evidence that the aggregates are protective rather than toxic UPS function is not impaired in the polyQ diseases UPS components are not re-distributed in polyQ diseases
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Degradation of PolyQ Proteins by the Ubiquitin Proteasome System There is disagreement as to the resistance of polyQ tracts to proteasomal degradation. There are various contradictory studies that show: polyQ tracts cannot be degraded, they can be degraded, they can be degraded but only slowly, and that once they have formed inclusions they cannot be degraded. PolyQ Tracts Cannot be Degraded by the UPS Normally, proteins labelled for degradation with ubiquitin enter the 26S proteasome where they are digested to give rise to peptides ranging from 2 to 26 residues long. These residues are then released from the proteasome and hydrolysed by cytoplasmic and nuclear peptidases. Some recent work has suggested that eukaryotic proteasomes cannot digest the polyQ sequences within polyQ proteins, even those of non-pathogenic lengths. PolyQbearing proteins entering the 20S proteasome core were only partially degraded. The polyQ sequence could not be digested, but was released back into the cell [30]. Interestingly, this appears to be an evolutionary phenomenon, as proteasomes from the archaebacteria Thermoplasma acidophilum can digest polyQ-bearing proteins, including the polyQ sequence. Accumulation of these polyQ proteins, and the continual but unavailing attempts of the UPS to degrade them, may lead to a reduction in the cell’s ability to degrade other proteins, and thus exert a toxic effect on the cell. PolyQ Tracts can be Degraded by the UPS In contradistinction to the results discussed in the previous section, a number of studies have shown that the UPS can degrade the polyQ proteins, but that this degradation is polyQlength dependent, such that the longer the polyQ tract the slower is the degradation. Both mouse and in vitro models [31] have been used to show that ataxin-1 can be degraded by the UPS, with both wild-type and mutant ataxin-1 being polyubiquitinated. However, although mutant ataxin-1 is three times more resistant to degradation than the wild-type protein, inhibition of the UPS resulted in accelerated aggregate formation, suggesting that UPSmediated proteolysis of at least some species of expanded polyQ proteins may be important. Others have confirmed that both normal and expanded polyQ-containing proteins are degraded by the UPS, with the rate of degradation being inversely proportional to the length of the polyQ tract [23]. These findings further suggest that the slow degradation of the expanded polyQ proteins and consequent re-distribution of the proteasome may lead to decreased availability and function of the UPS components in other parts of the cell. Concomitantly, it may be that the synthesis of expanded polyQ proteins is faster than the rate of their degradation by the UPS, and that this leads to the accumulation of the polyQ proteins in inclusions. Holmberg et al. (2004) [32] have shown that the proteasome is irreversibly bound to inclusions in cells that over-express either mutant huntingtin (Htt) or other simple expanded polyQ proteins, and that targeting the polyQ proteins for degradation by the proteasome led to only incomplete degradation both in vitro and in vivo. (These researchers used the N-end rule pathway where a ubiquitin moiety is linked to the N-terminal of a protein using a 40 amino-acid linker. The ubiquitin is readily cleaved off by ubiquitin hydrolases, leaving an unstable and readily ubiquitinated N-terminus on the protein [33]).
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PolyQ Proteins are not Degraded by the UPS once they have Formed Inclusions Contrary to the finding that mutant Htt and simple polyQ proteins could be targeted for partial degradation using the N-end rule pathway [32], in an experiment where ataxin-1 protein containing an expanded polyQ tract was targeted for degradation using an N-end rule degradation signal, soluble polyQ proteins were degraded by proteasomes, but once in aggregates they resisted degradation. Introduction of the degradation signal into mutant ataxin-1 not only reduced inclusion formation but decreased cellular toxicity [34], showing that increasing the efficiency of expanded polyQ protein degradation by the UPS is protective, and suggesting that decreased UPS activity may increase neurotoxicity.
Effects of Inhibiting Proteosomal Function on PolyQ Protein Aggregation Inhibition of the proteasome with lactacystin in a transfected SCA-3 cell model has been shown to lead to an increase in aggregate formation which is dependent on the length of the polyQ repeat, suggesting that the proteasome plays a role in suppressing aggregate formation [24]. Although the proteasome was inhibited, its 20S catalytic core subunit was seen to associate with the aggregates [24]. In keeping with this, lactacystin inhibition of proteosomal degradation in ataxin-1 transfected cells leads to increased aggregate formation [31]. These researchers also showed that in a SCA-1 mouse model lacking the ubiquitin ligase E6-AP there was a reduction in the number of inclusions formed in Purkinje cells and that these animals had otherwise severe SCA-1 pathology, suggesting that nuclear inclusions are not necessary for neurotoxicity but that impaired degradation by the UPS may contribute to SCA1 pathology. Moreover, addition of either of two different proteasome inhibitors (ALLN or lactacystin) dramatically increased the rate of aggregate formation by moderately expanded Htt (Q60), but had little influence on that of grossly expanded Htt (Q150) in a cellular model of HD [23].
Opposite Effects of Proteosomal Loss-of-Function Mutations and Over-Expression of chaperones Loss of Function Mutations Affecting the UPS Evidence for impairment of the UPS in the polyQ diseases also comes from observations in experiments in which the function of the UPS is disrupted by mutation of specific genes. Loss-of-function mutation in genes coding for various components of the UPS leads to neurodegenerative diseases in humans [Parkinson’s disease (PD)] [35], rodents [36, 37] and Drosophila [38]. Loss-of-function mutations in cell and mouse models of SCA-1 result in enhanced cytotoxicity of the protein [31], as do mutations in the UPS in a Drosophila SBMA model [39]. Supporting information for the importance of the UPS in preventing toxicity comes from the report that an aberrant form of ubiquitin (resulting from a dinucleotide deletion) that inhibits the proteasome, has been found in inclusions of HD and SCA-3 patients. When this form of ubiquitin was co-transfected into cells with polyQ proteins there
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was an increase in inclusion formation and cytotoxicity [40]. However, in contrast, in a recent study a SCA-1 mouse model was crossed with a mouse model in which a mutant form of ubiquitin, which interferes with ubiquitin chain assembly, is expressed. No exacerbation of SCA-1 pathology was observed in the double transgenic animals, and a protective effect with regards to Purkinje cell neurotoxicity was reported, suggesting that inhibiting proteolysis of the ataxin-1 protein was protective to the cells [41]. Over-Expression of Chaperones Conversely, reduction of polyQ protein misfolding should lead to a decrease in aggregation, and therefore perhaps in neurotoxicity. On co-expression of an expanded polyQ tract-bearing androgen receptor (the mutant protein resulting in SMBA) with the chaperone HDJ2/HSDJ, there was significant repression of inclusion formation [42]. Over-expression of the chaperone DnaJ (Hsp40 family) has also been shown to promote recognition of misfolded ataxin-1 and suppress inclusion formation [25]. Furthermore, over-expression of Hsp70 in a SCA-1 mouse model not only suppressed neuropathology but also improved the motor function in the animals [43]. Collectively, these studies suggest that aggregate formation and neurotoxicity may result from decreased or insufficient levels of function of UPS components, and that increased chaperone activity, by reducing aggregation, reduces toxicity.
Depletion and Redistribution of Ubiquitin Proteasome System Proteins and Chaperones A number of studies have investigated whether the 26S proteasome complex, its subunits, or chaperones associated with the UPS are depleted or re-distributed within the cell in the polyQ disorders. 11S and 19S Proteasome Subunits Show a Different Distribution Pattern to the 20S Core Subunit Various studies have described different distribution patterns of proteasomal subunits in cells containing inclusions. While most of the intranuclear inclusions in pontine nuclei examined in SCA-3 patients were immunopositive for the 11S and 19S proteasomal protein cap subunits, only a few were immunopositive for the 20S core proteasome subunit [44]. Furthermore, inclusions in a cell model of SMBA were seen to associate with the 19S proteasome cap but not the 20S core proteasome subunit [32]. On comparing the distribution of a 20S subunit in pontine nuclei in SCA-3 brains with control samples it was noted that this 20S subunit was predominantly cytoplasmic in the SCA-3 brains and predominantly nuclear in the control brains. Where there was nuclear staining for the 20S subunit in the SCA-3 brains, this was seen in the nuclear inclusions [44] and the levels of some of the subunits that make up the 20S core were markedly increased in the cytoplasm of SCA-3 pontine nuclei compare to controls, suggesting that there is either a re-distribution or an up-regulation of a 20S core subunit in SCA-3.
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20S, 11S, and 19S Proteasome Subunits are Found together in Inclusions, Possibly Resulting in Functional Proteasomal Impairment Other studies, however, have demonstrated that the 20S, 11S and 19S subcomplexes all associate with the inclusions and do not have different distribution patterns. A study of the intranuclear inclusions in human SCA-3 brain samples revealed that all the inclusions were positive for ubiquitin and a subset were positive for subunits of the 20S core, 19S cap, and 11S activator of the 26S proteasome, suggesting that the inclusions are heterogeneous at the molecular level [24]. These findings were also reflected in several transfected cell models, with the subunits of both the 20S core and 19S cap are seen to localise with the nuclear inclusions. Gel electrophoresis of these inclusions showed that the proteasomes could be separated from the polyQ proteins; thus the proteasome does not appear to be irreversibly bound to the inclusions [24] (see below). When truncated N-terminal Htt, containing different polyQ lengths, was expressed in cell and animal models the subunits of the 20S proteasome core and 19S cap were redistributed to aggregates [23]. This, along with the slower degradation of truncated N-terminal Htt with longer length polyQ tracts, decreased the proteasomes’ ability to degrade other proteins such as p53, which led to a disruption in the membrane potential of mitochondria and release of cytochrome c into the cytosol with activation of caspase-9 and caspase-3-like proteases [23]. Impairment of UPS Function by Aggregating Proteins Further evidence for impairment of UPS function by aggregated proteins in the polyQ diseases comes from Bence et al. (2005) [45]. Using a degron-GFP reporter (i.e. a GFP protein with a degron label, which targets the protein for degradation by the UPS), these researchers showed that the UPS was almost completely inhibited in cells expressing two unrelated aggregating proteins, Htt and cystic fibrosis transmembrane conductance regulator protein. Another study looking at cells expressing ataxin-1 or Htt showed that there was global (cysosolic and nuclear) impairment of the UPS, even when the production of the inclusions was targeted specifically to either the cytosol or the nucleus [46]. This study therefore demonstrated that there was impairment of the UPS in cellular compartments that lack detectable inclusions. Depletion and Re-Distribution of the Chaperones in the PolyQ Diseases Immunostaining of intranuclear inclusions in SCA-3 patients showed that certain inclusions (about 20%) were immunoreactive for the chaperone Hsp90α (an isoform of Hsp90), fewer were immunoreactive for the chaperone HDJ-2 (a member of the Hsp40 family), and that most of the inclusions were ubiquitin positive. While the inclusions did not stain for Hsp27, Hsp60, Hsp70, or Hsc70, these chaperones were shown to be present in the cytoplasm of cells in control tissues, showing that they are present in the cells but are not recruited to the inclusions. Aside from the intense labelling of some of the intranuclear inclusions there was no obvious redistribution of the Hsp on comparison to control tissues [44]. In a cell model of SMBA, cells transfected with the androgen receptor bearing an expanded polyQ tract were seen to form inclusions that were immunoreactive for Hsp90 and HDJ-2/HSDJ but unlike the SCA-3 model the inclusions were also positive for Hsp70. The
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Hsp70 staining appeared to be increased, suggesting induction of the heat shock response [32].
The Role of Endoplasmic Reticulum Stress Endoplasmic reticulum (ER) stress occurs when initial mediators—ER-resident type I transmembrane serine/threonine protein kinases (PERK and IRE1)—are autophosphorylated, leading to cytoplasmic signal transduction, and ultimately activation of the apoptotic pathway (see Chapter 13 for more information). ER stress has been implicated as another possible pathogenic pathway in the polyQ diseases. It has been proposed that the polyQ proteins interfere with the UPS, and that this in turn induces ER stress. This hypothesis is supported by a rat phaeochromocytoma (PC12) cell model of SCA-3, in which ER stress was seen in transfected cells expressing the mutant protein. As ER stress is induced by accumulation of unfolded protein in the ER, it seemed unlikely that the stress was as a direct effect of the polyQ protein, and other factors were therefore investigated. Expression of the mutant ataxin3 protein in mouse primary neurons led to a significant inhibition of proteasome activity. Linking these two findings are the observations that inhibition of proteasomal function in primary neurons leads to ER stress [47]. These findings suggest that poly-Q mediated depletion of the UPS led to deficient ER-associated degradation (ERAD), with accumulation of misfolded proteins within the ER and therefore ER stress [47].
Evidence for UPS Involvement in other Neurodegenerative Diseases Characterised by Protein Aggregation It should be noted that it is not only the polyQ diseases in which impairment of the UPS has been implicated in pathogenesis. Other neurodegenerative disorders, such as Alzheimer’s disease (AD), prion diseases and PD have also been shown to involve the UPS (see Chapters 22 and 27). Human brain sections from prion disease and AD patients were shown to have stronger staining for ubiquitin, a proteasomal subunit and Hsp72 in those brain regions areas not affected by these diseases, and weaker staining in those areas that tend to be affected [48], with the nuclei being strongly positive for proteasomal subunits: 20S, S4, S6, and S7, and ubiquitin. Other studies have also noted the presence of ubiquitin in the various types of intracellular inclusions seen in these diseases, with 20S and S6b proteasome subunits and Hsp70 all being found in neurofibrillary tangles in AD and in Lewy bodies in dementia with Lewy bodies [49, 50, 51, 52]. In another study, Ardley et al. (2003) [53] have shown that inhibition of proteasomal function results in accumulation of parkin (the protein mutated in the commonest form of recessively-inherited PD), with formation of non-toxic cytoplasmic inclusions. This inclusion formation was not reversed by subsequent removal of the proteasome inhibitor. This would suggest that the UPS is unable to degrade inclusions once they are formed, but can prevent formation occurring.
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These findings, along with the fact that other neurodegenerative disorders such as prion diseases, AD and PD appear to show impairment of the UPS [48], would seem to suggest that the UPS has a role to play in the pathogenesis of a number of neurodegenerative diseases characterised by protein misfolding/aggregation.
EVIDENCE AGAINST THE INVOLVEMENT OF THE UBIQUITIN-PROTEASOME SYSTEM IN THE POLYQ DISEASES Given that it is the role of chaperones to ensure the proper folding of proteins, and the UPS to remove misfolded proteins from the cell, it is hardly surprising that components of this system have been found to be associated with inclusions composed of misfolded proteins. Is it therefore possible that the UPS does not actually play a part in the pathogenesis of these diseases, but rather that it forms part of an ultimately inadequate cellular response to protein misfolding and aggregation.
PolyQ Protein Inclusions are Dynamic Structures, not Necessarily Irreversibly Binding their Associated Proteins There have been several studies looking at the nature of the intranuclear inclusions seen in the polyQ diseases. These have led to the discovery that the inclusions are not all alike, and that they are dynamic rather than static structures. A study looking at ataxin-1 inclusions noted that there were two different types of inclusions; fast and slow exchanging. Ataxin-1 was seen to move readily between the inclusion and the nucleoplasmic pool in the fastexchanging inclusions, whereas in the slow exchanging inclusions it did not [54]. Inclusions containing relatively high levels of ubiquitin and low levels of proteasomal components also contained both fast and slow exchanging ataxin-1. However, those inclusions with low levels of ubiquitin and high levels of proteasomal components contained mainly fast exchanging ataxin-1, implying that the proteasomal components are not irreversibly trapped in inclusions, but that there is a dynamic exchange with the surrounding environment. This concept of proteins moving in and out of inclusions is also supported by the fact that Hsp70 is only transiently bound to Htt, in a dynamic interaction [55, 56]. However, this might not be the case for all the proteins associated with the polyQ inclusions. Some proteins appear to become trapped irreversibly in the inclusions, while others have a dynamic relationship with them, and this may vary between the different polyQ proteins. For example, the CREBbinding protein (CBP) has been shown to be immobile when associated with Htt and ataxin-3 inclusions, but is mobile in relation to ataxin-1 inclusions [57].
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PolyQ Proteins can be Degraded by the Ubiquitin Proteasome System As previously discussed (section ‘Degradation of polyQ proteins by the ubiquitin proteasome system’) the ability of the UPS to degrade polyQ-bearing proteins has been investigated, with conflicting results. Various studies have noted that the UPS cannot, or can only slowly, degrade polyQ proteins, or that once in inclusions these proteins become more resistant to degradation. Here we will look at the evidence showing that the UPS can readily degrade the polyQ proteins. In an in vitro model of HD, cells expressing mutant Htt labelled for degradation through the N-rule pathway showed that the half-life of the protein depended on both the degradation signal and the length of the polyQ tract [58]. Mutant (expanded) Htt with a shorter half life, as a result of N-rule labelling, showed delayed formation of aggregates, but appeared to be more toxic to the cells, suggesting that the proteins may be readily degraded by the proteasome regardless of polyQ tract length, that UPS function is not impaired, and that the inclusions are protective, or at least inert. Furthermore, evidence for polyQ degradation by the UPS comes from Michalik et al. (2003) [59] who used a cell model transfected with polyQ proteins of lengths Q103 or Q25 and found that both were degraded efficiently and completely. Contrary to the evidence discussed in section ‘PolyQ tracts cannot be degraded by the UPS’ and section ‘PolyQ proteins are not degraded by the UPS once they have formed inclusions’, soluble polyQ proteins do appear to be degraded by proteasomes, but once the polyQ proteins form aggregates they tend to resist degradation, as shown with ataxin-1 [34] and Htt [58]. This, too, has been challenged, however: two studies using conditional transgenic mouse models of HD and SCA-1 have noted that if the gene encoding the mutant polyQ protein is silenced after inclusion formation has occurred, there is a reversal of pathology and clearance of inclusions from the affected cells [60, 61]. Of course this clearance may have been achieved by mechanisms other than UPS degradation, such as dynamic exchange with a progressively depleted soluble pool of polyQ protein. There is evidence that autophagy plays a part in removal of cytoplasmic inclusions in HD and SCA1 [5], and SMBA [4], and that lysosomes and autophagic bodies proliferate in HD and AD [62, 63]. However, the predominantly nuclear inclusions that are seen in the polyQ diseases are not able to be removed by autophagy.
There is no Impairment of the Ubiquitin Proteasome System or Redistribution of its Components in some Models of PolyQ Diseases Using a SCA-7 knock-in mouse model with a fluorescent ubiquitin reporter, Bowman et al. (2005) [14] showed that early on in the disease there was neuronal dysfunction without any impairment of the UPS, and that in the later stages of the disease there was an increase in both ubiquitin mRNA and protein levels. An in vitro assay confirmed that proteasomal activity in the vulnerable neurons remained normal. These researchers also confirmed the findings from human cases that intranuclear inclusions were found in all cells, not just those vulnerable in SCA-7, thus showing that even if UPS components are sequestered into inclusions, this by itself is unlikely to determine cytotoxicity. Despite the findings of Schmidt
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et al. (2002) [44] (see section ‘Depletion and Redistribution of ubiquitin proteasome system proteins and chaperones’) it has also been reported that there is no depletion of UPS components in the cytosol or nucleus of cells transfected with polyQ proteins targeted to either of these compartments [46]. There may, however, be depletion of other proteins via sequestration into the inclusions, as shown in a range of neurodegenerative diseases [64, 65], and this in turn may affect other processes in the cell. Studies on HD transgenic and age-matched control mice showed that there is no difference in proteasome activity, but that there is an age-dependant decrease in proteasome activity that could explain the formation of inclusions later on in life [66]. This is further supported by the observations that components of the UPS are suppressed at the level of transcription in aged brains [67], and there is an age-related decrease in proteasome activity seen in rat brains [68]. An in vivo study measuring enzymatic activity in HD mouse brains did not detect inhibition of any of the ubiquitin-proteasomal enzymes, suggesting that if impairment does occur it is not at the level of the catalytic core; in fact two of the enzymes showed increased activity in brain areas affected in HD [18]. Western blot analysis showed no real difference in proteasomal content, although there was an increase in some of the immunoproteasome subunits (LMP2 and LMP7), both of which were found in the neurons of control mice but at a much lower level compared to those levels seen in the HD mice. Some of the aggregates in the HD mice were also positive for LMP2 [22].
CONCLUSION, METHODOLOGICAL PROBLEMS PARTICULARLY AFFECTING CONTEMPORARY STUDIES, AND FUTURE DIRECTIONS Numerous studies have shown that the proteins of the UPS and chaperones are associated with the inclusions seen in the polyQ diseases, and indeed with protein aggregates in other neurodegenerative disorders. Both the role of the inclusions, and of the UPS in the pathogenesis of these diseases, are still unclear, however. The critical question that remains to be answered is whether the association between the UPS and polyQ-containing proteins is merely an appropriate, albeit ultimately ineffectual, response to such aggregation, or whether the association itself contributes to neurotoxicity, perhaps by interference with normal UPS function. Considerable evidence can be marshalled to support either view, as summarised in sections ‘Evidence for the involvement of the ubiquitin-proteasome system in the polyQ diseases’ and ‘Evidence against the involvement of the ubiquitin-proteasome system in the polyQ diseases’, respectively. There are studies showing that the inclusions irreversibly bind the UPS components and impair their functions and/or redistribute them within the various cellular compartments. Some have shown that the polyQ proteins cannot be degraded by the ubiquitin proteasome pathway and that inhibition of the UPS as a result of attempted degradation leads to increased aggregate formation and cellular toxicity. For each of these findings there are other, contradictory studies: inclusions have been shown to be dynamic, allowing movement of UPS and other proteins in and out of the inclusions; some have failed to demonstrate any impairment or redistribution of UPS proteins; and there is evidence that
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the polyQ proteins can be degraded by the ubiquitin proteasome system, perhaps even once they have formed aggregates. One point that has to be considered when assessing at these studies is that many of the animal models utilize extremely long polyQ tracts, very rarely encountered in human disease, and/or promoters that lead to higher than normal expression levels of the polyQ-containing proteins. Whilst these manipulations enable a disease phenotype, and possibly inclusions, to develop during the lifespan of a mouse, these models are not truly reflective of the (typically adult-onset) polyQ diseases in humans. For example, extremely long polyQ tracts are very rare except perhaps in SCA-7, and in this disorder they result in an infantile and lethal form of the disease that is multi-systemic, not just neuronal [69]. It may be that UPS involvement in these animal models occurs because of the increased length of polyQ and/or due to the higher than endogenous expression levels of the protein - the UPS being swamped as it tries to rid the cell of the misfolded, aggregated proteins. Another consideration is that many of the in vivo studies look at the brain as a whole, rather than just the affected cells. In studies suggesting that there is no UPS involvement in polyQ disease, it may be that involvement of the UPS in the small subset of neurons that is susceptible in a given disorder is masked by the lack of change in other neurons and in glia. This problem has been specifically addressed in a recent study in which a SCA-7 mouse model was used to show that there was no impairment of the UPS. The study looked specifically at the vulnerable neurons and not just at the brain as a whole to determine whether there was specific UPS impairment [14]. Certainly more studies need to be carried out to investigate what role, if any, the UPS has to play in cytotoxicity. The fact that there seems to be a difference in the dynamics of the inclusions and composition of associated proteins in different polyQ diseases might suggest that there are different mechanisms involved in each, and that the role/involvement of the UPS could be different in each disease. In addition, the age of onset and the length of the polyQ tract could determine the relative importance of that role. A number of studies have noted that the UPS appears to be affected in old age, with reported decreased levels of 26S proteasome activity with increasing age [70] (see Chapter 22). There is suppression of the UPS at the transcription levels in aged brains [67], and agedependant decreased proteasome activity in rodent brains [66, 68]. The chaperone system has also been reported to deteriorate with age [71]. There also appears to be an accumulation of ubiquitinated polyQ proteins in inclusions over time, which might suggest an age-related failure of the UPS [72]. This would appear to provide a possible explanation for the agerelated nature of these disorders, with onset of these diseases typically being seen at around 30-50 years old, followed by steady degeneration thereafter.
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In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 815-834 © 2007 Nova Science Publishers, Inc.
Chapter 33
THE ROLE OF THE UBIQUITIN PROTEASOME SYSTEM IN THE PATHOGENESIS OF PRION DISEASES Birkir Thor Bragason and Astridur Palsdottir∗ Institute for Experimental Pathology, Keldur, University of Iceland, Vesturlandsvegur, Reykjavik 112, Iceland.
ABSTRACT Prion diseases are a group of neurodegenerative diseases that affect humans and animals. They are distinct from other neurodegenerative disorders in that they can be infectious as well as familial or sporadic. Prion diseases are characterized by long incubation periods prior to onset of symptoms, and the pathology is limited to the central nervous system consisting mainly of vacuolation in neuronal cell bodies, neuronal cell death, deposition of protein aggregates, and astrocytosis. Prion diseases were originally classified as slow viral infections; however, there is mounting evidence to support the claim that the infectious unit is a protein. A small endogenous protein, the prion protein (PrPC; c: cellular), is a key factor in these diseases. The expression of PrPC is highest in neurons and its precise physiological role is not clear. It is processed through the secretory system to the plasma membrane where it is predominantly an extracellular glycosyl-phosphatidyl-inositol anchored protein that contains one disulfide bond and it is di-glycosylated. The protein aggregates detected in diseased individuals contain a structurally altered protease resistant form of PrPC, called PrPSc (Sc: scrapie). PrPSc is though to be the major part of the infectious unit. The neurotoxic mechanisms behind neuronal death in prion diseases are not clear. Loss of functional PrPC and/or PrPSc toxicity have been suggested; however, PrPC knockout mice are apparently normal, suggesting that loss of PrPC is not the major cause, and toxic effects of PrPSc are limited ∗
Correspondence concerning this article should be addressed to Dr. Astridur Palsdottir, BSc, PhD; Institute for Experimental Pathology, Keldur, University of Iceland, Vesturlandsvegur, Reykjavik 112, Iceland. Phone: +354 585 5100; Fax: +354 567 4714; E-mail:
[email protected].
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to PrPC expressing tissue. Therefore, alternate pathways of neurotoxicity have been proposed, e.g., transmembrane forms of PrPC, and an interplay between the ubiquitinproteasome system (UPS) and cytosolic PrPC or cytosolic PrPSc. As with other neurodegenerative diseases the UPS has been linked with prion diseases. There are, for example, reports of ubiquitinated PrPSc, increased polyubiquitin expression, and impaired proteasome activity in prion disease and disease models. The majority of PrPC is topologically located in the secretory system and the extracellular space. However, there are reports describing a small subset of PrPC in the cytosol, cytosolic PrPC, where it is subject to efficient ubiquitin-proteasome degradation. This subset of PrPC can either arise from inefficient translocation into the endoplasmic reticulum (ER) or retrotranslocation from the ER via the ER-associated-degradation (ERAD) pathway. Cytosolic PrPC has a tendency to aggregate if proteasome activity is inhibited. Initial reports of toxic effects of cytosolic PrPC on cells prompted speculation whether impairment of cytosolic PrPC degradation by the UPS due to, e.g., PrPC mutations or perturbed ubiquitin-proteasome activity, with a resulting rise in cytosolic PrPC concentration, could explain some of the neurotoxicity in prion diseases. However, the effects of cytosolic PrPC between studies are in conflict, with some studies reporting toxic effects and others reporting neuroprotective effects. A recent study of the effect of mild proteasome inhibition on viability in scrapie cell-culture models has shown that cytosolic aggresome formation of PrPSc, rather than PrPC, caused apoptosis, suggesting that accumulation of cytosolic PrPSc due to impairment of the UPS could be an important factor in the neurotoxic mechanisms at work in prion diseases.
Keywords: PrPC proteins, PrPSc proteins, Prion diseases, Protein transport, Post translational protein processing.
ABBREVIATIONS aa, amino acid; Bax, Bcl-2 associated X protein; Bcl-2, B-cell leukemia/lymphoma 2; BSE, bovine spongiform encephalopathy; CMW, cytomegalovirus; CNS, central nervous system; CJD, Creutzfeldt-Jakob disease; CtmPrP, transmembrane PrPC with the C-terminus in the extracellular space; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum associated degradation; GPI, glycosyl phosphatidyl inositol; Grb2, Growth factor receptor bound protein 2; GSS, Gerstmann-Straussler-Scheinker disease; Hsc, heat-shock cognates; Hsp, heat-shock proteins; NRAGE, neurotrophin receptor interacting MAGE homolog; PK, protein kinase; Prnp, prion protein gene; PrPC, normal cellular form of the prion protein; PrPSc, disease associated form of the prion protein; UPS, ubiquitin-proteasome system.
INTRODUCTION Prion Diseases Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative disorders that affect humans and animals. They are unique among
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 817 neurodegenerative disorders in that they are infectious as well as sporadic and hereditary. The human forms of prion diseases are categorized as sporadic [sporadic Creutzfeldt-Jakob disease (sCJD)], familial [familial CJD (fCJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI)], or acquired/iatrogenic [Kuru, iatrogenic CJD (iCJD), and variant CJD (vCJD)]. The familial forms represent 10-15% of human prion diseases, whereas sporadic CJD is the most common form and represents about 85% of all diagnosed CJD cases with an average incidence of 1 per million per annum [1]. The most prominent prion diseases in animals are scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and transmissible mink encephalopathy. In animals, there is no conclusive evidence of genetic, or sporadic, prion diseases; the animal diseases are therefore acquired by infection. However, due to the existence of sporadic/hereditary forms of disease in humans, the same phenomena in animals cannot be excluded. Prion diseases are characterized by long incubation periods prior to the onset of clinical illness ending fatally. The phenotype in humans consists of various neurological symptoms. The symptoms of sporadic CJD are, e.g., rapidly progressive dementia, pyramidal/extrapyramidal signs with myoclonus, and triphasic electroencephalogram discharges [1]. The underlying pathology is restricted to the central nervous system (CNS) and consists of: vacuolation of the neuropil within neuronal cell bodies and neurites, neuronal cell death, deposition of protein aggregates, and activation of astrocytes (astrocytosis) and microglia. The pathological changes result in characteristic spongiform changes due to vacuolar degeneration of grey matter in the CNS, hence the term spongiform. The infectious agent of prion diseases differs from other pathogens. The agent is resistant to treatment that abolishes the infectivity of many conventional agents such as treatment with heat or formalin (reviewed by Prusiner [2]). In addition, the agent is resistant to ionizing radiation and UV-radiation [3]. Several hypotheses about the nature of the prion disease agent have been proposed (reviewed by Prusiner [2]). The prevalent view, introduced in 1982 by Dr. Stanley Prusiner and his colleagues at the University of California, San Francisco, is that the infectious agent is a proteinaceous particle devoid of nucleic acids [2].
The Prion Protein The protein aggregates detected in prion diseases contain a modified form of an endogenous protein called the prion protein. This modified form of the prion protein was originally identified and characterized from infectious brain fractions of hamsters experimentally infected with scrapie by Prusiner and colleagues [4]. They coined the term ‘prion’, which is an abbreviation for proteinaceous infectious particle. To distinguish the endogenous, normal, form of the prion protein it is called PrPC (C: cell), whereas the modified, disease associated form, is called PrPSc (Sc: scrapie). The difference between these two forms of the prion protein lies in a post-translational change in structure from the predominantly alpha-helical form of PrPC to PrPSc which is mainly β-sheet [5]. In addition, the N-terminus of PrPSc, up to amino acid (aa) ~90, has been cleaved off. It is possible to distinguish between these two forms of the prion protein due to their different biochemical
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characteristics; PrPC is detergent soluble and protease sensitive, whereas PrPSc is detergent insoluble and protease resistant to a degree. PrPC is a small protein (253 aa in humans) encoded by the prion protein gene, PRNP. The prion protein gene in mammals consists of two to three exons. For example, in humans and hamsters it has two exons, whereas in sheep, cattle, mice, and rats, Prnp has three exons. In both cases the open reading frame is completely contained within the latter/last exon and encodes PrPC. The crucial role of this protein in prion diseases was made clear when it was demonstrated that Prnp0/0 knockout mice are ‘immune’ to infection with prion disease (scrapie) [6]. The primary PrPC expressing cell type in the CNS is the neuron; Prnp expression and protein are detected in glial cells, they are, however, in relatively low quantities compared to neurons [7]. In the periphery Prnp expression is detected in a wide range of cell types; the expression levels, however, are lower than in the CNS [8,9]. PrPC is targeted for translocation into the endoplasmic reticulum (ER) by an NH2terminal (N-terminal) signal peptide. In the ER a COOH-terminal (C-terminal) signal sequence facilitates glycosyl-phosphatidyl-inositol (GPI)-anchor membrane attachment [10]. Structural studies of recombinant PrPC show that the protein has a structured C-terminal part (aa 125-231) consisting of three alpha-helices and two anti-parallel β-sheets [11,12]. In contrast, the N-terminal region (aa 23-124) is flexible [13]. A characteristic feature of the Nterminus are repeats (5 in humans) consisting of 8-9 amino acids, notably histidine (P(Q/H)GGG(G/-)WGQ). After translocation into the endoplasmic reticulum, PrPC is processed through the secretory system during which it is glycosylated at two Asn-glycosylation sites and a single disulfide bond is formed. Fully processed, GPI anchored, PrPC on the plasma membrane is mainly located in detergent resistant microdomains, or ‘rafts’ (Figure 1) [14]. There are reports that indicate that in neurons, PrPC is located at the neuronal synapse [15]. PrPC cycles between the plasma membrane and endosomes [16]. As is the case for other GPI-anchored proteins, PrPC endocytosis by caveolae has been reported; however, there are also reports of endocytosis by clathrin-coated pits for chicken PrPC [17,18]. Like other membrane proteins the ultimate fate of PrPC seems to be degradation in the endo-lysosome system. Overall, the estimated time for PrPC synthesis is less than 2 hours, and its half-life has been estimated at 4.5-5 hours [19]. Despite its small size, the translocation of PrPC can yield several topological forms. In addition to the predominant GPI-anchored PrPC form, transmembrane forms have been detected [20]. In the transmembrane forms a conserved hydrophobic region of PrPC (aa ~110135 in human PrPC) serves as a transmembrane region spanning the lipid bilayer. Two topological orientations have been described, termed NtmPrP (C-terminus in the cytosol) and Ctm PrP (N-terminus in the cytosol). The precise physiological function of PrPC is not clear. There are several theories. For example, PrPC can bind Cu2+ via histidines in the N-terminal repeats [21]. This binding affects endocytosis [22] and a Cu2+ receptor function of PrPC has been proposed. It has also been suggested that PrPC may modulate Cu/Zn superoxide dismutase activity [23]. Reports of impaired synaptic inhibition in neurons from Prnp0/0 mice [25] suggest that PrPC may have a function in neuronal synapses. However, because other reports have not detected synaptic
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 819 abnormalities in Prnp0/0 mice [26,27], the functional importance of PrPC at neuronal synapses is still unclear. An anti-apoptotic role of PrPC has been suggested based on observations that PrPC expression protects human primary neurons against Bax-mediated apoptosis [28], and that expression of either PrPC or Bcl-2 rescues Prnp knockout neurons from apoptosis induced by serum deprivation [29]. In addition, a signalling role has been proposed, for example by interaction with caveolin-1 and stress-inducible-protein-1 resulting in Fyn activation and signalling via the cAMP/protein kinase (PKA) pathway, respectively [30,31].
Figure 1. A schematic overview of the cellular processing of PrPC and PrPSc. This figure is a simplified schematic overview of various PrPC processing pathways within the cell which are addressed in the text. It shows the processing of normal GPI-anchored PrPC through the secretory system and its localization in rafts on the cell surface. From the surface, PrPC is internalized by endocytosis and some of it is recycled to the membrane. The interaction of PrPC and PrPSc during infection is shown on the cell surface as well as during endocytosis. The figure shows retrotranslocation of the mutant PrPC forms PrP145, PrP160, and PrP217, from the endoplasmic reticulum (ER) for proteasome degradation. In light of the debate described in the text a question mark is set on the retrotranslocation of non-mutant PrPC from the ER. The generation of a cytosolic subset of PrPC by inefficient translocation into the ER is depicted. The consequences of an impaired ubiquitin-proteasome system, i.e., the accumulation of PrP forms in the cytosol with subsequent aggregation or alternative locations are shown. Finally, the location of transmembrane PrPC species in the ER and Golgi is indicated.
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Studies on scrapie infected cell cultures indicate that the PrPC → PrPSc transformation occurs on the cell membrane and/or after endocytosis in the endo-lysosome system [32,33]. Other locations of PrPSc formation have also been suggested such as the endoplasmic reticulum [34] and the cytosol [35]. The mechanism of transformation is not precisely defined. Reports suggest that the transformation requires an interaction between PrPC and PrPSc, e.g., antibodies specific for certain PrPC epitopes inhibit prion propagation, perhaps by impairing PrPC -PrPSc interaction [36,37]. Alternatively, the antibodies may interfere with interactions with other factors required for the transformation. Genetic studies suggest that a hitherto unidentified protein co-factor, called protein-X, is involved in the conversion [38]. During infection, PrPC-PrPSc interactions result in a structural transition of endogenous host PrPC into the PrPSc form in a process which imparts the biochemical characteristics of PrPSc on to the host PrPC [39].
THE ASSOCIATION OF THE UBIQUITIN-PROTEASOME SYSTEM AND PRION DISEASES As the overview of PrPC processing above details, PrPC is predominantly topologically located in the secretory system or in the extracellular space from which it is internalized by endocytosis and degraded in the endo-lysosome system. Therefore, it would seem that the cytosolic ubiquitin-proteasome system (UPS) is not significantly involved in the processing of PrPC. Indeed, in comparison to other neurodegenerative diseases, such as Parkinson’s disease, there is a relative paucity of data on the association of the UPS and prion diseases. However, there is some evidence that indicates the involvement of the UPS, to some degree, in prion diseases and in the processing of PrPC and PrPSc. First of all, there are several reports of increased ubiquitin reactivity in diseased tissue (discussed below). Second of all, there are reports that a cytosolic subset of PrPC and/or PrPSc are substrates of the UPS and that impairment of the proteasome resulting in an increased concentration of cytosolic PrP species affects the viability of certain neuronal cell types (discussed below). These observations have added yet another possible mechanism of neurotoxicity in prion diseases, which at present is not completely defined.
Ubiquitin Reactivity in Prion Diseases An increase in ubiquitin reactivity, compared to controls, has been described in several prion diseases, such as CJD [40-45], GSS [42,46], BSE [47], as well as in experimental models of scrapie in mice [48,49]. Some studies report an increase in this reactivity in association with a longer duration of disease, e.g., in CJD [41], and in the terminal stages of disease in scrapie infected mice [49]. This increased reactivity correlates with reports of an increased expression of polyubiquitin and Hsp70 genes, and a decline in proteasome function, in the terminal stages of disease in scrapie infected mice [49,50]. Furthermore, a systematic immunohistochemical analysis of the distribution of Hsp72, the 20S proteasome, and the ATPases of its 19S regulatory complex in the brains of CJD affected individuals vs.
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 821 controls [44] revealed that neuronal populations with low expression levels of these proteins were more vulnerable in disease indicating the importance of the UPS. Taken together, this data suggests that the UPS is involved in ‘responding’ to the disease state. The distribution of ubiquitin reactivity seen in CJD has been reported to fall mainly into two categories as described by Ironside et al. [42], i.e., ‘punctate’ reactivity in and around PrPSc amyloid plaques and a granular pattern in the neuropil often associated with spongiform changes. In addition ubiquitin reactive inclusions are seen within neurons, and also in ‘thread-like structures’ in the neuropil [42]. Similar ubiquitin reactivity has also been described in mouse models of scrapie [48], in association with PrPSc deposits in GSS [46], and dot-like ubiquitin reactive structures have been described in the neuropil in BSE [47]. In addition to the ubiquitinated intraneuronal inclusions described by Ironside et al. [42], intracytoplasmic ‘inclusion-like bodies’ that react with PrP antibodies, and are somewhat morphologically reminiscent of inclusion bodies described in other neurodegenerative diseases, have, for example, been documented in CJD [51,52] and BSE [47]. However, in these studies the precise intracellular location of the inclusions was not demonstrated, i.e., whether they were in the cytosol or in a cellular compartment such as the lysosome, in which PrPSc has been detected (see below). A recent paper described the co-localization of ubiquitin with PrP aggregates in neurons by con-focal microscopy [43] and another recent report has described ubiquitinated PrPSc in the terminal stages of infection in scrapie infected mice [49]. This is interesting because ubiquitination, and the degradation of ubiquitinated proteins by the proteasome machinery, are cytosolic processes (or nuclear, as proteasomes reside in both these locations, see Chapter 11) [53]. Therefore, the existence of ubiquitinated PrPSc implies that some part of PrPSc, at least, has been in contact with the cytosol, and subsequently been targeted for degradation in that compartment [54]. In light of the results by Kristiansen et al. [55], who detected cytosolic aggresomes of PrPSc in scrapie infected neuronal cell lines (discussed below), it would be interesting to further characterize the precise nature of these ‘inclusion-like bodies’ detected in CJD. The distribution of ubiquitin reactivity in the vicinity of spongiform changes is similar to that of PrPSc and the lysosomal proteinase cathepsin-D [42]. PrPSc has been detected in lysosomes [56] and, as mentioned, this compartment is considered to be important for the PrPC to PrPSc transformation in prion diseases. PrPSc has been detected in ‘late-endosomelike-organelles’ along with ubiquitin [57]. Although ubiquitination of cytosolic regions of transmembrane proteins can target them for endocytosis and lysosomal degradation [58], lysosomes are, in general, not responsible for the degradation of ubiquitinated proteins. However, ubiquitinated proteins from cytosolic aggregates can end up in lysosomes due to autophagic mechanisms in response to impairment of the UPS. This has, for example, recently been described in the case of aggregated huntingtin [59]. Again, in light of the results of Kristiansen et al. [55], this raises the question whether a similar process might sometimes take place in prion diseases as well, which could be one explanation for the observed co-existence of PrPSc and ubiquitin in endosomes-lysosomes. Indeed, autophagic vacuoles have been described in neuronal synapses in CJD [60].
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Neurotoxic Mechanisms in Prion Diseases For the past few years there has been an ongoing debate [61] regarding the intracellular processing of PrPC. This debate revolves around reports that a subset of PrPC is subject to retrotranslocation from the ER for proteasome degradation by the ERAD pathway (see Chapter 13). Accumulation of this cytosolic subset of PrPC, termed cytosolic PrPC, following proteasome impairment can affect the viability of some neuronal cells, and has been suggested as a possible factor in the neurotoxic mechanisms in prion diseases which have not been completely defined. Before the data surrounding this debate are described (see below) it is appropriate to briefly review some of the data regarding the neurotoxic mechanisms in prion diseases. The cell type that is lost in prion diseases is the neuron; degeneration of astrocytes is not seen [62]. Some experiments indicate that there is a selective loss of a subset of GABAergic inhibitory neurons that are immunoreactive for the calcium binding protein parvalbumin [6366]. Other reports suggest a selective loss of glutamatergic neurons [67]. Finally, several observations in human patients, as well as in mouse models of scrapie, suggest that this neuronal cell death is by apoptosis [68-73]. As described above, the disease process in prion diseases involves conversion of host PrPC into PrPSc by a post-translational process, supported by observations that the accumulation of PrPSc occurs without an increase in Prnp mRNA [74]. Inherent in this process is that for each produced PrPSc protein one PrPC is lost. Therefore two possible reasons for the neurodegeneration are loss of functional PrPC or toxic effects of produced PrPSc. However the changes in prion disease tissue have not been sufficiently explained with either of these possibilities (reviewed by Weissmann [75]). For example, there is a lack of phenotype in Prnp0/0 knockout mice [76] and transgenic mice that are rendered Prnp0/0 postnatally do not develop disease [77] suggesting that loss of PrPC function is not the major cause of disease. However, although not the major factor in pathogenesis and in light of the ideas about PrPC function mentioned above, loss of PrPC might increase susceptibility to, e.g., oxidative stress, growth factor deprivation, and apoptotic cascades. Regarding PrPSc, deposition of PrPSc aggregates is often highly associated with neuropathological changes [78], but this is not always the case, and in some instances little or no PrPSc is detected in diseased tissue [20,79,80]. Furthermore, experiments with mice show that Prnp0/0 brain tissue is ‘immune’ to PrPSc produced by infected Prnp+/+ tissue grafts in the same animal [81] and Prnp0/+ mice have a delayed onset of symptoms after infection despite accumulating high amounts of PrPSc [82,83]. Taken together, these results show that in order for PrPSc to have an effect, the target tissue must express PrPC, and that the disease process is correlated with PrPC expression. A recent study has demonstrated that in order for PrPSc aggregates to cause clinical disease the target tissue must express GPI-anchored PrPC, tissue that expresses non-GPI-anchored, secreted, PrPC is not affected [84], suggesting that perhaps toxicity is to some extent due to abrogation of a signalling role of PrPC. In order to explain the pathology in prion diseases alternative pathological forms of PrP have been suggested (reviewed by Chiesa and Harris [85]). As already mentioned, PrPC can take on two different transmembrane orientations. The CtmPrP form of PrPC has been associated with neurodegeneration in a case of GSS and in transgenic mice expressing Prnp
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 823 constructs with a propensity to form CtmPrP [20,86]. Furthermore, experiments indicate that PrPSc induces CtmPrP formation in such mice [86]. However, most PrPC mutations do not cause an increase in transmembrane forms [87], arguing against CtmPrP as a general cause of neurotoxicity in prion diseases. The mechanism by which CtmPrP causes neurodegeneration is unknown. Finally, localization of PrPC to other cellular compartments than the secretory system/plasma membrane, such as the cytosol, and aggregation of such forms due to impairment of the UPS has been linked to neurotoxicity in certain cell types (see below). These reports have sparked the debate mentioned above and resulted in some very interesting reports regarding the cell biology of PrPC, and the association of PrPC with the UPS, as discussed in detail below.
PrPC and PrPSc as Substrates of the Ubiquitin-Proteasome System There are several well documented polymorphisms/mutations in the open reading frame of the prion protein gene in mice, sheep, and humans. Most of the known mutations in the human gene are directly associated with familial forms of prion disease, whereas polymorphisms in mouse and sheep Prnp affect susceptibility to infection with prion diseases. The variation in the human prion protein gene falls into three categories (reviewed by Kovacs et al. [88]): (a) point mutations (some silent), most of which are located within, or near, the secondary structural elements in the C-terminus, (b) amber mutations (Y145stop and Q160stop), and (c) insertions of additional repeats in the flexible N-terminus. How the mutations in human PRNP cause disease is not precisely clear. They may affect the thermodynamic stability of PrPC facilitating the PrPC to PrPSc conversion; however, studies suggest that this is not a general mechanism [89] although it may apply to some mutations [90]. As already mentioned, mutations in the ‘transmembrane’ and its vicinity have been shown to cause an increase in CtmPrP [87]. Finally, some mutations can influence the intracellular processing of PrPC as has been demonstrated for the GSS associated mutations Y145stop [91] and Q217R [92]. In contrast to the ‘normal’ processing of PrPC through the secretory pathway described above, the majority of PrPC with the Y145stop mutation (PrP145) is retrotranslocated for degradation by the UPS and accumulates in the nucleus if the UPS is inhibited [91]. In addition to a nuclear localization, a mitochondrial localization of PrP145 has been reported, affecting mitochondrial membrane potential resulting in apoptosis [93]. Subsequently, it has been demonstrated that another truncated PrP mutant, Q160stop, is processed in a manner similar to PrP145, also showing a nuclear localization following proteasome inhibition [94]. Two nuclear localization signals in PrPC have been defined [95] which are, however, only active if the protein is non-glycosylated and non-GPI anchored. In contrast to PrP145, the majority of PrPC with the Q217R mutation (PrP217) is processed through to a site distal of the cis-Golgi, whereas a small non-GPI anchored subset of PrP217 is retained in the ER in association with the chaperone BiP followed by retrotranslocation for proteasomal degradation [92]. These reports of the processing of mutant PrP’s suggested that in some cases of prion diseases the neurotoxicity might be explained by the aberrant localization of
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PrPC to other locations than the secretory system and plasma membrane in the case of UPS impairment. Observations from the expression of recombinant PrPC in the yeast cytosol revealed that when PrPC is expressed in this compartment it displays characteristics reminiscent of PrPSc, notably detergent insolubility and proteinase-K resistance in a concentration dependant manner, i.e., these attributes are enhanced with higher expression [96]. Similar observations were made for PrP145 in the cytosol of mammalian cells [91]. This prompted studies into whether normal PrPC, like PrP145 and PrP217, is to some degree, generally processed for degradation by ERAD in which case such processing, coupled with the PrPSc like characteristics of PrPC when in this compartment, might be relevant for pathogenesis in the special case of impaired protein degradation. The question of PrPC retrotranslocation was addressed in two studies [97,98]. Their results showed that when mammalian cell lines expressing PrPC are treated with proteasome inhibitors, an unglycosylated PrPC form accumulated that was detergent insoluble and proteinase-K resistant to a degree. One of the studies showed that this PrPC form was ubiquitinated [98]. Furthermore, fluorescence microscopy of these cells revealed that the PrP form that accumulated in response to the proteasome treatment had an altered localization [97], i.e., it was localized in Hsc70 positive cytosolic aggregates, compared to that of PrPC in untreated cells, which resided in the secretory system and on the plasma membrane. The lack of glycosylation, and the size of the PrP species detected in these studies, indicated that the N-terminal and C-terminal signal peptides had been cleaved, and therefore that it had been subject to signal peptide cleavage within the ER and that its location within the cytosol was due to its processing by ERAD. Yedidia et al. [98] estimated that ~ 9% of normal PrPC produced is degraded in the cytosol by the UPS following retrotranslocation from the ER. In addition to these studies it has been reported that PrPC constructs with the CJD associated mutations V203I and E211Q, and the GSS associated mutation Q212P, aggregate in vimentin-positive aggresomes in cells treated with proteasome inhibitors [99]. Finally, treatment of PrPC expressing cells with cyclosporin-A, an inhibitor of the cyclophilin family of peptidyl-prolyl cis-trans isomerases, results in the formation of PrPC aggresomes [100]. Cumulatively, these studies suggested that PrPC was processed by ERAD under normal circumstances as well as in response to impaired PrPC folding due to mutations or inhibition of the folding machinery, in which cases it formed cytosolic aggresomes. The question whether retrotranslocated PrPC, and its PrPSc characteristics, could be associated with pathogenesis was addressed in two studies [35,101]. They reported that (a) the PrPSc like characteristics of cytosolic PrPC were ‘self-perpetuating’ in mammalian cells expressing PrPC following a brief period of proteasome-inhibitor treatment [35], and (b) that cytosolic PrPC was toxic to mouse neuroblastoma cells (N2a) and caused cerebellar degeneration characterized by the loss of cerebellar granular neurons in transgenic mice expressing a cytosolic PrPC construct on a normal background [101]. These results, along with those previously discussed [97,98], prompted the hypothesis that retrotranslocation of PrPC to the cytosol, accompanied by proteasome impairment, plays a role in generating PrPSc and thus in the pathogenesis of prion diseases. However, reports of PrPC retrotranslocation from the ER have been challenged [102]. In short, Drisaldi et al. [102] concluded from their experimental results that PrPC is not
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 825 retrotranslocated and that the observations described above were due to the experimental conditions used. They based their conclusions, among other things, on observations that (a) proteasome inhibition results in increased transcription from the cytomegalovirus (CMV) promoter, a common promoter in expression plasmids used in mammalian cells, and (b) that the PrPC form that accumulates in response to proteasome inhibition in cells expressing PrPC under the control of such a promoter contains an uncleaved N-terminal signal-peptide and is located at the cytosolic side of the ER, therefore indicating that it has not entered the ER and that the accumulation of this cytosolic form is due to inefficient translocation, possibly due to saturation of the translocation machinery due to elevated expression levels [102]. Therefore, they suggested that although some mutant PrP’s are retrotranslocated for degradation by the proteasome, such as described above [91,92], this mechanism does not apply to normal PrPC. Subsequent to the report by Drisaldi et al. [102], evidence has been presented for endogenous cytosolic PrPC following proteasome-inhibitor treatment of primary human neurons and cell lines, without overexpression of PrPC from vector constructs [103-105], suggesting that this subset is not merely due to overexpression from a strong promoter. In addition, cytosolic PrPC, in the absence of proteasome inhibition, has been described in hippocampal neurons of normal mice [106] and in transgenic mice that express a PrPC-EGFP fusion protein from a Prnp promoter [107]. A recent report [105] has presented evidence for an origin of endogenous cytosolic PrPC other than retrotranslocation, i.e., inefficient translocation of PrPC into the ER due to inherent characteristics of the PrPC N-terminal signal sequence resulting in a cytosolic subset of PrPC with an uncleaved N-terminal signal peptide. These results are in line with those of Drisaldi et al. [102] regarding the uncleaved N-terminal signal peptide; however, Rane et al. [105] show that the generation of this cytosolic subset is not due to saturation of the translocation machinery, but rather these characteristics of the N-terminal signal. This might explain the reports of endogenous cytosolic PrPC mentioned above. Rane and colleagues [105] do not rule out retrotranslocation of PrPC; however, their data indicate that the impact of retrotranslocation on the amount of cytosolic PrPC under normal circumstances is minor. Their estimates regarding the amount of cytosolic PrPC indicate that PrPC subject to proteasome degradation due to this inefficiency is approximately 20% of the total protein produced. They point out that the existence of inefficient PrPC translocation implies that it has been evolutionary conserved, and therefore, that it is of possible physiological importance. This conservation was demonstrated by experiments showing that the N-terminal signal sequences of PrPC from four species (human, cattle, hamster, and mouse) are equally ‘inefficient’ in terms of translocation [108]. Subsequent studies by this same group have revealed a marked heterogeneity in the efficiency of translocation by N-terminal signal sequences of various proteins destined for the secretory system [109], indicating that this may be a general mechanism to expand protein use to more than one compartment, as they have demonstrated for calreticulin [110]. This leads to the speculative question, raised by Rane and colleagues [105], whether the inefficiency of PrPC translocation could be due to a physiological role for cytosolic PrPC. The reports detailed above agree that PrPC in the cytosol is rapidly degraded and does not accumulate under normal circumstances. Interestingly, the reported effects of cytosolic PrPC vary, and seem to depend on the neuronal cell type. The stabilization of cytosolic PrPC by
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proteasome inhibition has been reported to cause apoptosis in PrPC expressing mouse neuroblastoma cells (N2a) [101,103,105]. On the contrary, such cytosolic PrPC has also been reported to protect against apoptosis under similar conditions in this cell type [55,111]. As mentioned above, transgenic mice expressing cytosolic PrPC on a normal background had neuropathological changes consisting of gliosis and selective neurodegeneration of cerebellar granular neurons, even though the promoter used resulted in expression of cytosolic PrPC in other neuronal cell types as well [101]. Finally, cytosolic PrPC does not seem to be toxic in human primary neuron cultures or human neuroblastoma cell lines, in fact it counteracts Baxmediated apoptosis in these cells [103].Therefore, not only are there conflicting results regarding the origin, and importance, of a proteasome degraded cytosolic subset of PrPC, but also regarding the effect of cytosolic PrPC on neuronal cells. The discrepancies regarding the effects of cytosolic PrPC suggest that its effect may be determined by the neuronal cell type, its state, and context. The studies described above paved the way for an interesting recent report regarding the processing of PrPSc in prion infected cell lines. Kristiansen et al. [55] utilized cell lines that can be infected with, and propagate, prions to study the intracellular processing of PrPSc formed in these cell lines. They found that when prion infected cells were subject to mild proteasome inhibition, chosen to represent levels of proteasome inhibition seen in vivo in disease or ageing, PrPSc formed cytosolic aggresomes in association with Hsc70, ubiquitin, proteasome subunits, and vimentin. Furthermore, the aggresome formation correlated with apoptosis in these cells characterized by activation of caspases-3 and -8. They also present evidence that PrPSc is associated with vimentin in scrapie infected mice, suggesting that aggresome formation also occurs in vivo. Of interest to the papers discussed above, the conditions of mild proteasome inhibition they used did not cause apoptosis in uninfected cells expressing PrPC.
CONCLUSIONS Despite its small size, the processing of PrPC seems to be fairly complex. This is, for example, demonstrated by its ability to take on several topological forms in respect to the plasma membrane. Furthermore, the precise function of PrPC has remained elusive. In addition to the functional question, there are several unanswered questions in the field of prion science (reviewed by Aguzzi and Polymenidou [112]), for example regarding the basis of neurotoxicity. Reports of the retrotranslocation of mutant PrP’s, and subsequently of normal PrPC, from the ER into the cytosol, and concomitantly of the toxic effects of the accumulation of cytosolic PrPC when the UPS is impaired, promised an explanation for neurotoxicity in prion diseases. However, due to the conflicting reports described above, the relevance of this scenario remains a matter of debate. Overall, retrotranslocation of PrPC may be a special case for certain PrP mutants, whereas inefficient translocation of PrPC into the ER may explain the majority of PrPC in the cytosol at any given time. Therefore, the cytosolic location and aggregation of mutant PrPC when the UPS is impaired could be relevant to neurotoxicity in
The Role of the Ubiquitin Proteasome System in the Pathogenesis of Prion Diseases 827 certain cases of disease but not necessarily a general mechanism. In fact, as mentioned, a non-translocated PrPC subset could have a physiological function rather than a toxic function. The recent description of cytosolic PrPSc aggresomes in scrapie infected cell lines has added yet another dimension to the debate, suggesting that cytosolic PrPSc, rather than PrPC, could be an important factor in prion disease neurotoxic mechanisms. This report raises some interesting questions and opens avenues of investigation, such as, how does PrPSc enter the cytosol? And, can these observations be connected to the increased ubiquitin-reactivity detected in prion diseases described above? For example, can such PrPSc aggresomes be detected by immunohistochemical analysis of prion disease tissue? Elucidations of the molecular pathways that mediate the effects of cytosolic PrPC, whether toxic or protective, and the effects of PrPSc, offer a challenge. The effects may be due to specific interactions with cytosolic proteins, or disruption of such interactions in the case of toxic effects. Therefore the identification of cytosolic PrPC binding proteins, and the characterization of their functional significance, may aid in the understanding of the pathological/protective effects. Several cytosolic PrPC interacting proteins have already been identified with various methods, such as Bcl-2 [113], Grb2 [114], and the neuronally expressed proteins Synapsin Ib [114] and NRAGE (neurotrophin receptor interacting MAGE homolog) [115]. Furthermore, pathogenic mechanisms elucidated in other neurodegenerative diseases characterized by aggregated proteins may, by inference, give valuable clues. For example, aggregated huntingtin has been demonstrated to disrupt functional pathways in cells by interfering with nuclear transcription factors, axonal transport, mitochondria, or the function of the UPS [59]. Considering the nuclear [91], or mitochondrial [93], localization of PrP145 mentioned above and reports of granular PrP deposits in the axons of neurons in CJD patients [43], the question arises whether aggregated PrPSc may have similar effects.
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In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 835-849 © 2007 Nova Science Publishers, Inc.
Chapter 34
ROLE OF THE UBIQUITIN PROTEASOME SYSTEM IN ANTIGEN PRESENTATION, AUTOIMMUNE DISORDERS AND INFLAMMATION IN THE CENTRAL NERVOUS SYSTEM Jakub Golab∗ and Dominika Nowis¥ Department of Immunology, The Medical University of Warsaw, Warsaw, Poland.
ABSTRACT The proteasome plays a pivotal role during proteolytic processing of cellular proteins required for the generation of antigenic peptides presented to cytotoxic T cells by major histocompatibility complex class I molecules. The process of peptide generation is greatly improved by formation of immunoproteasomes through replacement of three β subunits with β1i (also called LMP2), β5i (LMP7) and β2i (MECL-1) and expression of PA28, a heptameric activator complex. The assembly of immunoproteasomes is stimulated by interferon-γ, a cytokine that is produced shortly after viral infection and is one of the mediators that link innate and adaptive immune responses. Numerous infectious microorganisms developed sophisticated strategies to avoid presentation of their antigenic peptides including production of proteasome-modulating molecules. Recent studies indicate a unique mechanism of epitope generation by proteasomes referred to as peptide splicing. In the brain, microglial cells are the major antigen presenting cells. However, during inflammation virtually all cells in the central nervous system can be induced to express immunoproteasomes and to present antigens in association with MHC class I molecules. The proteasome-mediated generation of peptide ∗
¥
Correspondence concerning this article should be addressed to Dr. Jakub Golab, MD, PhD; Department of Immunology, The Medical University of Warsaw, Ul. Banacha 1a, F building, 02-097 Warsaw, Poland. Phone: *48-22-5992199; Fax: *48-22-5992194 E-mail:
[email protected]. Correspondence concerning this article should be addressed to Dr. Dominika Nowis, MD, PhD; Department of Immunology, The Medical University of Warsaw, Ul. Banacha 1a, F building, 02-097 Warsaw, Poland; Phone: *48-22-5992199; Fax: *48-22-5992194 E-mail:
[email protected].
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Jakub Golab and Dominika Nowis epitopes evolutionarily serves to present antigens derived from intracellular infectious microorganisms. However, proteolytic processing of intracellular proteins is by no means selective and includes processing of all proteins including self molecules that can become targets for cytotoxic T cells during several inflammatory and degenerative central nervous system diseases, such as multiple sclerosis and paraneoplastic neurological disorders. The role of ubiquitin-proteasome pathway in neuroinflammatory disorders extends beyond antigen processing for MHC class I presentation. Activation of NF-κB a key modulator of inflammatory reaction results from proteasomal degradation of its inhibitor – IκB. Proteasomes are also involved in the regulation of the activity of other transcription factors involved in the inflammatory responses including STAT proteins and Egr-1. Understanding of the underlying mechanisms involved in proteasomemediated inflammatory processes is important for the development of novel, mechanismbased drugs.
Keywords: Antigen presentation, Autoimmune diseases, Major histocompatibility complex, Proteasome, Ubiquitin.
ABBREVIATIONS AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; APP, amyloid precursor protein; BBB, blood-brain barrier; bFGF, basic fibroblast growth factor; β2m, β2 microglobulin; BiP, bding protein; CD, cluster of differentiation; CNS, central nervous system; COX, cyclooxygenase; CTL, cytotoxic T lymphocyte; DRips, defective ribosomal proteins; EAE, experimental autoimmune encephalomyelitis; ER, endoplasmic reticulum; ERAP, endoplasmic reticulum-resident aminopeptidases; IKK, IκB kinase; IL, interleukin; LMP, low molecular mass protein; LPS, lipopolisaccharide; MECL, multicatalytic endopeptidase complex; MHC, major histocompatibility complex; MS, multiple sclerosis; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; NK, natural killer; NES, nuclear-export signal; NGF, neuron growth factor; NIK, NF-κB kinase; NLS, nuclearlocalization sequence; NO, nitric oxide; PD, Parkinson’s disease; RHD, Rel homology domain; ROS, reactive oxygen species; SAPPα, Secreted amyloid precursor protein; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TAP, transporters associated with antigen presentation; TCR, T cell receptor; TNF, tumor necrosis factor.
INTRODUCTION Central nervous system (CNS) has been regarded as an immune privileged site. This concept has been supported by both anatomic and functional observations that include descriptions of a unique blood-brain barrier (BBB), lack or low expression of MHC class I and class II molecules and lack of lymphatic drainage. However, CNS should not be completely devoid of immune protection. There must exist mechanisms to control infections and tumor development. These mechanisms clearly operate in the CNS and it seems that the
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unique immunity within the brain results from an active tolerance rather than passive barrier mechanisms. Accumulating evidence indicates that MHC class I molecules are indeed expressed in different brain regions by both neuronal cells and supporting glia [1]. Inability to detect MHC molecules was rather an artifact caused by using aldehyde-fixed brain sections and antibodies developed for fluorescence-activated cell sorting. Moreover, neurons can be induced to express high levels of MHC molecules following various experimental procedures including axotomy [2], exposure to cytokines [3] or manipulation of electrical activity [4]. Moreover, viral, parasitic or bacterial infections [5], neuronal transplantation [6] or even seizures [4] can also induce expression of MHC class I molecules in the brain. Intriguingly, despite the expression of MHC molecules neurons are usually resistant to lysis by cytotoxic T lymphocytes (CTL) and soluble factors released by neurons or glia can interfere with CTL activity thereby creating an environment suitable for immunomodulation [7]. Interestingly, it seems that the role of MHC class I molecules in the CNS expands beyond the mere antigen presentation for the induction of protective immunity. These molecules seem to be involved in both structural and functional plasticity and are thought to participate in the weakening and strengthening of synapses [8,9]. At least in rodents MHC class I molecules are also possibly involved in olfaction [10]. The functional significance of the immune response in the brain can be underscored by the existence of various neuroinflammatory disorders. Some of these result from the failure of self tolerance and lead to development of autoimmune disorders such as multiple sclerosis or various paraneoplastic autoimmune reactions and primarily involve adaptive immunity. Other pathologies that involve the innate immune system include degenerative disorders of the central nervous system (CNS) and include Alzheimer’s disease (AD), Parkinson’s disease (PD) or stroke and can be triggered secondarily for example by local deposition of amyloid βprotein [11]. The importance of the immune reactions in the CNS will possibly be even greater in the nearest future due to the development of novel therapeutic approaches to treat these diseases. For example, the unsuccessful results of the recent double-blind placebocontrolled trials in patients with PD receiving allografts of fetal dopaminergic neurons might result from the development of a classical transplantation immunity and rejection of grafted tissue [12].
THE ROLE OF THE UBIQUITIN-PROTEASOME SYSTEM IN ANTIGEN PRESENTATION Major histocompatibility antigens (MHC) act as platforms presenting antigenic peptides to T cell receptors (TCR) on T lymphocytes. There are two groups of classical MHC molecules referred to as class I and class II molecules [13]. Class I MHC molecules are responsible for antigen presentation to ‘killer’ or cytotoxic CD8+ T cells (CTL), while MHC class II molecules present antigens to helper CD4+ T cells. Additionally, MHC class I molecules can be recognized by natural killer (NK) cells that depending on additional signals can become triggered or suppressed. MHC class I molecules present so called endogenous antigens i.e. peptides derived from proteins synthesized within the MHC-bearing cell. On the other hand MHC class II molecules present exogenous antigens derived from proteins
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endocytosed by antigen presenting cells. The genes encoding human MHC molecules (human leukocyte antigens – HLA) are the most polymorphic genes ever known and currently account to 2435 different alleles (IMTG/HLA Sequence Database: http://www.ebi.ac.uk/imgt/hla/). Since proteasomes are involved in the generation of antigenic peptides bound and presented by MHC class I molecules expressed by all nucleated cells, including neurons and other cells in the brain, the discussion in this chapter will be limited to this pathway of antigen presentation only.
General Description of MHC Class I Peptide Loading MHC class I molecules are constitutively expressed by most nucleated cells and present endogenous peptides of 8-11 amino acids [14]. All are composed of a heavy chain (composed of three immunoglobulin-like domains) and a constant β2-microglobulin (β2m). During assembly and folding heavy chains are extensively chaperoned by cytosolic BiP proteins and finally by endoplasmic reticulum (ER) lectin calnexin [15]. Final complexing with β2m results in an exchange of calnexin into a related calreticulin followed by a formation of a large complex of proteins that resides in the ER. This protein complex is frequently referred to as a loading complex as it promotes peptide loading into MHC class I peptide-binding groove [16]. The loading complex includes MHC molecule, calreticulin as well as tapasin, TAP and Erp57 molecules [17]. Tapasin functions as a transmembrane protein that interacts with TAP and MHC molecules thus stabilizing the loading complex – it is required to retain these molecules in the ER until optimal peptides have been loaded [18,19]. Erp57 is a thiol oxidoreductase that associates with MHC class I molecules and facilitates disulfide bond formation thus influencing the conformation of these molecules [20]. Additionally Erp57 is a cysteine protease that facilitates trimming of peptides that are being loaded onto MHC class I molecules (see below). Peptide transporters (TAP1 and TAP2 proteins) shuffle peptides from the cytosol to the ER thereby supplying antigens for presentation [21]. TAP proteins transport peptides of 7 to more than 20 amino acids so some of the longer peptides can be trimmed within ER by a number of aminopeptidases (see below). The major source of peptides for antigen presentation is the ubiquitin-proteasome system but some peptides are generated in the cytosol by TPPII [22].
Where do the Peptide Antigens Come from? This is certainly not a trivial question. Unfortunately, very little is known about the peptide processing in the CNS. An average cell contains approximately 2.6 x 109 proteins and produces some 4 millions new proteins every minute [23]. To avoid chaos there must exist sophisticated mechanisms that control targeting as well as turnover of cellular proteins. Proteolytic degradation is responsible for the elimination of damaged, unfolded or incomplete proteins. It is also engaged in the regulation of their function. Therefore, proteolysis might serve as an abundant source of peptide libraries derived from the majority of cellular proteins that would be sampled by MHC class I molecules (Figure 1). Normal cells infected by viruses
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or intracellular parasites would contain both self and foreign proteins with the latter presumed to be processed and presented in the most immunogenic manner. For many years it was thought that most peptides destined for presentation by MHC molecules would derive from old or used proteins targeted for degradation as a part of protein turnover. It was therefore unexpected to observe that the majority of antigenic peptides are derived from proteins degraded immediately after synthesis [24]. A rich source of antigens are peptide ligands derived from cryptic transcription products, such as open reading frames contained within 5’ and 3’ untranslated regions, alternative open reading frames, introns, or intron-exon junctions [25-27]. It is known that protein synthesis is error prone. These erroneous proteins are referred to as defective ribosomal products (DRips) and they represent between 30% to as many as 80% of newly synthesized proteins [23,28]. Since accumulation of these proteins could have disastrous consequences for the cell they are rapidly ubiquitinated and degraded by proteasomes. Therefore, MHC molecules have an immediate access to peptides derived from current profile of protein expression pattern. This strategy makes sense from the perspective of immunity. CTL are part of the surveillance system that look for MHC molecules presenting peptide antigens derived from viral or mutated proteins and exert cytotoxic activity towards the cells having such abnormal antigens. This surveillance mechanism is exquisitely sensitive as CTL can respond to as little as one MHC class I peptide complex at the cell surface [29]. This system also needs to be very rapid. In extreme conditions a viral cycle (a time necessary to enter the cell, replicate and release new progeny) is just about 4 hours. Early detection of pathogen-derived peptides is of paramount importance especially that viruses use a cohort of genes that interfere with antigen presentation [30]. Despite these pressing demands, the frequency of peptides available for MHC class I binding is extremely low. Out of 8 x 105 proteasomes that degrade 2.5 substrates per minute only one for each 500-3000 viral translation products degraded is suitable for presentation [23]. We still do not understand this apparent insufficiency in the generation of antigenic peptides.
The Role of Proteasomes in the Generation of Antigenic Peptides Individual MHC class I molecules can bind to a limited set of peptides restricted predominantly by particular anchor residues of which one is always located at the carboxy (C) terminus [31,32]. While proteasomes form a central proteolytic system it is clearly insufficient to generate final peptides for MHC class I binding. Nonetheless, proteasomes are the only cellular proteases that generate correct C-terminus of peptides for presentation and there is no further need for C-terminal processing [33,34]. Final trimming of oligopeptides requires additional proteases that include TPPII, and other aminopeptidases such as leucine aminopeptidase, bleomycin hydrolase, puromycin-sensitive aminopeptidase, thimet oligopeptidase as well as endoplasmic reticulum-resident aminopeptidases ERAP1 and LRAP [31]. There is a number of unresolved and sometimes paradoxical observations regarding the mechanism of peptide generation by proteasomes. They seem to cut proteins blindly irrespective of their potential further utility for the cell. Purified proteasomes can even
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Figure 1. The pathway for presentation of antigenic peptides in association with MHC class I molecules. The majority of peptides presented by MHC class I molecules derive from defective ribosomal products (DRips). These incorrectly produced polypeptides undergo ubiquitination followed by proteasomal degradation. The peptides frequently undergo further proteolytic trimming with cytoplasmic and endoplamic reticulum-associated proteases. Transporters associated with antigen processing (TAP) translocate peptides from the cytosol to the lumen of endoplasmic reticulum where they are loaded into grooves of MHC class I pockets by the chaperone complex. Then the peptide antigen-loaded MHC molecules are transported to the surface of the cells where they can be sampled by CD8+ T cells.
destroy immunodominant epitopes [35]. Although they are required for the generation of antigenic peptides they in fact destroy many more epitopes than they generate [36,37]. Purified proteasomes degrade proteins mainly into 2-25 amino acid-long peptides. Only a
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small fraction of these (10%) are of correct size to fit into MHC class I molecules, with the majority (65%) being too short for this. The remaining 25% of peptides are too long to fit into MHC class I grooves [31,36,37]. This latter pool of peptides undergoes N-terminal processing to generate antigens suitable for MHC loading. Until recently it was thought that peptides generated by proteasomes are continuous sequences derived from cellular proteins generated by simple proteolysis. However, it now seems that antigenic peptides can be the products of cutting and pasting reactions analogous to exon-intron splicing events during mRNA maturation. It was shown that antigenic peptide derived from basic fibroblast growth factor (bFGF) is in fact a nonamer consisting of residues 172 to 176 fused to residues 217 to 220 of the protein [38]. Similarly, melanocyte glycoprotein gp100-derived nonamer corresponds to residues 40 to 52 with amino acids 43 to 46 excised from the original sequence [39]. It seems that proteasomes can degrade proteins in such a way that the terminal amino group of one peptide can attack the acyl-enzyme intermediate (a complex between the remaining peptide end and a proteasomal β subunit) thus regenerating a peptide bond between two previously separated amino acids.
Fine-Tuning of Antigen Presentation with Proteasomes An effective immune response results from a series of positive feedback loops that include improved antigen presentation. Under the influence of certain cytokines such as IFNγ and TNF the cells improve their antigen-presenting capabilities [40]. They not only express more MHC and co-stimulatory molecules but have more dexterous machinery to process and load peptides into MHC class I peptide-binding grooves. IFN-γ induces the synthesis of proteasomal β subunits: β1i (LMP2), β2i (MECL1) and β5i (LMP7) that are incorporated into newly assembled proteasomes instead of their counterparts: β1, β2 and β5 [41,42]. These so called immunoproteasomes have different cleavage site preferences as well as a different cleavage rate. Additionally, IFN-γ induces the synthesis of a proteasomal activator PA28, a heptameric complex composed of three PA28α and four PA28β subunits that assemble with the outer α ring of the immunoproteasomes [43,44]. IFN-γ is also inducing phosphorylation of the α7 subunit of the 20S proteasome thereby facilitating disassembly of 26S proteasomes [45]. The significance of this unexpected modification is unknown but it may either suggest that free 20S proteasomes are somehow engaged in antigenic peptide generation or alternatively this modification is part of a negative regulation of antigen presentation. The significance of these modifications in the CNS is unknown. So far PA28αβ activator has not been detected in the brain [46]. Immunoproteasomes are not any better in peptide generation than regular proteasomes [40]. However, it was shown that immunosubunits are necessary for the generation of influenza-derived antigens or in the development of an effective response against hepatitis B virus [47,48]. Interestingly, it was recently shown that immunoproteasomes are incapable of generating several immunogenic antigens from self proteins [49]. This mechanism might be extremely important in negative regulation of potentially devastating autoimmune disorders that could develop during protective antiviral response.
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The overall improved antigen presentation by immunoproteasomes seems to result from the capacity to generate longer polypeptides, with a correct C terminus but an extended N terminus that might facilitate TAP transport to endoplasmic reticulum where the peptides would undergo final trimming by aminopeptidases [35]. This process is greatly facilitated by PA28 that induces conformational changes in the α subunits thereby leading to the opening of the central gates of the proteasome and a faster exit of the cleavage products from the catalytic cavity [40,50].
THE ROLE OF PROTEASOMES IN THE REGULATION OF INFLAMMATORY RESPONSES IN THE BRAIN The role of the ubiquitin-proteasome system in the development of immune response expands beyond just antigen presentation. In fact defective degradation of ubiquitinated proteins may result in up-regulation of cyclooxygenase 2 (COX-2) expression and commencement of neuroinflammation [51]. Several transcription factors involved in the regulation of inflammatory response are regulated by the ubiquitin-proteasome system. These primarily include NF-κB, which is activated by proteasome-mediated degradation of its inhibitor IκB. However, other transcription factors that induce expression of inflammationassociated molecules are degraded by this proteolytic system indicating that the regulation of inflammation is more complex than originally thought. SOCS-1, potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines targets Jak1 to a perinuclear distribution resembling the microtubule organizing complex where it is degraded by proteasomes [52]. Egr-1 and STAT1 are transcription factors that induce the expression of proinflammatory genes are negatively regulated by proteasomal degradation [53,54].
Regulation of NF-κB Activity The term NF-κB (nuclear factor kappa B) covers a family of inducible transcription factors that regulate the host immune and inflammatory responses and cellular growth properties [55]. NF-κB was first identified in the nuclei of mature B lymphocytes as a transcription factor binding an 11-bp DNA sequence in the κ-light chain enhancer [56]. The NF-κB family mediates the transcription of over 180 target genes, including genes for cell adhesion molecules, cytokines, chemokines and antiapoptotic factors [57]. NF-κB consists of homodimers or heterodimers of a family of proteins sharing a 300-acid common Rel homology domain (RHD). RHD allows DNA-binding, dimerization and nuclear localization of NF-κB [57,58]. The Rel family includes the following members: p105/50 (NF-κB1), p100/52 (NF-κB2), p65 (RelA), RelB and c-Rel [58]. Each member of the NF-κB family, except for RelB, can form homodimers, as well as heterodimers with one another [59]. The major activated form of NF-κB consists of the p65 subunit associated with either a p50 or p52 subunit [59]. In a stable state, NF-κB binds IκB. IκB is an inhibitory molecule that sequesters NF-κB in cellular cytoplasm in an inactive state, covering its RHD [57]. There are at least six IκB proteins: IκBα, IκBβ, IκBε, IκBδ, IκBγ and Bcl-3. The first three of these are
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stimulus-dependent regulators, while the others have distinct functions [60]. These proteins have several ankyrin repeats, a 33-amino-acid motif that mediates protein-protein interactions [59]. IκB masks dual nuclear-localization sequences (NLSs) on NF-κB subunits. There is a dynamic balance between the nuclear and cytosolic amount of inactive NF-κB-IκB complexes. IκBα covers only one of the two NLSs in the NF-κB dimer. At the same time, the nuclear-export signal (NES), a part of IκB, functions to expel the NF-κB-IκB complex from the nucleus. IκBβ, by contrast, covers both NLSs on the NF-κB dimers and preserves them in the cytoplasm [59,61]. It is well established that IκBα regulates transient, while IκBβ regulates persistent NF-κB activation. In response to a stimulus, IκBα is quickly degraded and resynthesized. The newly formed IκBα subunit has its own NLS that allows it to enter the nucleus, displace the NF-κB form its DNA binding site and transport it back to the cytoplasm [59]. The proinflammatory cytokines such as TNF or IL-1, signal to NF-κB by activating the IKK complex (IκB kinase). IKK is composed of three subunits. The subunit γ of IKK called NEMO (NF-κB essential modulator) is a regulatory component. Subunits α and β, serving as kinases, phosphorylate IκB on Ser32 and Ser36 in IκBα and Ser33 and Ser37 in IκBβ [62]. Phosphorylated IκB is recognized by the β-TrCP component of the SCF ubiquitination complex that in consequence leads to the IκB ubiquitination and degradation by the 26S proteasome. β-TrCP is critical for the preserving IκB phosphorylation prior to ubiquitination. Degradation of IκB by IKK frees NF-κB to stably translocate to the nucleus where it induces expression of target genes [61,63]. The role of proteasomes in the regulation of NF-κB activity is more complex. 26S proteasome not only degrades IκB proteins but also mediates the proteolytic cleavage of the p105 precursor to the p50 subunit of NF-κB1. Moreover, the activity of the IKK kinase depends on the formation of unusual polyubiquitynated chains linked by Lys63 [64]. Cellular response to a wide range of different stimuli leads to NF-κB activation. Among them, there are cytokines (TNF superfamily, IL-1, IL-18], inducers of the reactive oxygen species (ROS) such as hydrogen peroxide, infectious agents (bacterial as well as viral), inducers of apoptosis, carcinogens, tumor promoters and diverse kinds of stress (change in the cellular pH, hypoxia, presence of heavy metal ions) [56]. These stimuli reveal the role of NF-κB in cellular adaptation to stress. There are two ways of NF-κB activation. The classical signaling pathway is mediated by IKKβ and leads to the phosphorylation and degradation of IκB. The stimuli that result in this pathway include predominantly proinflammatory agents (TNF, IL-1, LPS and double-stranded RNA). The nonclassical pathway of NF-κB activation involves IKKα and results in p100 phosphorylation and its cleavage to the p52 subunit. IKKα is activated by the upstream kinase NIK. This signaling pathway is activated by the lymphotoxin β receptor (LTβR) in the stromal cells to produce the B-lymphocyte chemoattractant required for the proper lymphoid organs development. [61,63,65]. NF-κB is also modified posttransationaly. For its transcriptional activity, NF-κB requires the phosphorylation of p65 by the MAP kinases [61].
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The Proteasome-Mediated Regulation of NF-κB Activity in Neuro-Inflammation and Neurodegenerative Disorders NF-κB is a crucial transcription factor for glial and neuronal cell functions. It is involved in the processes of neuronal plasticity, neurodegeneration and neuronal development [66]. Interestingly, in the CNS neurons, a high level of constitutive NF-κB has been detected, probably due to the electrical activity within neurons and synaptic transmission, which are the potent stimuli for the NF-κB activation [67]. Inducible activity of NF-κB is present in presynaptic as well as postsynaptic sites and in the neuronal cytoplasm [66]. As stated above, NF-κB is a critical regulator of neuronal apoptosis and plays an extremely important role in survival of neurons exposed to cell injury, mostly by upregulating a wide spectrum of antiapoptotic and antioxidant genes [68]. On the other hand, by promotion of production of cytotoxic agents (such as NO) NF-κB may lead to the apoptosis of surrounding cells. In the microglia, activation of NF-κB stimulates the production of ROS and excitotoxins, which are toxic to the environment but at the same time activated microglia produce neurotrophic factors (e.g. NGF, bFGF, TNF), which are essential for the proper neuronal function and prevention of their apoptosis [67]. The role of NF-κB in the initiation and progression of the neurodegenerative disorders is complex. In Alzheimer’s disease (AD), increased NF-κB activity is detected in the immediate vicinity of amyloid plaques. Amyloid β-peptide (Aβ) as well as glycated tau proteins can activate NF-κB [62,67]. This activation may, actually, be neuroprotective, while TNF preserves neurons from Aβ-induced cell death in the NF-κB-dependent manner [67]. Secreted amyloid precursor protein (sAPPα) can also activate NF-κB and is regarded as a neuroprotectant [66]. Moreover, decreased NF-κB activity is linked to the early-onset inherited form of AD. Cells expressing mutation in the Presenilin-1 gene have an insufficient level of activated NF-κB, that leads to their death. Based on the data from in vitro and in vivo studies, the activation of NF-κB in the amyloid deposits seems to be a cytoprotective response [67]. NF-κB is involved in the pathogenesis of Parkinson’s and Huntington diseases and amyotrophic lateral sclerosis (ALS). The increased NF-κB activity in the affected neurons may represent, similarly to AD, an early protective response [67]. NF-κB is highly active at sites of inflammation. The NF-κB-mediated production of excessive amount of proinflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases, COX-2 and iNOS play a crucial role in the exacerbation of the pathologic processes [69]. In multiple sclerosis (MS), a neuronal disorder with strong inflammatory background, NF-κB activity is detected at high levels in microglia of active plaques [67]. In the experimental autoimmune encephalomyelitis (EAE), an animal MS model, the positive role of NF-κB inhibition (predominantly by the use of proteasome inhibitors) has been reported [70]. Moreover, some predisposing alleles in the inhibitors of NF-κB genes leading to the excessive activation of NF-κB increase MS risk [71]. Activation of NF-κB is also crucial for the progression of brain infections caused by viruses, including, for example, human polyoma JC, EBV and measles viruses [66,68]. NF-κB alone is required for HIV replication in astrocytes and is involved in the pathogenesis of HIV-induced encephalitis [66]. The use of agents inhibiting NF-κB activity in the therapy of the neurological disorders is complicated.
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They may attenuate the inflammatory reactions but on the other hand, even exacerbate the neurodegenerative processes and inhibit memory and learning ability [67].
CONCLUSIONS The role of the ubiquitin-proteasome system in antigen presentation is relatively well known. However, most of the identified details of the protein processing machinery derive from studies on fibroblasts and professional antigen presenting cells such as dendritic cells and macrophages. Very little is known about potential similarities or differences in antigen presentation between peripheral tissues and brain. Elucidation of these differences should prove useful in designing effective therapeutic strategies aimed at thwarting the pathological immune responses in the course of autoimmune and neurodegenerative diseases. The role of the ubiquitin-proteasome system in the regulation of innate or inflammatory disorders is much more complex. Proteasomes are engaged in the degradation of inhibitors (IκB) of transcription factors (NF-κB) thereby augmenting the inflammatory response. Therefore, the use of proteasome inhibitors might seem justified in the treatment of several neuroinflammatory diseases. However, proteasomes are also engaged in the degradation of egr-1, STAT proteins which also drive inflammatory responses. Moreover, inhibition of NFκB pathway might result in impaired neuroprotection conferred by this transcription factor.
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[42] Ortiz-Navarrete V, Seelig A, Gernold M, Frentzel S, Kloetzel PM, Hammerling GJ: Subunit of the '20S' proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex. Nature 1991, 353: 662-664. [43] Dick TP, Ruppert T, Groettrup M, Kloetzel PM, Kuehn L, Koszinowski UH, Stevanovic S, Schild H, Rammensee HG: Coordinated dual cleavages induced by the proteasome regulator PA28 lead to dominant MHC ligands. Cell 1996, 86: 253-262. [44] Groettrup M, Soza A, Eggers M, Kuehn L, Dick TP, Schild H, Rammensee HG, Koszinowski UH, Kloetzel PM: A role for the proteasome regulator PA28alpha in antigen presentation. Nature 1996, 381: 166-168. [45] Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ: Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. Biochem J 2004, 378: 177-184. [46] Rechsteiner M, Hill CP: Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol 2005, 15: 27-33. [47] Sibille C, Gould KG, Willard-Gallo K, Thomson S, Rivett AJ, Powis S, Butcher GW, De Baetselier P: LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes. Curr Biol 1995, 5: 923-930. [48] Sijts AJ, Ruppert T, Rehermann B, Schmidt M, Koszinowski U, Kloetzel PM: Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes. J Exp Med 2000, 191: 503-514. [49] Morel S, Levy F, Burlet-Schiltz O, Brasseur F, Probst-Kepper M, Peitrequin AL, Monsarrat B, Van Velthoven R, Cerottini JC, Boon T, Gairin JE, Van den Eynde BJ: Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity 2000, 12: 107-117. [50] Stohwasser R, Salzmann U, Giesebrecht J, Kloetzel PM, Holzhutter HG: Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28. Eur J Biochem 2000, 267: 6221-6230. [51] Li Z, Jansen M, Pierre SR, Figueiredo-Pereira ME: Neurodegeneration: linking ubiquitin/proteasome pathway impairment with inflammation. Int J Biochem Cell Biol 2003, 35: 547-552. [52] Vuong BQ, Arenzana TL, Showalter BM, Losman J, Chen XP, Mostecki J, Banks AS, Limnander A, Fernandez N, Rothman PB: SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome. Mol Cell Biol 2004, 24: 9092-9101. [53] Bae MH, Jeong CH Kim SH, Bae MK, Jeong JW, Ahn MY, Bae SK, Kim ND, Kim CW, Kim KR, Kim K: W. Regulation of Egr-1 by association with the proteasome component C8. Biochim Biophys Acta 2002, 1592: 163-167. [54] Yokosawa N, Yokota S, Kubota T, Fujii N: C-terminal region of STAT-1alpha is not necessary for its ubiquitination and degradation caused by mumps virus V protein. J Virol 2002, 76: 12683-12690. [55] Yamamoto Y, Gaynor RB: Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J Clin Invest 2001, 107: 135-142.
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[56] Garg A, Aggarwal BB: Nuclear transcription factor-kappaB as a target for cancer drug development. Leukemia 2002, 16: 1053-1068. [57] Aradhya S, Nelson DL: NF-kappaB signaling and human disease. Curr Opin Genet Dev 2001, 11: 300-306. [58] Delhalle S, Blasius R, Dicato M, Diederich MA: Beginner's Guide to NF-κB Signaling Pathways. Ann N Y Acad Sci 2004, 1030: 1-13. [59] Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol 2002, 2: 725-734. [60] Baeuerle PA: IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell 1998, 95: 729-731. [61] Dixit V, Mak TW: NF-kappaB signaling. Many roads lead to madrid. Cell 2002, 111: 615-619. [62] Makarov SS: NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today 2000, 6: 441-448. [63] Pomerantz JL, Baltimore D: Two pathways to NF-kappaB. Mol Cell, 2002 10: 693-695. [64] Wojcik C, Di Napoli M: Ubiquitin-proteasome system and proteasome inhibition: new strategies in stroke therapy. Stroke 2004, 35: 1506-1518. [65] Mercurio F, Manning AM: NF-kappaB as a primary regulator of the stress response. Oncogene 1999, 18: 6163-6171. [66] O'Neill LA, Kaltschmidt C: NF-kappa B: a crucial transcription factor for glial and neuronal cell function. Trends Neurosci 1997, 20: 252-258. [67] Mattson MP, Camandola S: NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest 2001, 107: 247-254. [68] Baldwin AS Jr: Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest 2001, 107: 3-6. [69] Tak PP, Firestein GS: NF-kappaB: a key role in inflammatory diseases. J Clin Invest, 107: 7-11, 2001. [70] Elliott PJ, Zollner TM, Boehncke WH: Proteasome inhibition: a new anti-inflammatory strategy. J Mol Med 2003, 81: 235-245. [71] Miterski B, Bohringer S, Klein W, Sindern E, Haupts M, Schimrigk S, Epplen JT: Inhibitors in the NFkappaB cascade comprise prime candidate genes predisposing to multiple sclerosis, especially in selected combinations. Genes Immun 2002, 3: 211-219.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 851-863 © 2007 Nova Science Publishers, Inc.
Chapter 35
THE FUNCTIONAL ROLE OF PROTEASOME ANTIBODIES IN NEUROLOGICAL DISORDERS Anette Storstein1,∗ and Christian Vedeler1,2 1
2
Department of Neurology, Haukeland University Hospital, and Institure of Clinical Medicine, University of Bergen, N-5021 Bergen, Norway.
ABSTRACT The proteasome is involved in a number of critical intracellular processes. A major function of the proteasome is non-lysosomal degradation of intracellular proteins, in particular defect or damaged proteins. Targeted proteins are attached to ubiquitin, a process which is catalysed by three enzymes, E1 – E3. In human brain, the activity of proteasome is varying in different regions and with age. The normal function of proteasome in the nervous system is as essential as in other tissues, and perhaps even more, due to the limited capability of renewal of neurons and glial cells. Indeed, inhibition of proteasome alone has been shown to induce neuron death in vitro. There is considerable interest concerning the role of the ubiquitin-proteasome pathway in pathological neurodegeneration. This is partly due to the observation that proteasome activity decreases with normal aging of the brain. The main reason, however, is the accumulation of disease-related proteins in aggregates within neurons or glial cells that is a major feature of many neurodegenerative diseases, as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease. Besides proteolysis, another vital task of the proteasome is the processing of intracellular proteins to be presented by the MHC class I molecules to cytotoxic T lymphocytes on the surface of the cell. Antibodies to proteasome have been identified in patients with autoimmune diseases as systemic lupus erythematosus and Sjögren syndrome. Proteasome antibodies have recently also been identified in serum from patients with immune-mediated neurological diseases, as multiple sclerosis (MS) and paraneoplastic cerebellar ∗
Correspondence concerning this article should be addressed to Dr. Anette Storstein, Department of Neurology, Haukeland University Hospital, N-5021 Bergen, Norway. Phone: + 47-559-75044; FAX +47-559-75165 E-mail:
[email protected].
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Anette Storstein and Christian Vedeler degeneration (PCD). In addition, autoreactive T cells to proteasome have been identified in MS patients. The presence of circulating proteasome antibodies suggests a more widespread affection of the immune system than indicated by the organ-specific nature of MS and PCD. A humoral response to proteasome can be triggered by the elevated proteasome levels that are found in some autoimmune diseases secondary to tissue damage. Primary damage of cells is a plausible explanation for an immune response targeted to an intracellular organelle. PCD is associated with a systemic tumour, and apoptosis of dying tumour cells could result in cross-presentation of intracellular antigens to the immune system and evoke immune responses, whereas the mechanism of initiation of systemic immune responses to proteasome in MS is unknown. The functional role of antibodies to proteasome in chronic inflammatory neurological disease remains to be determined. If the antibodies are indeed of pathogenic importance, as has been shown for antibodies to intracellular targets in SLE, the action of these antibodies might mimic the action of synthetic proteasome inhibitors. Such inhibitors exert their action through disturbance of protein breakdown, inhibition of antigen presentation and inhibition of proliferation, as well as the induction of apoptosis. In animal models, proteasome inhibitors also have potent anti-inflammatory effects. Thus, immune responses to proteasome in chronic inflammatory disease can potentially be both harmful, through interference with normal proteasome function, and beneficial, by suppressing inflammation. This review article aims to evaluate the current literature on antibodies to proteasome in neurological diseases, and to discuss the potential importance of these responses.
Keywords: Neurodegenerative disease, autoimmune diseases, proteasome complex, paraneoplastic neurological syndromes.
antibody formation,
ABBREVIATIONS ANA, Anti-nuclear antibodies; dsDNA, double-stranded DNA; CNS, Central nervous system; CSF, Cerebrospinal fluid; ELISA, Enzyme-linked immunosorbent assay; MHC, Major histocompatibility complex; MS, Multiple sclerosis; PCD, Paraneoplastic cerebellar degeneration; PEM, Paraneoplastic encephalomyelitis; SLE, Systemic lupus erythematosus; UPS, Ubiquitin-proteasome system.
INTRODUCTION The proteasome and its unique role in the maintenance of normal function in all eukaryotic cells has received a lot of scientific attention during the later years. The ubiquitinproteasome system (UPS) is crucial in the degradation of intracellular proteins and the processing of antigenic peptides for presentation by MHC class I molecules. The integrity of UPS function in the central nervous system (CNS) is particularly important due to the essential function and vulnerability of neurons, and their limited capability for renewal. The role of the UPS in the CNS in normal state seems to go far beyond proteolysis; for instance, UPS is crucial in the development of the nervous system [Chapter 15]. The role of the
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proteasome in neurodegeneration is of specific interest to neurologists and neuropathologists, as these diseases are associated with the accumulation of misfolded proteins, forming intracellular aggregates [Chapter 23]. Disturbance of the UPS is now believed to participate in the pathogenesis of neurodegenerative disorders as Parkinson’s disease, prion diseases and Huntington’s chorea. Another central topic of research interest is the use of proteasome inhibitors, which is emerging as a major treatment strategy in some types of cancer [1], but which also seems to have therapeutic potentials in the field of cerebrovascular disease [2, and Chapters 39 and 41]. The aim of this chapter is to discuss the current literature on naturally occurring antibodies to proteasome. The number of reports on such antibodies in neurological disease is limited, however, the research on humoral responses to proteasome in connective tissue disorders has been more extensive. Some features are shared by neurological and rheumatological autoimmune disorders, in particular the presence of antibodies to intracellular antigens, and we will draw some parallels between these groups of diseases. This review aims to evaluate the potential functional importance of proteasome antibodies and their role in autoimmune diseases as systemic lupus erythematosus (SLE) and multiple sclerosis (MS).
CIRCULATING PROTEASOME The levels of circulating proteasome in plasma are particularly high in autoimmune diseases as Sjögren syndrome, myositis, autoimmune hepatitis and SLE [3]. These levels correlate with disease activity and cellular damage, and are higher in patients with systemic disease than in patients with milder and more limited affection [4]. Thus, the level of circulating proteasome has been suggested as a marker of disease severity and activity in autoimmune disease [4], and humoral immune responses to proteasome could simply reflect that cellular damage results in the release of proteasome and exposal of normally hidden antigenic epitopes, resulting in the formation of antibodies. High levels of circulating proteasome have also been found in patients with metastatic malignant melanoma [5]. As malignant melanoma is a tumour type particularly prone to trigger immune responses and often contains inflammatory infiltrates, circulating proteasome in these patients could reflect immune activation secondary to tumour growth. Elevated circulating proteasome levels are also found in patients with solid tumours, leukemia and myeloproliferative syndromes [6,7], and similar to what has been found in Sjögren syndrome and myositis, the level of plasma proteasome correlates with disease activity. This is an interesting observation, which could explain why proteasome antibodies are more common in paraneoplastic cerebellar degeneration (PCD) than in paraneoplastic encephalomyelitis (PEM). Patients with PCD often have ovarian cancer which is disseminated at the time of diagnosis [8]. On the other hand, patients with PEM usually harbour very small lung tumours that can be initially undetectable [9]. Finally, a marked increase of circulating proteasome levels are detected in patients with septic states and in trauma patients, and these levels were significantly higher than in patients who had undergone abdominal surgery [10].
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DISEASES ASSOCIATED WITH PROTEASOME ANTIBODIES There are several reports on naturally occurring antibodies to the ubiquitously expressed proteasome. The research has mainly focused on antibody prevalence in different autoimmune diseases. Although there are a few reports on antibodies to proteasome in neurological diseases, most of the reports concern systemic autoimmune diseases (Table 1). One disorder that has consistently been associated with proteasome antibodies is SLE. SLE is of interest to neuroimmunologists and neurologists for several reasons. Not only is SLE regarded as a prototype of systemic autoimmune disease, but in addition, affection of the nervous system is very common in SLE [11,12]. Antibodies to proteasome have been detected in a high proportion of patients with SLE, varying from to 35% [13] to 58% [14]. Circulating antibodies to proteasome are also found in patients with polymyositis and dermatomyositis [13,14] and primary Sjögren syndrome [15,16]. Proteasome antibodies are also reported to be found in patients with diabetes mellitus type I, which, in contrast to many of the other diseases with proteasome antibodies, is an organ-specific autoimmune disease. However, in this group of diabetes patients, the proteasome antibodies are associated with a higher risk of developing other autoimmune diseases [17]. Proteasome antibodies have not been identified in patients with rheumatoid arthritis, systemic scleroderma, primary sclerosing cholangitis and autoimmune thyroid diseases [18]. Thus, proteasome antibodies certainly seem to be most prevalent in systemic autoimmune diseases, of which, in particular, SLE and Sjögren syndrome are associated with B cell hyperreactivity [19,20]. Table 1. The reported frequencies of proteasome antibodies in different diseases, detected by immunoblotting or ELISA. The frequency of proteasome antibodies in healthy controls is reported to be 0% [15, 16, 22] to 2% [14] Disease Systemic lupus erythematosus Primary Sjögren syndrome Sarcoidosis Polymyositis/dermatomyositis Behcet’s disease Rheumatoid arthritis Vasculitis Multiple sclerosis Paraneoplastic cerebellar degeneration Paraneoplastic encephalomyelitis Cancer without paraneoplastic disease
Percentage of positive patients [ref] 35% [18] 58% [14] 16% [16] 39% [15] 7% [16] 19% [16] 62% [14] 19% [16] 0% [15] 5% [14] 0% [16] 13% [22] 66% [16] 43% [22] 11% [22] 0% [22] 15% [22]
The research of anti-proteasome immune responses in inflammatory disease primarily affecting the nervous system is so far quite limited. However, in a recent study, antibodies to proteasome were detected in the serum of more than 60% of patients with MS [16]. The prevalence of proteasome antibodies was about the same in patients with relapsing-remitting or and primary progressive MS, but higher in patients with secondary progressive disease
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(58% and 50% versus 80%). However, the numbers of patients in the two latter groups are quite small, and the authors concluded that the presence of antibodies to proteasome was not restricted to a particular subgroup of MS patients. Proteasome antibodies were found in the cerebrospinal fluid (CSF) of about 80% of the patients with serum antibodies. Interestingly, of the patients who were tested at the time of their first attack, about 67% harboured proteasome antibodies [16]. These patients had not yet been subjected to immunomodulating therapy as interferon, which could have influenced the prevalence of proteasome antibodies. The early detection of antibodies in MS patients are in line with studies of patients with SLE, in whom the majority have detectable anti-nuclear antibodies several years prior to the onset of clinical disease [21]. The presence of proteasome antibodies has also been investigated in paraneoplastic neurological syndromes. In paraneoplastic cerebellar degeneration (PCD), patients with a malignant tumour of the breast, lung or ovary mount a cellular and humoral immune response to antigens shared by malignant cells and normal Purkinje cells of the cerebellum. Antibodies to proteasome were detected in the serum of 45% of PCD patients with breast or ovarian cancer [22]. In contrast to MS patients, however, the humoral response to proteasome in PCD seems to take place mainly in the systemic compartment, as proteasome antibodies were not found in the CSF, even in seropositive patients. In PEM, which is usually associated with small cell lung cancer, the prevalence of serum proteasome antibodies was significantly lower than in PCD [22]. Low levels of antibodies like anti-nuclear antibodies (ANA) are detected in a percentage of healthy individuals [23]. However, antibodies to proteasome is consistently not found in normal control sera [15,16,22]. In addition, antibodies to proteasome have not been detected in patients with cancer of the gastrointestinal tract [15], ovary or lung [22] without concomitant autoimmune disease. Thus, the anti-proteasome response seems so far to be limited to patient groups with manifest autoimmune disease. The prevalence of serum antibodies to proteasome in patients in preclinical stages of autoimmune disease has not been investigated. The eukaryotic proteasome contains a number of specific subunits [24]. The humoral responses to proteasome have been found to be heterogenous, and when antisera are tested by ELISA or immunoblotting, they recognize different subunits of both α and β subtype. Many patients have antibodies to several subunits, indicating a polyclonal activation [15,16,22]. One possible explanation for this polyclonal response is inter- or intramolecular epitope spreading within the proteasome, resulting in antibodies directed to different proteasome subunits [25]. In addition, crossreactivity between the two proteasome subtypes, may account for some of the variation in reactivity patterns [15]. The polyclonality can be of relevance for the possible functional importance of the antibodies, as they can bind to different parts of the proteasome. Relative levels of individual components of the UPS vary between different regions of the brain [26], and polyclonal antibodies could therefore also have varying local effects depending on the expression of the antigens. Interestingly, antibodies to components of the proteasome activator complex PA28 (antiPA28α and anti-Ki antibodies) have been detected in patients with SLE (23%) and Sjögren syndrome (24%) [27]. The anti-Ki antibodies may associate with particular clinical subsets [28]. The functional importance of the anti-PA28α and anti-Ki antibodies is uncertain. The
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high prevalence of such antibodies in these patient groups indicates that the proteasome can be the target of different antibodies, which may interact with the function of the proteasome on several levels. The onset of production of antibodies to a ubiquitously expressed target antigen as the proteasome could be initiated by defects in the clearing of apoptotic cells, as suggested in SLE [29], where dying cells are thought to be a major source of antigenic material to trigger autoantibody formation [30]. It is likely that the proteasome is exposed to the immune system by the same mechanism. The anti-proteasome immune response found in many of these patients indicates an antigen-driven immune response. However, the correlation of high levels of circulating proteasome and of antibodies to proteasome in the same individuals is an issue that has not been investigated. Such an association would strengthen the hypothesis that proteasome antibody production is brought on by and is a secondary marker for tissue damage.
Figure 1. Representative immunoblot of 20S proteasome, purified from human red blood cells, separated by 14% SDS-PAGE. The left lane represents standard molecular weights. In second lane from the left, the blot is probed with serum from a patient with paraneoplastic cerebellar degeneration, showing that the serum (diluted 1:100) reacts with a proteasomal protein with a molecular weight of 25kDa. In lane 3, there is no reaction with the cerebrospinal fluid (diluted 1:20) of the same patient; lane 4 shows negative reaction with serum from a healthy individual. Reprinted from: Journal of Neuroimmunology, volume 165; Storstein A, Knudsen A, Vedeler CA: ”Proteasome antibodies in paraneoplastic cerebellar degeneration” , pages 172-178. Copyright (2005), with permission from Elsevier.
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DETECTION OF PROTEASOME ANTIBODIES The most common method by which antibodies to proteasome are detected is Western blotting, or immunoblotting. In this assay, purified proteasome complex or recombinant proteasome subunits is separated by SDS-PAGE electrophoresis, and probed with human serum or cerebrospinal fluid [13,14,16,22] (Figure 1). The sera often react with several proteasome proteins of different molecular weights, thus indicating a polyclonal response directed at various proteasome subunits [16]. Immunoblotting of recombinant proteasome subunits can identify the different antibody specificities in positive sera. Recombinant antigens can also be used for epitope mapping to detect the antigenic epitope in the different proteasome subunits [16]. Some authors have used enzyme-linked immunosorbent assays (ELISA) to detect proteasome antibodies. This assay also employs purified proteasome to coat multititer plates; sera are then added and allowed to react, and bound antibodies are detected enzymatically by a microplate reader. The specificity of the ELISA assay can be increased by using proteasome-specific monoclonal antibodies (sandwich ELISA) [14]. Sandwich ELISA is also the preferred technique for detection of circulating proteasome [4]. Finally, proteasome antibodies can also be detected by the use of immunohistochemistry, allowing human sera to react with preparations of cell cultures [22] (Figure 2).
A
B
Figure 2. Cancer cells, prepared as slides and stained by serum from a patient with paraneoplastic cerebellar degeneration and high levels of proteasome antibodies. (A) Multilocular intracellular staining produced by serum diluted 1:2000. (B) When the serum has been pre-absorbed with 20S protein, the staining is abolished, showing that antibodies in the serum react with 20S to form antigen-antibody complexes that do not stain the cancer cells. Reprinted from: Journal of Neuroimmunology, volume 165; Storstein A, Knudsen A, Vedeler CA: ”Proteasome antibodies in paraneoplastic cerebellar degeneration”, pages 172-178. Copyright (2005), with permission from Elsevier.
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PATHOGENIC ROLE OF PROTEASOME ANTIBODIES The proteasome antibodies are targeted to an intracellular antigen that is located in the nucleus as well as the cytoplasm, and which is ubiquitously expressed in all eukaryotic cells. The pathogenic role of antibodies to intracellular antigens has been a controversial issue for a long time. Many systemic autoimmune diseases are associated with antibodies to intracellular targets. It is generally believed that nuclear and cytoplasmic antigens are inaccessible to circulating antibodies and that such antibodies are highly unlikely to be of pathogenic importance. However, some antibodies are able to penetrate the cell membrane, among them anti-dsDNA antibodies, anti-RNP antibodies and anti-SSB/La antibodies [31]. Even if the antibodies are successful in traversing the cell membrane, it is still debated whether antibodies, once internalized, can initiate cell damage, and the evidence for this is mainly experimental. However, recent research has shown that humoral responses directed to intracellular targets may indeed participate actively in the pathogenesis of autoimmune disease. In SLE, anti-dsDNA antibodies antigens seem to be both of pathogenic importance as well as important markers of disease [21,32]. It has also been hypothesized that antibodies to intracellular antigens may exert functional effects in systemic sclerosis and in rheumatoid arthritis [33]. Thus, although the pathogenicity of antibodies to intracellular targets is still a debated issue, future research may change the present impression of these antibodies as pure diagnostic markers. Whether antibodies to proteasome participate in the pathogenesis of autoimmune diseases, neurological or otherwise, has not been clarified. In general, there are certain criteria that need to be fulfilled for an antibody to be considered as pathogenic. First, the antibodies should be associated with a high degree of disease specificity, which is not the case for the proteasome antibodies. Even if autoimmunity is a common feature in the diseases in which proteasome antibodies are found, the clinical spectrum is still very wide. Second, the proteasome antibodies do not seem to cluster with distinct clinical phenotypes. Third, there is no certain correlation between the level of proteasome antibodies and disease activity, although this has not been systematically investigated. Finally, the pathogenic effects of antibody-antigen interactions in vivo should be reproducible in experimental systems. However, no such models have been established for proteasome antibodies. Which arguments are in favour of functional effects of proteasome antibodies? The antibodies have been detected at a very early stage of disease, i.e. in MS, suggesting that they are a contributing factor, and not only a secondary response to the inflammation and cell damage [16]. Additionally, the proteasome antibodies are of the IgG and IgM subclass [16]; and the IgG subclasses of proteasome antibodies in PCD are IgG1 and IgG2 [22]. These results indicate an immune response that is antigen-driven and T cell-dependent, as is often the case of pathogenic antibodies. If antibodies to proteasome exert functional effects inside the blood-brain barrier, intrathecal antibody synthesis is to be expected. In the study by Mayo and coworkers, antibodies to proteasome were also detected in the CSF of about 80% of the seropositive MS patients [16]. Although it is noted that the concentration of proteasome antibodies, based on the total IgG content, was enriched in the CSF of one single patient, the report does not comment on intrathecal antibody production in the seropositive patients. Intrathecal IgG
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production is very common in MS, shown by multiple oligoclonal IgG bands detected by isoelectric focusing of the CSF [34]. The specificity of the IgG represented by these bands is not known, but it remains to be seen whether some are directed against proteasomes. The lack of intrathecal proteasome antibodies in PCD strongly suggests that they do not participate in the pathogenesis of Purkinje cell death in this disorder. On the other side, the anti-proteasome response in PCD may be a part of the immune responses directed at tumoural antigens, such as antigens presented by ovarian tumours. In ovarian cancer, the majority of patients have advanced malignancy at the time of diagnosis [35]. Thus, the antibodies to proteasome found in PCD patients could reflect tumour burden and cell death. In patients with PEM, the associated tumour is usually very small and often occult, perhaps resulting in a less extensive presentation of antigenic tumour material to the immune system. This may partly explain why proteasome antibodies are less common in PEM than in PCD [22]. Coexisting cellular responses to proteasome have only been investigated in MS. Autoreactive T cells to proteasome were detected in 40% of analysed MS sera, but proliferation of peripheral blood mononuclear cells was not detected in patients without proteasome antibodies [16]. The presence of autoreactive T cells is an argument in favour of a pathogenic role of the anti-proteasome immune response in MS, but must be regarded with some caution, as these results have not been confirmed by others.
FUNCTIONAL EFFECTS OF PROTEASOME ANTIBODIES The functional effects of proteasome antibodies are uncertain. This is partly due to the low serum levels of proteasome antibodies in autoimmune disease, which is usually in the range of 1/100 – 1/2000 [22]. Furthermore, the antibodies are directed to an intracellular target, leaving doubt as to their pathogenicity. The polyclonality of the proteasome antibodies and the complexity of proteasome function still suggest that there are multiple potential molecular pathways for the antibodies to exert their effects. Also, conformational diversities could allow the antibodies to recognize more than one target epitope. The UPS has a complex and central role in the maintenance of neurons, their excitability and outgrowth, in neuroprotection and in neurometabolism [2]. The UPS is also important in synaptic function and synaptic plasticity [36], perhaps also by its close association with MHC class I function and antigen expression [37]. Thus, interaction with the proteasome can have disastrous effects in neurons, which the role of proteasome dysfunction in neurodegenerative disorders has clearly demonstrated [38]. In these diseases, proteasome inhibition seems to participate in neuronal death, probably through multiple effects, including elevated intracellular levels of protein oxidation. Whether antibodies could inhibit proteasome function by similar mechanisms as found in neurodegenerative diseases, is not known. However, such effects could work in orchestra with the multitude of other humoral and cellular immune responses in immune-mediated diseases like MS, to increase neuronal vulnerability. The potential effects of proteasome antibodies in a pro-inflammatory environment are likely to differ from effects under normal conditions. In systemic autoimmune inflammation, there is usually an upregulation of interferon-inducible proteasome subunits, whereas in Sjögren syndrome,
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deficiency of a specific proteasome subunit has been identified [39]. To relate this finding to the specificity of proteasome antibodies in Sjögren syndrome would thus be of great interest. We have shown that proteasome antibodies stain living cancer cells, indicating IgG internalisation, but viability studies did not reveal any direct cytotoxic effects of such antibodies on cancer cells in vitro [22]. Whether natural antibodies could bind and inhibit the pathways of the UPS remains uncertain. However, even if the lack of cytotoxicity suggests that the antibodies do not interfere with vital cellular mechanisms concerning growth and differentiation, the antibodies could still affect other functions as degradation and presentation of antigens on MHC class I molecules, potentially influencing the presentation of intracellular antigens to the immune system. The UPS has particular functions within the nervous system, in particular concerning synaptic transmission, and antibody effects inside the CNS may thus differ from the effects observed in cell cultures of neuronal as well as nonneuronal origin. By suppression of the activation of nuclear factor-kappa B (NF-кB), synthetic inhibitors of the UPS can mediate anti-inflammatory effects, which is of potential great therapeutic interest in CNS inflammation [40]. If antibodies to proteasome can act in the same inhibitory fashion, their presence in inflammatory CNS diseases as MS could be favourable. An interesting observation is that the prevalence of proteasome antibodies is even higher in MS than in systemic inflammatory diseases like SLE. There are immunological similarities between MS and SLE, and there could possibly be shared pathways and mechanisms in the pathogenesis of these diseases. Nevertheless, the proteasome antibodies may not reflect a specific immune response. The spectrum of autoimmune diseases in which proteasome antibodies have been detected indicates that these antibodies reflects a global abnormality in the B cell regulation and function in certain patient groups that have a predilection for autoimmune disease and a lowered threshold for the production of autoantibodies [16,41]. It is thus possible that the proteasome antibodies represent a bystander immune response without direct pathogenic effects.
CONCLUSION Proteasome antibodies are detected in patients with systemic and organ-specific autoimmune diseases. The significance of these antibodies is uncertain. It is likely that they reflect cellular damage and release of intracellular antigens and represent bystander immune responses. A primary pathogenic function seems less probable, however, in an inflammatory micro-environment, the antibodies may still exert functional effects. This is an issue that needs to be clarified in experimental models. An interesting aspect of proteasome antibodies is why they are present in some patients and not in others, and whether proteasome antibodies are related to the severity and prognosis of disease. The limited number of reports on proteasome antibodies does not allow for conclusions in this matter, but further studies should aim to investigate possible correlations of the presence and level of proteasome antibodies and the long term prognosis of MS and other immune-mediated diseases.
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[15] Feist E, Kuckelkorn U, Dorner T, Donitz H, Scheffler S, Hiepe F, Kloetzel PM, Burmester GR: Autoantibodies in primary Sjogren's syndrome are directed against proteasomal subunits of the alpha and beta type. Arthritis Rheum 1999; 42:697-702. [16] Mayo I, Arribas J, Villoslada P, Alvarez DoForno R, Rodriguez-Vilarino S, Montalban X, de Sagarra MR, Castano JG: The proteasome is a major autoantigen in multiple sclerosis. Brain 2002; 125:2658-2667. [17] Kordonouri O, Meyer K, Egerer K, Hartmann R, Scheffler S, Burmester GR, Kuckelkorn U, Danne T, Feist E: Prevalence of 20S proteasome, anti-nuclear and thyroid antibodies in young patients at onset of type 1 diabetes mellitus and the risk of autoimmune thyroiditis. J Pediatr Endocrinol Metab 2004; 17:975-981. [18] Mayo I, Arizti P, Pares A, Oliva J, Doforno RA, de Sagarra MR, Rodes J, CAstano JG: Antibodies against the COOH-terminal region of E. coli ClpP protease in patients with primary biliary cirrhosis. J Hepatol 2000; 33:528-536. [19] Hansen A, Lipsky PE, Dorner T: Immunopathogenesis of primary Sjogren's syndrome: implications for disease management and therapy. Curr Opin Rheumatol 2005; 17:558565. [20] Tsubata T: B cell abnormality and autoimmune disorders. Autoimmunity 2005; 38:331337. [21] Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, Harley JB: Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 2003; 349:1526-1533. [22] Storstein A, Knudsen A, Vedeler CA: Proteasome antibodies in paraneoplastic cerebellar degeneration. J Neuroimmunol 2005; 165:172-178. [23] Pisetsky DS: Anti-DNA and autoantibodies. Curr Opin Rheumatol 2000; 12:364-368. [24] Brooks P, Fuertes G, Murray RZ, Bose S, Knecht E, Rechsteiner MC, Hendil KP, Tanaka K, Dyson J, Rivett J: Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem J 2000; 346:155-161. [25] Vanderlugt CL, Miller SD: Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol 2002; 2:85-95. [26] Klimaschewski L: Ubiquitin-dependent proteolysis in neurons. News Physiol Sci 2003; 18:29-33. [27] Matsushita M, Takasaki Y, Takeuchi K, Yamada H, Matsudaira R, Hashimoto H: Autoimmune response to proteasome activator 28alpha in patients with connective tissue diseases. J Rheumatol 2004; 31:252-259. [28] Cavazzana I, Franceschini F, Vassalini C, Danieli E, Quinzanini M, Airo P, Cattaneo R: Clinical and serological features of 35 patients with anti-Ki autoantibodies. Lupus 2005; 14:837-841. [29] Nagy G, Koncz A, Perl A: T- and B-cell abnormalities in systemic lupus erythematosus. Crit Rev Immunol 2005; 25:123-140. [30] Gaipl US, Voll RE, Sheriff A, Franz S, Kalden JR, Herrmann M: Impaired clearance of dying cells in systemic lupus erythematosus. Autoimmun Rev 2005; 4:189-194. [31] Lim PL, Zouali M: Pathogenic autoantibodies: Emerging insights into tissue injury. Immunol Lett 2005; 103:17-26.
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[32] Arbuckle MR, James JA, Kohlhase KF, Rubertone MV, Dennis GJ, Harley JB: Development of anti-dsDNA autoantibodies prior to clinical diagnosis of systemic lupus erythematosus. Scand J Immunol 2001; 54:211-219. [33] Benoist C, Mathis D: A revival of the B cell paradigm for rheumatoid arthritis pathogenesis? Arthritis Res 2000; 2:90-94. [34] Lunding J, Midgard R, Vedeler CA: Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index. Acta Neurol Scand 2000; 102:322-325. [35] Bjorge T, Engeland A, Sundfor K, Trope CG: Prognosis of 2,800 patients with epithelial ovarian cancer diagnosed during 1975-94 and treated at the Norwegian Radium Hospital. Acta Obstet Gynecol Scand 1998; 77:777-781. [36] Upadhya SC, Hegde AN: Ubiquitin-proteasome pathway components as therapeutic targets for CNS maladies. Curr Pharm Des 2005; 11:3807-3828. [37] Darnell RB: Immunologic complexity in neurons. Neuron 1998; 21:947-950. [38] Hattori N, Mizuno Y: Pathogenetic mechanisms of parkin in Parkinson's disease. Lancet 2004; 364:722-724. [39] Krause S, Kuckelkorn U, Dorner T, Burmester GR, Feist E, Kloetzel PM: Immunoproteasome subunit LMP2 expression is deregulated in Sjogren's syndrome but not in other autoimmune disorders. Ann Rheum Dis 2006; 65:1021-1027. [40] Wojcik C, Di Napoli M: Ubiquitin-proteasome system and proteasome inhibition: new strategies in stroke therapy. Stroke 2004; 35:1506-1518. [41] Davidson A, Diamond B: Autoimmune diseases. N Engl J Med 2001; 345:340-350.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 865-876 © 2007 Nova Science Publishers, Inc.
Chapter 36
THE UBIQUITIN PROTEASOME SYSTEM IN THE PATHOBIOLOGY OF HUMAN GLIOMAS Marco Piccinini1, Maria Teresa Rinaudo1, and Davide Schiffer2,∗ 1
Department of Medicine and Experimental Oncology, Section of Biochemistry, University of Turin, Turin, Italy; 2 Centre of Neuro-bio-oncology, Foundation Policlinico di Monza/University of Turin, Vercelli, Italy.
ABSTRACT In higher eukaryotic cells, the 26S proteasome is the central component of the ubiquitin-proteasome system (UPS), in which it provides for the degradation of cytoplasmic and nuclear proteins, usually tagged with ubiquitin oligomers, and their resolution into short peptides. This pathway is involved in the control of a large array of cellular processes including protein turnover, digestion of damaged, mutant and viral proteins, cell cycle regulation, cell division, differentiation and development. Furthermore, it is also implicated in DNA repair, stress, immune and inflammatory responses, apoptosis, cell surface receptor modulation, transcription factor processing and activation, etc. Proteins belonging to different molecular pathways playing an important role in glioma progression or regression may undergo degradation or processing via the UPS, and consequently be inactivated, or conversely activated following proteasome inhibition. In GBM there is a striking shift of the balance constitutive/immunoproteasome towards the latter; paralleled by depression of the chymotrypsin-like activity. This is in opposition to its expected enhancement, being this activity higher in the immunoproteasome with respect to the standard proteasome. A better understanding of this discrepancy as well as of the enhanced apoptosis associated
∗
Correspondence concerning this article should be addressed to Dr. Davide Schiffer, MD; Centro Ricerche di Neuro-bio-oncologia, Fondazione Policlinico di Monza, Via Pietro Micca, 29, 13100 Vercelli, Italy. Phone: +390161-3691; Fax: +39-0161-369109 ; E-mail:
[email protected].
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Keywords: nervous tissue neoplasms, glioblastoma enhanced pathways, ubiquitinproteasome system, apoptosis, proteasome inhibitors, standard proteasome, immunoproteasome.
ABBREVIATIONS AFX, transcription factor also known as FOXO4; Akt, protein kinase B PKB; BAD, proapoptotic tumor suppressor protein; Bcl-2, bcl-Associated Death Protein; CDK, cyclin dependent kinase; c-Myc, proto-oncogene protein c-myc-transcription factor product of the oncogene c-myc; E2F, transcription Factor of E2 gene; E2F4, transcription repressor E2F4; EGFR, epidermal growth factor receptor; Fas,. transmembrane fas receptor also defined CD95 or Apo1 antigen; FKHR, forkhead transcription factor; FRAP, FKBP12 rapamycinassociated protein; GBM, glioblastoma; LLnL, Leu-Leu-norLeu-al; L+R, ligand+receptor; MAPK, mitogen activated protein kinase; Mdm2, E3-like ubiquitin-protein ligase product of mdm2 gene; MG132, Z-Leu-Leu-Leu-al ; mTOR, mammalian target of rapamycin protein kinase; NFκB, transcription nuclear factor κB; p14, cell cycle regulator or tumor suppressor gene; p21WAF1, CDK inhibitor p21; p27, CDK inhibitor p27Kip1; PDGFR, platelet derived growth factor receptor; PIP3, phosphatidylinositol-3-phosphate; pRb-P, retinoblastomaassociated protein-phosphorylated; PS341, Z-Ile-Glu(octaBut)-Ala-Leu-al; PTEN, phosphatase and tensin homolog deleted on chromosome 10; Ras, product of Ras protooncogene, a monomeric G-Protein; SV40, tumor virus SV40; TNFα, tumor necrosis factor α; TP53, transcription protein 53; TRAIL, TNF-related apoptosis-inducing ligand; UPS, ubiquitin-proteasome system.
THE UBIQUITIN SYSTEM AND GLIOBLASTOMA In higher eukaryotic cells, the proteasome constitutes the central protease of the ubiquitin-proteasome system (UPS), playing a role in the cytoplasmic and nuclear degradation at neutral pH of most intracellular proteins, preferentially if tagged with ubiquitin oligomers (see Chapters 3, 6 and 7). Originally, the UPS was described as a way to provide for the digestion of misfolded, damaged, mutant, or viral proteins and their transformation into short peptides further degraded to single amino acids by specific peptidases. However, evidence is growing that this pathway is also involved in the degradation or processing of a variety of proteins, frequently short-lived regulatory proteins, implicated in different vital cellular processes including cell cycle regulation, cell division, differentiation and development, DNA transcription and repair, apoptosis, modulation of cell surface receptors, ion channels and secretory pathways, response to stress and extracellular challenges, immune surveillance, inflammation, etc [1]. Therefore, direct or indirect aberrations of the UPS result in a variety of pathologies, including malignancies.
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Proteins involved in different molecular pathways, playing an important role in glioma progression or regression, may undergo degradation or processing by the UPS and consequently be inactivated or conversely activated following proteasome inhibition. In this regard, there are many examples. The key regulation of the cell cycle check-point G1-S is exerted by p53 together with Mdm2 and p14ARF. The N-terminus of Mdm2 binds to the transactivation domain of p53 and inhibits its transcriptional activity; furthermore, Mdm2 regulates p53 protein level, because p53 is targeted for nuclear export and cytoplasmic degradation following ubiquitination by Mdm2 which in the UPS functions as an E3 ubiquitin ligase. Interestingly, Mdm2 is frequently amplified in glioblastomas. Another example of UPS intervention is given by p27/Kip.1 which regulates G1-S transition inhibiting cyclin E- and A-CDK2 complexes; p27/Kip.1 is expressed in G0 and G1 and decreases in cells entering into S-phase. It is post-translationally regulated by degradation into the UPS. SCF complexes (Skip1, Cul-1, F-box protein) are a class of ubiquitin ligases ensuring the specific recognition and ubiquitination of different substrates through different F-box proteins. Skp2 belongs to this group of proteins and is required for G1-S transition targeting p27/Kip.1 for ubiquitination and degradation. In glioblastoma, an inverse relationship is appreciable between Skp2 and p27/Kip.1. Among other proteins degraded into UPS, IκBα must be mentioned preventing NFκB translocation to the nucleus.
PATHWAYS OF ASTROCYTIC GLIOMA PROGRESSION AND ALTERATIONS AT THE GENE LEVEL In astrocytic gliomas, a progression is realized through anaplasia, which indicates loss of the phenotypic features of a certain stage of differentiation, with regression to a more immature stage. A diffuse astrocytoma grade II, through anaplastic astrocytoma grade III, may transform into a IV grade glioblastoma (GBM). In the course of anaplasia, pathologic aspects typical of the different stages are associated with genetic alterations, as depicted in Table 1. Table 1. Progressing anaplasia and genetic alterations Tumor stage Astrocytoma
Associated pathology Proliferation Apoptosis
Anaplastic Astrocytoma
Cell cycle deregulation
Glioblastoma
Necroses Angiogenesis Clonal selection
Genetics TP53 mutations PDGFR over-expression 7p, 22q losses CDKN2A/p16 deletion RB mutation CDK4 amplification 9q, 13q, 19q, 11p losses EGFR amplification/truncation PTEN mutations pRb pathway alterations
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Figure 1. PDGFR stimulation leads to apoptosis if p53 is wild type and to astrocytoma if it is inactivated.
Figure 2. pRb pathway. Complexes of different cyclins and kinases regulate the phosphorylation of pRb and activation of the transcription factor E2F leading to DNA gene expression.
Figure 3. Amplified or truncated EGFR keep active Ras pathway and proliferation.
Two types of GBM have been identified: primary GBM arising as such from the beginning and secondary GBM originating from the transformation of a pre-existing astrocytoma. It is worth mentioning that the two GBM types show different molecular assets, with TP53 mutations prevailing in the secondary type and EGFR amplification, PTEN mutations and other genetic alterations in the primary type [2]. Tumour progression is due to genotypic instability followed by genotypic and then phenotypic heterogeneity, giving rise to new clones characterized by increasing proliferation rate and mutability, which substitute the predecessors in a process of selection by competition. Inactivation of tumour suppressor genes and accumulation of mutations are the basis of this event. The genetic alterations are distributed across several molecular pathways [3,4], which include proteins susceptible to degradation by UPS. These pathways are: PDGFR stimulation of glia (Figure 1) which may lead to apoptosis with wild type p53 and to tumour development if p53 is inactivated by different ways; cell cycle regulation through cyclins and the relevant kinases with the
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consequent phosphorylation of pRb and activation of the transcription factor E2F (Figure 2); EGFR signalling keeping the Ras pathway active for proliferation with amplification or truncation of the receptor (Figure 3); p14-Mdm2-p53 loop which can inactivate p53 (Figure 4); PTEN–Akt circuit which can make a switch between cell proliferation and apoptosis (Figure 5).
Figure 4. p53 can be inactivated by Mdm2 and exported to the cytoplasm where it is degraded by the proteasome; Mdm2 is regulated by p14.
Figure 5. Mutated PTEN frees Akt which is a switch between apoptosis and proliferation.
PROTEASOME INHIBITORS IN GLIOBLASTOMACELL LINES AND EXPLANTS Proteasomes, as the central proteolytic machinery of the UPS, play a pivotal role in controlling cell proliferation and differentiation as well as programmed cell death (apoptosis) in a variety of normal and tumour cells [5-10]. Their implication in the regulation of apoptosis largely relies on the activation of NFκB (see Chapter 21). Constitutively, NFκB is prevented from nuclear translocation because sequestered in the cytoplasm following its binding with the inhibitory protein IκB. NFκB as an active transcription factor, is a dimer
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made up by p50 and p65/Rel subunits; p50 arises through processing of the inactive precursor p105. Both p105 and IκB enter UPS provided they have been phosphorylated and tagged with ubiquitin oligomers. The former will be processed into mature p50, whereas the latter will be fully degraded. Once processed and IkB-free, p50 binds with p65 and the heterodimer translocates to the nucleus, where it transactivates a series of genes encoding proteins implicated in promoting cell proliferation and immune and inflammatory responses, as well as in awakening the antiapoptotic surveillance system [11-13]. The latter event partly relies on caspase-3 and -8 degradation by proteasomes [14]; however, caspase-mediated degradation of some proteasome subunits occurs in early stages of apoptosis [15,16], revealing that the execution of the apoptotic progression is finely tuned by a network of signals that upregulate or slow down its progression. The involvement of proteasomes in the development of gliomas is supported by: (i) the status of NFκB, which is strongly activated in glioblastoma specimens and accumulates in the nucleus in parallel with tumour progression [17,18]; (ii) a number of findings carried out in conditions of proteasome function blockade by cell permeable inhibitors (see Chapter 40). For example, glioma cells exposed to proteasome inhibitors, whether peptide aldehydes, such as LLnL and MG132, or non-peptide molecules, such as lactacystin and epooxomicin [19,20], undergo apoptosis. This event is associated with activation of caspase-3 [21-23]. Another selective and strong proteasome inhibitor is PS-341, also called bortezomib, a boronic acid dipeptide, which exhibits prominent effects in vitro and in vivo against several solid tumours [24], and is the first approved drug in the proteasome inhibitor class of anticancer agents [25]. In human GBM cell lines and in primary GBM explants, PS-341 arrests cells in G2/M with a concomitant decrease in the percentage of cells in S phase. These events are associated with increased expression of p21WAF1, p27Kip.1, and cyclin B1 proteins, decreased levels of CDK2, CDK4 and the transcription factor E2F4. All these events take place along with reduced transcriptional activity of NFκB [17,18,26]. Furthermore, in GBM cells, PS-341 enhances TRAIL- and TNFα-induced cell death and apoptosis, suggesting that it may be considered for an effective therapy in patients with gliomas [26]. It is debated how TNFα and TRAIL signaling cascades, on the one hand, lead to NFκB activation, which is known to promote the expression of antiapoptotic genes, while on the other hand, they favour apoptosis. TRAIL- and TNFα-induced apoptosis implies activation of the caspase cascade as well as of NFκB [27-29]. Since caspases are proteasome substrates, it is possible that the proteasome inhibition, such as that determined by PSI-341, together with TRAIL- and TNFα -induced caspase activation gives rise to accumulation of caspases, because no longer degraded by the proteasome. However, in murine cortical cell lines, activation or conversely attenuation of the apoptotic program occurs depending on whether MG132-induced blockade of proteasome function was partial or complete [30]. Furthermore, in glioma cells, proteasome inhibitor-induced mitochondrial-independent caspase-3-dependent apoptosis relies on c-Myc protein stabilization, in turn responsible for a transient increase of Fas ligand (FasL) message to stimulate the apoptotic signaling pathway. In fact, in these cells, c-Myc protein accumulation is associated with a markedly increased expression of FasL mRNA along with slightly increased Fas-CD95 receptor mRNA; these events precede in time the activation of caspase-3. Indirectly, what above described
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strengthens the involvement of proteasomes as anti-apoptotic factors in malignant glioma cells [23]. Lastly, in human and rat GBM cell lines, the UPS has been implicated in the stability of the eukaryotic Elongation Factor-2 (eEF-2) kinase, a highly conserved calcium/calmodulindependent enzyme involved in the regulation of protein translation and cell proliferation. Rapid changes in the activity and quantity of the kinase are observed upon cell growth stimulation and up-regulasted kinase activity appears as a feature of malignant cell growth. In GBM cells, eEF-2 kinase behaves as a relatively short-lived protein, with a half-life of less than 6 hours. Treatment of these cells with MG132 results in accumulation of the kinase in ubiquitin-tagged forms, with a consequent prolonged protein half-life. In this regard, MG132 is considered as an effective agent in the treatment of certain forms of cancer, including GBM [31].
PROTEASOMES IN HUMAN GBM In contrast with the large array of findings that indirectly strengthen the involvement of proteasomes in tumorigenesis, there is little known evidence that proteasomes are functionally and structurally modified in malignant cells, including glioma cells. This gap of knowledge likely results from the lack of an adequate control tissue. In human cortical tissue, as well as in unstimulated mouse microglial cells, the 20S proteasome is expressed as a constitutive (standard) proteasome and as an immunoproteasome. The former comprises the coordinated assembly of 14 α and 14 β subunits, arranged in an α7β7β7α7 stoichiometry. In the latter, the active constitutive subunits β1 β2 and β5 are replaced by their interferon (INF)-γ inducible homologue counterparts, defined as iβ1/LMP2, iβ2/MECL-1 and iβ5/LMP7 [32-34]. These changes give rise to modifications of the peptidase activity of the proteasome (see Chapter 34). In fact, replacement of β1 with LMP2 implies suppression of the peptidyl-glutamyl peptide hydrolysing (PGPH) activity, a feature of β1, and substitution with chymotrypsin-like activity which is shared by LMP7; replacement of β2 with MECL-1 implies enhancement of the trypsin-like activity [35-37]. As a consequence, the proteolytic activity of the proteasome shifts towards the generation of peptides with a hydrophobic or basic amino-acidic residue at their C-terminus; these peptides are preferentially taken up by MHC-class I proteins for presentation to cytotoxic T lymphocytes, with a consequent immune response [35]. However, in contrast to proteasomes from other sources, in particular from professional antigen presenting cells, 20S proteasomes from brain cortical regions and unstimulated microglial cells are characterized by low levels of the three inducible subunits [33] and by low levels of LMP2 and LMP7, but undetectable MECL-1 [32], respectively. Neurons and resting microglial cells are poor or destitute of immunoreactivity, particularly the former [32,38-41]; however, in brains under appropriate stimuli, microglial cells become the major antigen presenting cells and respond to pathologic events [32,41]. In the human brain, the 20S proteasome PGPH activity is higher than in the kidney, a tissue largely involved in the immune response, whereas the opposite is true for the chymotrypsin-like and trypsin-like activities, both involved in the generation of antigenic
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peptides [33]. In primary microglia cultures lengthily treated with INF-γ, MECL-1 and LMP7 subunits accumulate and in parallel of the constitutive counterpart β2 disappears [32]. Lastly, in human brain, the 20S proteasome, besides as a single independent particle, also exists as a complex in association with the multimeric protein 11S regulator (11SReg), also defined as proteasome activator 28 (PA28) [34]. The regulator comprises two types of subunits, 7 α and 6 β, arranged in two caps, one at the top and the other at the bottom of the proteasome unit. Expression of the regulator is under the control of INF-γ and its regulatory role consists of enhancing the proteasome peptidase activity, namely the chymotrypsin-like activity [42], thus favouring the generation of peptides with increased affinity for MHC class I molecules. The 20S proteasome in various tissues is mainly expressed as a high molecular mass protein complex, derived from its ATP-dependent association with the regulatory component, the 19S complex or PA700; this assembly gives rise to the 26S proteasome, the central protease of UPS. The 26S proteasome has been purified and widely characterized in bovine, but not human brain [43], although reasons exist to believe that in human brain it has properties comparable to those of other tissues (Piccinini et al. unpublished observations). The 26S proteasome is likely to be expressed also in mouse glial cells [32], although a final confirmation is still lacking. The characterization of the proteasome catalytic core in human GBM has recently been attained by a comparison between the structural and functional properties of the 20S proteasome isolated and purified from fresh surgery specimens of tumour tissue and those of the 20S proteasome from fresh peritumoral, histologically normal tissue. In GBM, the 20S proteasome has properties largely similar to those of control tissue, as well as of human and bovine brain cortical regions and glial cells [32-34,43]. In human GBM, proteasome structural properties are preserved, since in all GBM surgical specimens examined, the 20S proteasome was constituted by the three active subunits β1, β2, and β5, as well as by their counterpart IFN-γ inducible subunits, LMP2, MECL-1 and LMP7. However, in the proteasome from 70% GBM specimens, the three inducible subunits are much more expressed than in controls (Figure 6); surprisingly, this feature is associated with a strongly depressed chymotrypsin-like activity, as opposed to the unvaried trypsin-like one which is. This discrepancy between proteasome functional and structural properties is also a feature of tissues in conditions of stress, such as those induced by oxygen radicals [44], and thus it can be taken as a marker of the metabolic disorders underlying the tumour malignancy. The incorporation of the 11SReg in the 20S proteasome is instead unaltered in all GBM specimens [34]; however, because of the influence of the regulator on proteasome chymotrypsin-like activity, it can be concluded that the aforementioned role of 11SReg does not take place in GBMs, particularly in those where the INF-γ inducible subunits accumulate, and again, this may be a mark of malignancy.
CONCLUSION Collectively, the reported observations indicate that in GBM, the UPS plays a great role as an anti-apoptotic system by eliminating controlled cell death and promoting factors favouring cell proliferation. This role largely relies on the efficiency of the proteasome. As
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for the UPS, gliomas do not behave differently from other tumours. Therefore, throwing light on the discrepancy between proteasome functional and structural properties in gliomas and ascertaining whether this discrepancy also occurs in other tumours might uncover a common dysfunction of a huge number of cell processes. This could be a crucial step in designing multifunctional therapeutic drugs common to all tumours. Interestingly, proteasomes have been revealed to be quite sensitive to cytostatic drugs [45,46].
Figure 6. The 20S proteasome from human glioblastomas (G-20S) and control specimens (C-20S). Panel A: Resolution of G-20S (lanes 1, 3) and C-20S (lanes 2, 4) by non-denaturing gel electrophoresis and visualization by gel overlying with the fluorogenic peptide Suc-L-L-V-Y-AMC, substrate of the proteasome chymotrypsin-like activity (lanes 1, 2) or Coomassie Blue staining (lanes 3, 4). Panel B: Resolution of G-20S (lane 1) and C-20S (lane 2) by denaturing gel electrophoresis (SDS-PAGE) and protein band visualization by silver staining. Panel C: G-20S (lanes 1, 3, 5) and C-20S (lanes 2, 4, 6) inducible subunits LMP2, LMP7 and MECL-1 resolved by SDS-PAGE, electroblotted on PVDF membranes and identified by immunodecoration by selective antibodies. Blots were re-probed by a monoclonal antibody to the 20S proteasome constitutive α5 subunit for equal protein loading. (From Piccinini et al. 2005; [34])
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Marco Piccinini, Maria Teresa Rinaudo and Davide Schiffer Collins VP: Brain tumours: classification and genes. J. Neurol Neurosurg Psychiatry 2004; 75:(Suppl II) ii2-ii11. Lecker SH, Solomon V, Mitch WE, Goldberg AL: Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states. J Nutr 1999; 12:227S-237S. Kisselev AF, Goldberg AL: Proteasome inhibitors: from research tools to drug candidates. Chem Biol 2001; 8:739-758. Glickman MA, Ciechanover A: The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002; 82:373-428. Almond JB, Cohen GM: The proteasome: a novel target for cancer chemotherapy. Leukemia 2002; 16:433-443. Husom AD, Peters EA, Kolling EA, Fugere NA, Thompson LV, Ferrington DA: Altered proteasome function and subunit composition in aged muscle. Arch Biochem Biophys 2004; 421:67-76. Ciechanover A: The ubiquitin proteolytic system: from a vague idea, through basic mechanisms, and onto human diseases and drug targeting. Neurology 2006; 66 (2 Suppl 1):S7-S19. Baldwin AS Jr: The NF-kB and IkB proteins: new discoveries and insights. Annu Rew Immunol 1996; 14:649-681. Karin M, Ben-Neriah Y: Phosphorylation meets ubiquitination: the control of NF[kappa]B activity. Annu Rev Immunol 2000; 18:621-663. Baud V, Karin M: Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 2001; 11:372-377. Kim S, Choi K, Kwon D, Benveniste EN, Choi C: Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death. Cell Mol Life Sci 2004; 61:10751081. Friedman J, Xue D: To live or die by the sword: the regulation of apoptosis by the proteasome. Dev Cell 2004; 6:460-461. Sun XM, Butterworth M, MacFarlane M, Dubiel W, Ciechanover A, Cohen GM: Caspase activation inhibits proteasome function during apoptosis. Mol Cell 2004; 14:81-93. Hayashi S, Yamamoto M, Ueno Y, Ikeda K, Ohshima K, Soma G, Fukushima T: Expression of nuclear factor-kappa B, tumor necrosis factor receptor type 1, and c-Myc in human astrocytomas. Neurol Med Chir (Tokyo) 2001; 41:187-195. Nagai S, Washiyama K, Kurimoto M, Takaku A, Endo S, Kumanishi T: Aberrant nuclear factor-kappaB activity and its participation in the growth of human malignant astrocytoma. J Neurosurg 2002; 96:909-917. Fenteany G, Staendaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL: Inhibition of the proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 1995; 268:726-731. Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, Crews CM: Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl Acad Sci USA 1999; 96:10403-10408.
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[21] Wagenknecht B, Hermisson M, Eitel K, Weller M: Proteasome inhibitors induce p53/p21-independent apoptosis in human glioma cells. Cell Physiol Biochem 1999; 9:117-125. [22] Wagenknecht B, Hermisson M, Groscurth P, Liston P, Krammer PH, Weller M: Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release. J Neurochem 2000; 75:2288-2297. [23] Tani E, Kitagawa H, Ikemoto H, Matsumoto T: Proteasome inhibitors induce Fasmediated apoptosis by c-Myc accumulation and subsequent induction of FasL message in human glioma cells. FEBS Lett 2001; 504:53-58. [24] An J, Sun YP, Adams J, Fischer M, Belldegrun A, Rettig MB: Drug interactions between proteasome inhibitor bortezomib and cytotoxic chemotherapy, tumor necrosis factor (TNF) alpha, and TNF-related apoptosis-inducing ligand in prostate cancer. Clin Cancer Res 2003; 39:4537-4545. [25] Mani A, Gelmann EP: The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005; 23:4776-4789. [26] Yin D, Zhou H, Kumagai T, Liu G, Ong JM, Black KL, Koeffler HP: Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM). Oncogene 2005; 24:344–354. [27] Franco AV, Zhang XD, Van Berkel E, Sanders JE, Zhang XY, Thomas WD, Nguyen T, Hersey P: The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAIL)induced apoptosis of melanoma cells. J Immunol 2001; 166:5337-5345. [28] Dai Y, Rahmani M, Grant S: An intact NF-kappaB pathway is required for histone deacetylase inhibitor-induced G1 arrest and maturation in U937 human myeloid leukemia cells. Cell Cycle 2003; 2:467-472. [29] Dai Y, Rahmani M, Grant S: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNKand NF-kappaB-dependent process. Oncogene 2003; 22:7108-7122. [30] Suh J, Lee YA, Gwag BJ: Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures. J Neurochem 2005; 9:684-694. [31] Arora S, Yang JM, Hait WN: Identification of the ubiquitin-proteasome pathway in the regulation of the stability of eukaryotic elongation factor-2 kinase. Cancer Res 2005; 65:3806-3810. [32] Stohwasser R, Giesebrecht J, Kraft R, Muller EC, Hausler KG, Kettenmann H, Hanisch UK, Kloetzel PM: Biochemical analysis of proteasomes from mouse microglia: induction of immunoproteasomes by interferon-gamma and lipopolysaccharide. Glia 2000; 29:355-365. [33] Piccinini M, Mostert M, Croce S, Baldovino S, Papotti M, Rinaudo MT: Interferongamma-inducible subunits are incorporated in human brain 20S proteasome. J Neuroimmunol 2003; 135:135-140. [34] Piccinini M, Rinaudo MT, Anselmino A, Ramondetti C, Buccinna B, Fiano V, Ghimenti C, Schiffer D: Characterization of the 20S proteasome in human glioblastomas. Anticancer Res 2005; 25:3203-3210.
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[35] Tanaka K, Kasahara M: The MHC class I ligand-generating system: roles of immunoproteasomes and the interferon-γ-inducible proteasome activator PA28. Immunol Rev 1998; 163:161-176. [36] Rock KL, Goldberg AL: Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu Rev Immunol 1999; 17:739-779. [37] Noda C, Tanahashi N, Shimbara N, Hendil KB, Tanaka K: Tissue distribution of constitutive proteasomes, immunoproteasomes and PA28 in rats. Biochem Biophys Res Commun 2000; 277:348-354. [38] Früh K, Yang Y, Arnold D, Chambers J, Wu L, Waters JB, Spies T, Peterson Per A: Alternative exon usage and processing of the major histocompatibility complexencoded proteasome subunits. J Biol Chem 1992; 267:22131-22140. [39] Neumann H, Cavlie A, Jenne DE, Wekerle H: Induction of MHC class I genes in neurons. Science 1995; 269:549-552. [40] Mavria G, Hall KT, Jones RA, Blair GE: Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes. Biochem J 1998; 330:155-161. [41] Xiao B G, Link H: Immune regulation within the central nervous system. J Neurol Sci 1998; 157:1-12. [42] Rechsteiner M, Realini C, Ustrell V: The proteasome activator 11S REG (PA28) and class I antigen presentation. Biochem J 2000; 345:1-15. [43] Piccinini M, Tazartes O, Mostert M, Musso A, DeMarchi M, Rinaudo MT: Structural and functional characterization of 20S and 26S proteasomes from bovine brain. Mol Brain Res 2000; 76:103-114. [44] Carrard G, Dieu M, Raes M, Toussaint O, Friguet B: Impact of ageing on proteasome structure and function in human lymphocytes. Int J Biochem Cell Biol 2003; 35:728739. [45] Piccinini M, Tazartes O, Mezzatesta C, Ricotti E, Bedino S, Grosso F, Dianzani U, Tovo PA, Mostert M, Musso A, Rinaudo MT: Proteasomes are a target of the antitumour drug vinblastine. Biochem J 2001; 356:835-841. [46] Piccinini M, Mostert M, Rinaudo MT: Proteasomes as drug targets. Curr Drug Targets 2003; 4:657-671.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 877-898 © 2007 Nova Science Publishers, Inc.
Chapter 37
THE UBIQUITIN PROTEASOME SYSTEM IN THE PATHOBIOLOGY OF HUMAN PITUITARY TUMORS Mădălina Muşat2, Márta Korbonits1 and Ashley B. Grossman1,∗ 1
2
Department of Endocrinology, St. Bartholomew’s Hospital, London, UK; Department of Endocrinology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania.
ABSTRACT Pituitary tumors are usually benign lesions, but their tumorigenic process may constitute a model of the initial stages of carcinogenesis. Two major theories have been subject to most investigation: hormonal (usually hypothalamic factors) and/or growth factor over-stimulation, or a molecular defect within the pituitary itself. Oncogenes and tumor suppressor genes involved in other types of tumor do not appear to play a major role in the pathogenesis of pituitary tumors. In addition, germline genetic disorders, in which pituitary tumors are a common feature, have not shed much light on the pathogenesis of the more common sporadic tumors. An increasing number of reports point to deregulation of the cell cycle in these tumors, while transgenic disruption of the cell cycle machinery frequently leads to pituitary tumors in animal models. Cell cycle progression during G1, S and G2 phases is normally regulated by the fluctuation in the concentration of cyclins, cyclin-dependent kinases (CDKs) and their inhibitors, while securin, separin and cohesin regulate progression through M phase. This is mainly achieved through the programmed degradation of these proteins within the ubiquitinproteasome system (UPS), but also by transcriptional regulation and subcellular compartmentalization. Alterations of these processes result in uncontrolled proliferation, aneuploidy and tumorigenesis. Aberrations of one or more components of the ∗
Correspondence concerning this article should be addressed to Prof. Dr. Ashley B. Grossman, Centre for Endocrinology, 5th Floor King George V Building, St.Bartholomew's Hospital, West Smithfield, London, EC1A 7BE,UK. Phone: +44-207-6018343; Fax: +44-207-6018505; E-mail:
[email protected].
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Mădălina Muşat, Márta Korbonits and Ashley B. Grossman pRb/p16/cyclinD1/CDK4 pathway have been shown in 80% of pituitary tumors. We have shown that low levels of nuclear p27 in human pituitary tumors associate with increased degradation of the protein through the UPS. Human securin, identified as the product of pituitary tumor transforming gene (PTTG), is over-expressed in human pituitary tumors. This can cause aneuploidy and inhibition of p53 actions towards cell cycle arrest, DNA repair and apoptosis. PTTG also contributes to pituitary tumorigenesis by modulation of angiogenesis. Degradation of PTTG is ubiquitin-dependent and promotes the initiation of anaphase and exit from mitosis. Incomplete PTTG degradation through the anaphase-promoting complex/cyclosome (APC/C) secondary to PTTG overexpression results in doubling of chromosome numbers. Whether the cell cycle changes reported in pituitary tumors are truly causal remains uncertain and it is more likely that alteration in signaling pathways feed into the cell cycle which then executes an aberrant set of instructions that result in cell proliferation. Excessive regulatory hormone stimulation can lead to an increased number of cells in the pituitary in various physiological or pathological states. Animal models also provide data that in the presence of excessive hypothalamic hormone stimulation, adenoma formation can occur. Hormonal (usually hypothalamic factors) and/or growth factor over-stimulation of the pituitary is dependent on signaling through membrane and/or nuclear receptors. A number of these receptors such as protein G- coupled receptors, tyrosine-kinase receptors, growth hormone, glucocorticoid and estrogen receptors are down-regulated via degradation through the ubiquitin proteasome system. Various anomalies of receptor expression observed in pituitary tumors may be explained through excessive or incomplete degradation, which may then cause aberrant signaling in different proliferative pathways to result in tumor formation. Increasing research in the field of ubiquitin-proteasome degradation of various proteins involved in pituitary proliferation is likely to provide new insights into pituitary tumorigenesis.
Keywords: human pituitary tumors, ubiquitin-proteasome system, cell cycle.
ABBREVIATIONS Akt, Protein kinase B; APC/C, Anaphase-Promoting Complex/Cyclosome; CKS1, CDC2-associated protein; CREB, cAMP Response Element-Binding; D2R, dopaminergic receptor type 2; ER, estrogen receptor; GH, Growth hormone; GHR, Growth hormone receptor; GHRH, GH-releasing hormone; GR, glucocorticoid receptor; IGF-I, insulin-like growth factor I; ICER, inducible cAMP early repressor; Jab1, Jun Activation DomainBinding Protein; MAPK, mitogen-activated protein kinase; MEN-1, multiple endocrine neoplasia type 1; MIF, Macrophage inhibitory factor; PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PRKAR1A, protein kinase A regulatory subunit 1α; PRLR, prolactin receptor; PTEN, protein and tensin homolog deleted on chromosome 10; PTTG , pituitary tumor transforming gene; SKP2, S-phase kinase interacting protein 2; TRK, tyrosine kinase receptor; UPS, ubiquitin-proteasome system.
The Ubiquitin Proteasome System in the Pathobiology of Human Pituitary Tumors 879
INTRODUCTION Pituitary tumors account for an average of 10% of intracranial tumors. Usually small, benign lesions, they may be clinically important in that they can affect the whole endocrine system as well as being locally invasive into the cavernous sinuses, optic chiasm or brain. Despite extensive research in the past 30 years, the molecular basis of pituitary tumorigenesis remains controversial. This review will concentrate on molecular changes in these tumors, and particularly alterations in the ubiquitin-proteasome system (UPS). There are two major theories which have been subject to most investigation: hormonal (usually hypothalamic factors) and/or growth factor over-stimulation, or a molecular defect within the pituitary itself. In the presence of excessive hypothalamic hormone stimulation, as in longstanding untreated end-organ failure, reactive pituitary adenomas can occur. This is also suggested by transgenic animal models that develop pituitary tumors when excessively exposed to hypothalamic releasing factors [1]. However, there is substantial evidence in favor of the monoclonal nature of pituitary tumors [1]. This argues for an intrinsic molecular defect as the primary initiating event in tumor formation, although there may be hypothalamic factors that accelerate or modulate the process of tumorigenesis. Thus, the question arises as to the nature of the precise molecular pathology underlying such tumors. Human genetic disease can in some instances be associated with endocrine neoplasia, and it was therefore thought likely that the identification of the genetic cause of multiple endocrine neoplasia type 1 (MEN-1), in which some 40% of patients develop pituitary tumors, would explain the somatic mutation in the majority of sporadic pituitary tumors. This has proved not to be the case, with somatic mutations of the MEN-1 gene, menin, accounting for no more than 1–2% of sporadic tumors [2]. We have also shown that the expression of menin mRNA is not different in pituitary adenomas compared with normal tissue [3]. Similarly, Carney syndrome is an autosomal dominant genetic disorder in which there is an approximately 10% prevalence of somatotroph adenomas. The genetic basis for one type of this disease is now known and it involves a mutation of the protein kinase A regulatory subunit on chromosome 17. Several studies have, however, been unable to demonstrate somatic mutations of this gene in sporadic pituitary tumors, nor alterations in the level of its mRNA expression [4]. At present, therefore, human germline disorders have shed little light on the pathogenesis of the much more common sporadic tumors, although a locus at 2p16, positionally identified as the probable locus for both the second type of Carney syndrome, or the gene for familial acromegaly at 11q13 [5], may be more enlightening when finally identified.
CELL CYCLE ALTERATIONS IN PITUITARY TUMORS An alternative approach to the problem of pituitary tumorigenesis is through data derived from animal models, particularly those involving gene additions and knockouts. Transgenic disruption of the cell cycle machinery frequently leads to pituitary adenomas in animal models. The cell cycle is the process by which cells grow, replicate their genome and divide. Its control system operates through cyclical interaction of proteins that induce and coordinate
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proper progression through the cycle. Driving the cell cycle is mainly dependent on the fluctuations in the concentration of cyclins, cyclin-dependent kinases (CDK) and their inhibitors (CDKI) achieved through the programmed transcription and degradation of these proteins by proteolysis within the UPS (Figure 1). Mitogenic Signals (e.g. bFGF)
G1
G0
CDK inhibitors (e.g. p27) P
Metaphase checkpoint
Rb
Cyclins - CDKs P P
Securin M Separin APC
P
Rb PP
G1checkpoint
P
G2 checkpoint Cyclins - CDKs
S
G2
Figure 1. Cell cycle control. Cell cycle progression through G1, S and G2 is mainly dependent on the fluctuation in concentrations of cyclins, CDKs and CDKI. In early G1, in response to mitogenic signals, cyclin-CDKs complexes phosphorylate Rb, resulting in activation of transcription factors which participate in the generation of molecules required for G1/S transition. Beyond G1 checkpoint the presence of mitogens is no longer required for the cell to enter a new round of division. At G2 checkpoint the quality of the new synthetized DNA is checked before entering mitosis. Progression through M phase is ensured by the interaction between separin, securin and cohesin. At metaphase/anaphase transition, when all chromosomes are attached to the spindle, APC is activated to trigger separin degradation allowing progression through anaphase.
To ensure proper progression through the cycle, cells have developed a series of checkpoints where feedback signals conveying information about the downstream processes can delay progress into a new phase until they have successfully completed the previous one, and also provide regulation by signals from the environment, such as mitogens, growth factors, etc. The major checkpoint in mammalian cells is in G1, known as the restriction point. As mammalian cells undergo a period of mitogen dependence before entering the cell cycle, the transition beyond the restriction point represents a commitment to a new round of division, regardless of the presence of mitogens (Figure 1). The three retinoblastoma family members pRb/p105 [6,7] p107 [8,9] and Rb2/p130 [10], negatively control cell cycle progression between G1 and S phases. Before G1 phase progression is initiated, Rb is underphosphorylated and thus able to repress cell cycle progression. Cyclin-CDK mediated phosphorylation of Rb is the most likely mechanism that turns off the anti-proliferative
The Ubiquitin Proteasome System in the Pathobiology of Human Pituitary Tumors 881 actions of Rb at G1/S transition. In early G1, in response to mitogenic signals, CDK4 & 6 in cyclin D complexes partially phosphorylate Rb, resulting in partial activation of E2F/DP transcription factors which participate in the generation of molecules required for G1/S transition, including cyclin E. CDK2 sequentially activates E-type cyclins (cyclin E1-E2). The cyclin E-CDK2 complex completes Rb phosphorylation which now releases the transcription factors allowing them to carry out specific tasks in cell-cycle progression, such as cyclin A synthesis. CDKI counteract CDK actions, either by blocking their activation, or by impairing substrate/ATP access [11]. There are two types of CDKI. The INK family (INK4a/p16 [12]; INK 4b/p15 [13]; INK4c/p18 and INK4d/p19 [14] exert inhibitory activity by binding to CDK4 and CDK6. These proteins exert their actions by competing with D-type cyclins for CDK subunits and thus preventing phosphorylation of pRb and inhibiting progress through G1/S. Members of the WAF/KIP family (WAF1/p21; KIP1/p27; KIP2/p57) form heterodimeric complexes with G1/S CDKs and inhibit kinase activity of CDK2-cyclin E complexes [15,16]. The UPS plays a central role in the regulation of cell growth and proliferation by controlling the abundance of key cell cycle proteins. Increasing evidence indicates that unscheduled proteolysis of many cell cycle regulators contributes significantly to tumorigenesis and is indeed found in many types of human cancers. Aberrant proteolysis with oncogenic potential is elicited by two major mechanisms: defective degradation of positive cell cycle regulators (i.e., proto-oncoproteins) and enhanced degradation of negative cell cycle regulators (i.e., tumor suppressor proteins) [17]. In many cases, increased protein stability is the result of mutations in the substrate that prevent the recognition of the protein by the ubiquitin-mediated degradation machinery. Alternatively, the specific recognition proteins mediating ubiquitination (ubiquitin ligases) are not expressed or harbor mutations rendering them inactive. In contrast, the over-expression of a ubiquitin ligase may result in the enhanced degradation of a negative cell cycle regulator [17]. The regulation of the G1/S transition appears to be one site of particular sensitivity in the provenance of pituitary tumors. In particular, aberrations of one or more components of the pRb/p16/cyclin D1/CDK4 pathway seem to be a frequent event (80%) in pituitary tumor formation [18,19]. Loss of pRb and p16 protein expression in these tumors was suggested to be mostly due to methylation in their gene-promoter region [20-22]. Cyclin D1 is overexpressed in aggressive and non-functioning pituitary tumors [23,24], and this occurs in the absence of the cyclin D1 gene CCND1 allelic imbalance (i.e. gene amplification) suggesting that there are additional mechanisms responsible for deregulating cyclin D1 expression in human pituitary tumorigenesis [23]. Abnormal cyclin D degradation through the UPS could theoretically be involved [17,25], but this has not yet been shown to be aberrant in pituitary tumors. More data have been gathered on the ubiquitination of cyclin E and p27 in pituitary tumors. p27-/- mice show an increased growth rate due to increased cellularity, testicular and ovarian cell hyperplasia and infertility, and hyperplasia of the pituitary intermediate lobe, with nearly 100% mortality caused by such a ‘benign’ pituitary tumor. Although the p27 gene was not found to be mutated in human pituitary tumors [26], and its mRNA expression was similar in tumor samples in comparison with normal pituitaries, the load of p27 protein expression in pituitary adenomas, especially in corticotroph adenomas and pituitary
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carcinomas, was shown to be much lower than that in normal pituitary tissue [27-29]. On the contrary, cyclin E expression was increased in corticotroph adenomas [24], which may be related to the particularly low levels of nuclear p27 in these tumors. Both p27 and cyclin E are degraded through the UPS. The E3 enzyme, known as ubiquitin ligase/SCF complex, catalyses the transfer of ubiquitin (Ub) groups to a lysine residue in the target protein and also controls the specificity of the ubiquitination (Figure 2). The SCF complex consist of 3 core subunits (Cul, Skp1, Roc/Rbx1) that couple to one of several F-box proteins, named after their F-box motif - a highly conserved sequence of amino acids.. While the F-box is the SCF-binding domain, the F-box protein also has a substrate-binding domain to ensure specificity. In the case of p27, the specific F-box protein is Skp2 (S-phase kinase interacting protein 2, named as it was discovered through its interaction with cyclin A-CDK2 complex). Skp2 cooperates with Cks1 (CDC2-associated protein) to undergo allosteric alterations allowing it to bind phosphorylated p27 (Figure 2). The highest level of p27 ubiquitination occurs at the G1/S transition, targeting lysine residues 134, 135 and 165 on the p27 molecule [30]. Ub
Ub
E1
Ub Ub Ub Ub Ub Ub Ub
Ub
Rbx 1
SCF complex (E3)
Cul 1
Ub Ub Ub
26S Proteasome
E2
Skp1 F box
Skp2
p27 P
P
Substrate recognition domain
Cks1 COOH tail
Figure 2. Degradation of p27 by the UPS. Ubiquitination is a specific process that is signalled by a degradation signal – degron – in the substrate protein. In response, a cascade of enzymes generically termed E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin ligase), catalyse the addition of ubiquitin polymers to the protein substrates. The ring finger type E3 that contains a SCF complex consists of 3 core subunits (Cul, Skp1, Roc/Rbx1) that couple to one of several F-box or D-box proteins. While the F-box is the SCF-binding domain, the F-box protein has also a substrate-binding domain to ensure specificity. In the case of p27, the specific F-box protein is Skp2 (Sphase kinase interacting protein 2,). Skp2 cooperates with Cks1 to undergo allosteric alterations allowing it to bind phosphorylated p27 (adapted from Nakayama et al. [92]).
At the G1/S transition, the increasing cyclin E-CDK2 activity is responsible for nuclear phosphorylation of p27 on Thr 187 [31]; p27 therefore can bind to cyclin E-CDK2 in two conformations: in a tight state, in the presence of high ATP concentrations under which the kinase activity is inhibited [32], and secondly in a loose state, at low concentrations of ATP, under which CDK2 phosphorylates p27. Thus, once cyclin E-CDK2 is activated, it can trigger p27 degradation accounting for the irreversibility of the subsequent entry to S phase
The Ubiquitin Proteasome System in the Pathobiology of Human Pituitary Tumors 883 [33]. p27 mutants (Thr187/Ala) which are resistant to phosphorylation by cyclin E-CDK2 are resistant to ubiquitination [32]. p27 mutants that can be phosphorylated, but cannot bind the cyclin E-CDK2 complex, have also been claimed to be refractory to ubiquitination. In most pituitary adenomas phosphorylation at Thr187 occur in a similar manner to that seen in the normal pituitary [34]. However, this appears to be greatly increased in corticotroph adenomas. Jab1, which enables p27 to be exported from the nucleus and would thus enhance its cytoplasmic degradation, was not obviously over-expressed in adenomas sufficient to account for diminished nuclear p27 [34]. Macrophage inhibitory factor (MIF), which has been reported to bind and hence inactivate Jab1, was also not changed in a direction that would explain the loss of nuclear p27 [35]. Increased Skp2 expression could play at least a part in p27 depletion, but overall levels of Skp2 mRNA and protein were not significantly different between normal pituitary tissue and pituitary adenomas [36]. However, tumors with low p27 protein expression did show significantly higher Skp2 expression than samples with normal p27 protein expression, suggesting that Skp2 may play a role in at least part of this process [36]. No difference was observed in Cks1 mRNA levels between normal pituitaries and pituitary adenomas; Cks1 protein expression was not assessed [37]. PTEN (protein and tensin homolog deleted on chromosome 10), the tumour suppressor function of PTEN as shown by the analysis of hereditary cancer in Cowden syndrome, controls PI3K (phosphatidylinositol-3-kinase) action and also collaborates with the transcription factor p53 in DNA repair, apoptosis, senescence and inhibition of angiogenesis It has been suggested that PTEN-deficiency in mouse embryonic stem cells causes a decrease of p27 levels with a concomitant increase in Skp2. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction in Skp2 [38]. The study of PTEN expression in normal and tumorous human pituitary revealed a direct correlation between nuclear PTEN and p27 levels (Figure 3) [39]. The degradation of PTEN is most likely mediated by a proteasome-dependent pathway, as there is evidence that PTEN is polyubiquitinated [40]. Post-translational regulation of PTEN has also been reported in different cell types [41,42]. Under-expression of nuclear PTEN in the pituitary tumors compared to normal tissue may possibly relate to enhanced protein degradation leading to the increased proliferation of the tumors. Although the nuclear ubiquitin ligase Skp2 is implicated in p27 degradation, proteolysis of p27 at the G0-G1 transition proceeds normally in Skp2(-/-) cells [43]. These data suggest the existence of a Skp2-independent pathway for the degradation of p27 at G1 phase. Kamura et al. have described a previously unidentified E3 complex, KPC (Kip1/p27 ubiquitinationpromoting complex), consisting of KPC1 and KPC2 [43]. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27 and is localized to the cytoplasm. Over-expression of KPC promoted the degradation of p27, whereas a dominant-negative mutant of KPC1 delayed p27 degradation [43]. The nuclear export of p27 seems to be necessary for KPCmediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27 degradation. KPC thus probably controls degradation of p27 in G1 phase after export of the latter from the nucleus [43]. There are no data with respect to KPC function in pituitary tumors.
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80
*
60 * Percentage of immunopositive cells
p27 P-Akt PTEN Skp2
40
20
0 NP
Tumors
Figure 3. Immunohistochemical expression of p27, phospho-(Ser 473) Akt, PTEN and Skp2 in human pituitary. Normal pituitary (NP) have a higher expression of p27 than pituitary tumors, which positively correlates with PTEN expression. On the contrary, phospho-Akt is overexpressed in pituitary tumors compared to normals (*p 20μM). Competitive inhibitors that are largely specific for individual β subunits of the 20S proteasome but also inhibit intracellular cysteine proteases. The compound is also a weak, low micromolar inhibitor of the chymotryptic activity of the 20S proteasome by binding the proteasome subunit β5 and β5i.
The Ubiquitin Proteasome System in Cerebral Ischemia
943
Table 2. Animal models of cerebral ischemia and proteasome inhibitor treatment Study
Year
Treatment
Model of ischemia (Strain) 90 min MCAo 1 & 7 day recovery (inbred SH, rats)
Buchan AM et al. [195]
2000
CVT-634 (50 mg/kg, i.p.)
Phillips JB et al. [101]
2000
MLN-519 (0.003-0.1 mg/kg, i..v.)
Temporary MCAo 24 & 72 h recovery (Sprague-Dawley, rats)
Zhang L et al. [ 202]
2001
MLN-519 (1.0 mg/kg, i.v.)
Embolic stroke model 7 day recovery (Wistar, rats)
Williams AJ et al. [153]
2003
MLN-519 (1.0 mg/kg, i.v.)
Temporary MCAo 24 h recovery (Sprague-Dawley, rats)
Berti R et al. [193]
2003
MLN-519 (1.0 mg/kg, i.v.)
Temporary MCAo 3-72 h recovery (Sprague-Dawley, rats)
Results Smaller infarct of 13±2% (P95% for 24 h or more) [50]. Toxicity following MLN-519 administration does not occur until doses reach 8 mg/kg or higher in rats. Specific side effects at this dose include gastrointestinal problems and lowering of blood pressure [50]. In brain-injured animals, the neuroprotective effects of MLN-519 (1.0 mg/kg) did not significantly alter physiological parameters (i.e. blood pressure, heart rate, blood gases, or body temperature) following MCAo injury. Overall, the timing of drug administration, cell-type affected and dose used may all be important factors in assessing effective therapy with proteasome inhibitors. Based on neuroprotective efficacy of MLN-519, with effective doses ranging from 0.03-1.0 mg/kg, a safety index (toxic/neuroprotective dose) can be estimated to at least 8 or higher. Overall, patients may benefit from transient proteasome inhibition protocols shown to be safe and effective in clinical trials. However, care providers may want to consider the presence of an active neurodegenerative disease in a patient as preclusion to the therapeutic use of proteasome inhibitors due to the potential exacerbation of an already dysfunctional UPS-related disease.
MLN-519: Clinical Trials Phase I clinical trials with MLN-519 in normal human volunteers have indicated that 20S proteasome activity can be safely reduced by 70-80% (1.6 mg/m2) following a single bolus injection [50]. The effects of proteasome inhibition with MLN-519 on blood proteasome activity were transient and up to 2 additional doses could be administered at 24 h intervals following initial dosing without treatment-emergent symptoms or abnormality of laboratory tests [50]. Minor adverse events included a transient altered taste sensation, discomfort in injection arm, headache, or flu-like symptoms but were not dose-related and were observed in both MLN-519 (28%) and placebo (41%) treated subjects [50]. These clinical data indicate that proteasome inhibition is achievable to levels that have been associated with significant neuroprotective effects in animal models of brain injury. Given the safety profile of MLN519, treatment could potentially be delivered as early as possible during the acute post-injury period (i.e. in the home or ambulance by trained medical staff). Thereafter, ‘clot-busters’ such as tissue plasminogen activator could be administered, if appropriate, in the hospital. This pharmaceutical ‘cocktail’ approach to the treatment of acute brain injury has been proposed as a strategy to optimize therapy due to the multitude of physiological factors and molecular perpetrators that act in concert towards progression of injury to the brain. In addition, therapies that target the more advanced phase of the injury could be used to induce regeneration of damaged neural tissue (e.g. growth factors or stem cell therapy) to help promote the overall functional recovery of the brain-injured patient.
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THE PROTEASOME AND NF-ΚB MEDIATED INFLAMMATION The proteasome plays a key role in the NF-κB-mediated inflammatory response (Figure 2). The transcription factor NF-κB is constitutively expressed within the cytosol but is normally bound to an inhibiting molecule IκB. A variety of inflammatory signals can induce the phosphorylation of IκB, targeting it for degradation through the UPS. The release and translocation of NF-κB into the nucleus can then stimulate inflammatory gene expression. As such, proteasome inhibition represents one method of inhibiting the activation of the NF-κBmeditated inflammatory response [8,9].
Figure 2. Proteasome and the NF- B pathway. Stimulation of the NF-κB pathway by activation signals, such as inflammatory cytokines and reactive oxygen species, activate intracellular IκB kinases. IκB kinases induce the phosphorylation and subsequent ubiquitination of the IκB molecule. Ubiquinated IκB is then targeted for degradation by the proteasome. Release of constitutively expressed NF-κB from its inhibitory molecule, IκB, allows activation and translocation of NF-κB to the nucleus. Nuclear NF-κB is responsible for expression of numerous pro-inflammatory genes.
The activation of NF-κB within the brain has been reported to occur in a variety of CNS injury models including spinal cord injury [52], traumatic brain injury [53], and both global [54] and focal brain ischemia [55-58]. Following MCAo injury in rats, the activation of NFκB has been reported as early as 3 h post-injury and was sustained out to at least 72 h in the ipsilateral cerebral cortex and striatum [59]. Based on cellular morphology, early activation of NF-κB appears in large multipolar neurons and astrocytes (3 h) while at 12-24 h activated NF-κB appears in small round or rod-shaped cells suggestive of infiltrating leukocytes or microglia [59]. The activation, nuclear translocation, and DNA binding of NF-κB following
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experimental ischemic brain injury has been reported in several other studies as well and has been implicated in the promotion of brain injury [56,60]. The inhibition of NF-κB activity has shown therapeutic efficacy in several experimental inflammatory disease models including neuronal injury [61]. Mice lacking the p50 subunit of NF-κB (p50 knockout) have been reported to have reduced brain injury following focal brain ischemia as compared to wild type controls [55]. Inhibition of NF-κB expression with pyrrolidine dithiocarbamate reduces the injury associated with focal or global ischemia with a post-injury therapeutic window of up to 6 h [58,60]. However, depending on the drug and dose used, inhibition of NF-κB can be toxic to the normal [51] or ischemic brain [57]. The inhibition of NF-κB in specific cell types may have contrasting effects on outcome due to the complex array of molecular events involved in NF-κB signaling and the role of NF-κB activation in different cell types [62]. NF-κB activation has also been linked to cerebral remodelling and may promote neuroplasticity during the advanced stages of recovery from brain injury [63]. A critical factor in assessing the inhibition of NF-κB as a therapeutic treatment is to evaluate the toxic versus therapeutic dose range and assessment of cellspecific activity. Progress towards understanding the role of NF-κB regulation on cell physiology is an active area of research and is elegantly reviewed in a series of recent review articles [62,64-68].
TREATMENT OF CEREBRAL ISCHEMIA WITH MLN-519: MECHANISM OF ACTION In ischemia/reperfusion injury, such as that encountered during stroke, there are delayed biochemical events leading to ‘abnormal’ de novo gene expression and protein synthesis. These changes in cellular pathology are marked by increases in activated microglia and leukocyte infiltration of neutrophils and macrophages initiated by intra-luminal adhesion molecule attachment and diapedesis across the blood brain barrier. As a neuro-inflammation causality promoting secondary brain damage in peri-infarct zones this process, which has been relatively well defined in both experimental and clinical studies of ischemic brain injury, represents a ‘delayed cellular target’ for achieving neuroprotection [69]. Inflammatory mediators, including cytokines, adhesion molecules and infiltrating leukocytes all play a major role coordinating the pro-inflammatory response to an ischemic event. As such, sitespecific targeting of anti-inflammatory mechanisms through regulation of these inflammatory proteins represents an exciting and promising research platform for explorations in neuroprotection strategies against cerebral ischemic episodes. In this regard, specific, nontoxic inhibitors of the 20S proteasome and proteasome-related systems (i.e. NF-κB) with compounds such as MLN-519 indeed represent viable opportunities as a stroke therapy [9]. Based on pre-clinical evidence, the neuroprotective effect of MLN-519 is strongly associated with an anti-inflammatory mechanism of action although other potential mechanisms of action have not been ruled out including the direct inhibition of injury-induced protein degradation [9].
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Inhibition of NF-κB Activity Proteasome inhibition has been associated with a concomitant reduction in activation of the transcription factor NF-κB. For example, MLN-519 has been reported to inhibit the formation of NF-κB-DNA complexes in activated T-cells in vitro and a significant dosedependent reduction of super antigen mediated T-cell proliferation [70]. Treatment of focal cerebral ischemia with MLN-519 has also been directly associated with an attenuation of activated NF-κB. A single injection of MLN-519 (1.0 mg/kg, i.v.) given 2 h following a MCAo in rats was associated with a significant reduction in immunoreactivity for the activated form of NF-κB from 3-72 h following ischemic brain injury as compared to vehicle treatment, particularly in endothelial cells [71]. Similarly, in a subsequent study, a 6 h delayed injection of MLN-519 reduced optical density of activated NF-κB immunoreactivity predominately noted in endothelial cells (47% reduction) and leukocytes (45% reduction) as compared to neurons and glial cells (34% reduction) [48]. Two interesting aspects of these studies were apparent: (i) NF-κB activation was not completely inhibited with MLN-519 and (ii) the inhibitory effect favored vascular cells and peripheral leukocytes over resident brain cells (i.e. neurons/glia). This cell-specific effect of MLN-519 is most likely related to the poor brain penetrability of MLN-519 [11,46].
Inflammatory Gene Expression Microarray gene studies of the injured rat brain following transient focal brain ischemia have mapped the post-injury expression profile of multiple inflammatory genes post-injury. In particular, a host of pro-inflammatory cytokines, chemokines and cellular adhesion molecules are upregulated with the peak expression levels between 4-24 h post-injury [2,59]. Direct evidence has indicated that several of these pro-inflammatory markers are toxic to cells and can promote cell death although the role of inflammation in brain injury is still controversial [41,72]. However, several preclinical studies have indicated the therapeutic efficacy of treatment of brain injury with anti-inflammatory agents [31,41]. A wide selection of anti-inflammatory drugs are currently available that target one or more mediators of the inflammatory cascade. However, treatment of brain-injured subjects with anti-inflammatory agents aimed at reducing injury severity may not be optimal. One potential complication is related to the redundancy in function of the multitude of mediators involved in inflammation [41]. In response, recent approaches have focused on modulation of transcriptional factors, such as NF-κB, that control the gene expression of multiple proinflammatory molecules [55]. The use of compounds such as proteasome inhibitors is one method to block the activation of NF-κB and reduce expression of a host of inflammatory genes, representing a novel approach to treatment of ischemic brain injury [8-10,22]. MLN-519 treatment of MCAo injury in rats has been associated with an attenuation of the increase in both cytokine and cell adhesion molecule expression (Figure 3). MLN-519, delivered 2 h post-MCAo, reduced IL-1β expression from 6-24 h (33-43% decrease) but appeared to have little effect on the expression of TNF-α and IL-6 although a slight but significant reduction of TNF-α was measured at 24 h [71]. Immunohistochemical staining for
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each of these cytokines revealed a strong presence in perivascular cells [71]. Interestingly, by delaying the initial injection of MLN-519 to 6 h post-injury, significant reductions were measured in both TNF-α (46%) and IL-6 (58%) at 24 h post-injury [1]. It appears that delaying the initial post-injury injection of MLN-519 corresponds to a differential effect on cytokine expression as compared to injections initiated at the time of reperfusion (2 h). Importantly, however, a 6 h delayed injection of MLN-519, which corresponds to the time of peak cytokine expression, was still able to significantly reduce inflammatory gene mRNA levels and was associated with a neuroprotective outcome. Other models of inflammatory disorders have also indicated the therapeutic efficacy of proteasome inhibitors to reduce inflammatory gene expression [11]. In particular, in a model of streptococcal induced polyarthiritis in rats, significantly lower serum levels of the pro-inflammatory factors IL-1 and IL-6 were measured in PS-341 treated rats as compared to the increase in these factors measured following vehicle treatment [73].
Figure 3. Reduction of inflammatory gene expression with MLN-519. Effect of MLN-519 treatment (1.0 mg/kg, delivered i.v. starting 6 h post-occlusion) on cytokine and cell adhesion molecule mRNA expression after 2 h of transient middle cerebral artery occlusion in the rat. (Data derived from report by [1]).
Similar to the effects on cytokine expression, a 2 h delayed injection of MLN-519 was associated with dramatic decreases in the cellular adhesion molecule mRNA expression of ICAM-1 at 3 h post-injury, while E-selectin was decreased at 12 and 24 h as compared to vehicle treated animals [71]. Reductions in ICAM-1 (58%) and E-selectin (72%) were also verified at 24 h post-injury with a 6 h delayed injection of MLN-519 [1].
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Immunohistochemical staining of these cellular adhesion molecules indicated expression was predominately within infiltrating leukocytes and endothelial cells [71].
Inflammatory Cell Infiltration The upregulation of pro-inflammatory cytokines, chemokines, and cellular adhesion molecules following brain injury has been linked to formation of cerebral edema and infiltration of inflammatory cells into the injured brain [74-76]. The resulting diapedesis of inflammatory cells involves the interaction of several cell surface molecules under control of NF-κB including integrins, selectins, and members of the immunoglobulin superfamily (IgCAM). Cell permeable inflammatory messengers such as chemokines are released from the injured brain and attract circulating peripheral leukocytes to the injured endothelium where they bind newly expressed P- and E-selectin. The interaction between cell surface integrins and IgCAMs then promote the diapedesis of peripheral leukocytes across the blood brain barrier and into injured brain regions where they participate in the neuro-inflammatory process [74,75]. Neuronal injury following an ischemic attack can evolve over several hours to days postinjury [77,78]. Strong evidence now exists indicating that active, delayed injury processes are involved in, and ultimately determine, the eventual degree of cell survival following injury [39]. The injury-induced inflammatory response is a complex and multi-step process involving inflammatory gene upregulation, release of chemotaxic agents into the blood stream, and eventual activation/recruitment of peripheral leukocytes to the site of injury. Diapedesis of inflammatory cells into the injured brain begins with an initial phase of neutrophil infiltration, which peaks at 24 h post-injury, followed by macrophage infiltration at 72 h post-injury [78]. Inflammatory cells not only dispose of cellular debris but also are a major source of post-injury toxins including reactive oxygen species and pro-inflammatory cytokines [41]. Microvascular occlusion may also occur due to the intravascular collection of inflammatory cells around the site of injury [75]. Although inflammatory cell-mediated phagocytosis is inherent to the natural healing process, pro-inflammatory molecules can promote further injury to the vulnerable penumbral regions that surround an ischemic lesion in a ‘feed-forward’ process of inflammatory cell-mediated injury [41,54]. Proteasome inhibitors have shown efficacy in reducing inflammatory cell infiltration in a variety of inflammatory-related disorders. Reduction of inflammatory infiltrate has been observed following streptococcal induced polyarthiritis in rats when the proteasome inhibitor PS-341 was administered after onset of the disease and was associated with minimal degradation of articular cartilage of the joints [73]. In an animal model of pulmonary eosinophilia induced by allergen exposure, intratracheal administration of MLN-519 induced a significant dose-dependent reduction of leukocytes in lung lavage fluid [79]. MLN-519 has also been shown to decrease CNS infiltration of T-cells in a mouse model of autoimmune encephalomylelitis (an experimental model of multiple sclerosis) and was associated with an improvement in clinical disease score with fewer relapses [80]. Additionally, MLN-519 has been reported to significantly reduce the inflammatory infiltrate of lesional human plaquestage psoriasis skin grafts in mice [70].
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Treatment of ischemic brain injury with proteasome inhibitors has also been shown to affect the diapedesis of cells into the injured brain. An interesting aspect of treatment with proteasome inhibitors is that different cell types may react differently to proteasome inhibition particularly following injury [81]. For example, the sensitivity of human endothelial cells to proteasome inhibition increases 10-fold following exposure to hypoxic conditions [82]. Studies evaluating the treatment effects of MLN-519 following rat MCAo injury have repeatedly indicated the neuroprotective reduction in brain infarction is associated with a reduction in both neutrophil and macrophage infiltration in both cortical and subcortical brain regions with up to a 10 h delayed injection of MLN-519 [1,45,83]. Overall, MLN-519 treatment reduced neutrophil infiltration by 36-60% as evaluated 24 h post-injury [45,48] and reduced both neutrophil (32-79%) and macrophage (up to 32-80%) infiltration as evaluated at 72 h post-injury [1,45]. Similarly, following embolic stroke in rats, significant reductions in inflammatory cells were also observed with delayed injections of MLN-519 (initiated 2, 4, or 6 h post-injury) [46]. This dramatic effect of MLN-519 treatment on inflammatory cell infiltration may be directly related to reducing endothelial cell-induced attraction of peripheral leukocytes, providing a key anti-inflammatory mechanism of action related to the improved outcome following experimental cerebral ischemia.
CONCLUSION Based on experimental data, the proteasome appears to play a crucial role in the progression of several different disease states including CNS disorders. In many cases, neurodegenerative disorders are associated with a loss of function of proteasome activity or the UPS in general. These types of disorders may benefit from strategies to reestablish normal levels of proteasomal activity. However, the proteasome is also a therapeutic target in several types of disease due to the role it plays in promotion of the aberrant gene expression associated with disease progression. Pre-clinical data supports the use of proteasome inhibitors to treat inflammatory related disorders such as injury to the CNS due to cerebral ischemia. In fact, the proteasome inhibitor MLN-519 appears to provide an excellent therapeutic window for the treatment of ischemia/reperfusion brain injury in rats. The neuroprotective effects of MLN-519 have been associated with an improvement in histopathological and functional recovery with a 6-10 h therapeutic treatment window with no significant detrimental changes observed in physiological parameters between vehicle and drug-treated animals. MLN-519 treatment also correlated to a reduction in blood proteasome activity by 80-90% with a dosing schedule shown to be safe in normal, healthy human volunteers. The mechanism of action of proteasome inhibition to treat ischemic brain injury appears to involve an anti-inflammatory effect via reduction of NF-κB mediated inflammatory gene expression. MLN-519 treatment has been shown to reduce activated NF-κB immunoreactivity, attenuate inflammatory gene expression, and reduce infiltration of inflammatory neutrophils and macrophages in the injured rat brain. Additionally, the 6-10 h therapeutic window of MLN-519 correlates well to peak increases in cytokine and cellular adhesion molecule mRNA upregulation. As such, one of the primary mechanisms of action of
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MLN-519 in ischemic brain injury may involve the inhibition of cellular adhesion moleculemediated infiltration of peripheral inflammatory cells into the brain, which represents a potentially ‘non-neuronal’ mechanism of neuroprotective activity. Overall, pre-clinical brain injury studies support the therapeutic use of proteasome inhibitors, which represent a new approach for the treatment of CNS disorders such as clinical stroke through attenuation of the inflammatory-mediated neuropathology associated with ischemia/reperfusion injury to the brain. The superior neuroprotective treatment profile of novel compounds such as MLN-519 to treat brain injury may ultimately play a key role, alone or in conjunction with other drug therapies, as part of an overall therapeutic regiment to promote recovery of the brain-injured patient.
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[62] Hayden MS, Ghosh S: Signaling to NF-kappaB. Genes Dev 2004, 18:2195-2224. [63] Barger SW, Moerman AM, Mao X: Molecular mechanisms of cytokine-induced neuroprotection: NFkappaB and neuroplasticity. Curr Pharm Des 2005, 11:985-998. [64] Bonizzi G, Karin M: The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004, 25:280-288. [65] Chen LF, Greene WC: Shaping the nuclear action of NF-kappaB. Nat Rev Mol Cell Biol 2004, 5:392-401. [66] Karin M, Yamamoto Y, Wang QM: The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 2004, 3:17-26. [67] Kucharczak J, Simmons MJ, Fan Y, Gelinas C: To be, or not to be: NF-kappaB is the answer--role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 2003, 22:8961-8982. [68] Ruland J, Mak TW: Transducing signals from antigen receptors to nuclear factor kappaB. Immunol Rev 2003, 193:93-100. [69] Wang X: Investigational anti-inflammatory agents for the treatment of ischaemic brain injury. Expert Opin Investig Drugs 2005, 14:393-409. [70] Zollner TM, Podda M, Pien C, Elliott PJ, Kaufmann R, Boehncke WH: Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model. J Clin Invest 2002, 109:671-679. [71] Berti R, Williams A, Velarde L, Elliott P, Adams J, Yao C, Dave J, Tortella F: Effect of the proteasome inhibitor MLN519 on expression of inflammatory molecules following middle cerebral artery occlusion and reperfusion in the rat. Neurotox Res 2003, 5:505514. [72] Emerich D, Dean R, Bartus R: The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct? Exp Neurol 2002, 173:168-181. [73] Palombella VJ, Conner EM, Fuseler JW, Destree A, Davis JM, Laroux FS, Wolf RE, Huang J, Brand S, Elliott PJ, Lazarus D, McCormack T, Parent L, Stein R, Adams J, Grisham MB: Role of the proteasome and NF-kappaB in streptococcal cell wallinduced polyarthritis. Proc Natl Acad Sci U S A 1998, 95:15671-15676. [74] Yoshimoto T, Houkin K, Tada M, Abe H: Induction of cytokines, chemokines and adhesion molecule mRNA in a rat forebrain reperfusion model. Acta Neuropathol (Berl) 1997, 93:154-158. [75] Danton GH, Dietrich WD: Inflammatory mechanisms after ischemia and stroke. J Neuropathol Exp Neurol 2003, 62:127-136. [76] Frijns CJ, Kappelle LJ: Inflammatory cell adhesion molecules in ischemic cerebrovascular disease. Stroke 2002, 33:2115-2122. [77] Clark R, Lee E, Fish C, White R, Price W, Jonak Z, Feuerstein G, Barone F: Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study. Brain Res Bull 1993, 31:565572. [78] Clark R, Lee E, White R, Jonak Z, Feuerstein G, Barone F: Reperfusion following focal stroke hastens inflammation and resolution of ischemic injured tissue. Brain Res Bull 1994, 35:387-392.
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[79] Elliott PJ, Pien CS, McCormack TA, Chapman ID, Adams J: Proteasome inhibition: A novel mechanism to combat asthma. J Allergy Clin Immunol 1999, 104:294-300. [80] Vanderlugt CL, Rahbe SM, Elliott PJ, Dal Canto MC, Miller SD: Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519. J Autoimmun 2000, 14:205-211. [81] Wojcik C: Regulation of apoptosis by the ubiquitin and proteasome pathway. J Cell Mol Med 2002, 6:25-48. [82] Zund G, Uezono S, Stahl GL, Dzus AL, McGowan FX, Hickey PR, Colgan SP: Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes. Am J Physiol 1997, 273:C1571-1580. [83] Williams A, Lu X-C, Hartings J, Tortella F: Neuroprotection assessment by topographic electroencephalographic analysis: effects of a sodium channel blocker to reduce polymorphic delta activity following ischaemic brain injury in rats. Fund Clin Pharm 2003, 17:581-593.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 1013-1037© 2007 Nova Science Publishers, Inc.
Chapter 42
CLINICAL EXPERIENCE OF PROTEASOME INHIBITORS IN CENTRAL NERVOUS SYSTEM DISEASES Imtiaz M. Shah1,∗ and Mario Di Napoli2,3 1
2
Mansionhouse Unit, Victoria Infirmary, Glasgow, Scotland. G41 3DX; Neurological Service, San Camillo de’Lellis General Hospital, Rieti I-02100, Italy; 3 Neurological Section, SMDN – Center for Cardiovascular Medicine and Cerebrovascular Disease Prevention, Sulmona (L'Aquila) I-67039, Italy.
ABSTRACT The proteasome is an enzyme, which is present within all cells, from yeast to humans. It has a central role in the proteolytic degradation of the vast majority of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in controlling inflammatory processes, cell cycle regulation, and gene expression. Agents that inhibit the proteasome have been shown to be active in numerous animal models of inflammation and cancer. Two proteasome inhibitors are under clinical evaluation. MLN-519 is being studied for the treatment of reperfusion injury that occurs following cerebral ischemia and myocardial infarction. The other, Bortezomib (Velcade®), has recently been licensed for the clinical treatment of multiple myeloma. It is also undergoing further evaluation for the treatment of chronic lymphocytic leukemia and a variety of solid tumors. The proteasome may also have an important role in the evolution of HIV-related disorders including AIDS and inflammatory disorders. Therapeutic strategies using proteasome inhibitors for the treatment of these conditions have now entered preclinical development. MLN-519 is a small-molecular-weight lactacystin analogue developed by Millenium (LeukoSite) for the potential treatment of inflammatory disease and stroke using a novel ubiquitin proteasome enzyme inhibitor ∗
Correspondence concerning this article should be addressed to Dr. Imtiaz M. Shah, MSc MRCP; Mansionhouse Unit, Victoria Infirmary, Glasgow. Scotland. G41 3DX. Phone: +44 141 434 0902; Fax: +44 141 201 6305; Email:
[email protected].
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approach. The reperfusion that follows an ischemic event provides both positive and negative factors that affect the overall outcome of the cerebral tissue. The ischemic endothelium upregulates the expression of cell adhesion molecules, which then attract the circulating leukocytes. Once bound to the endothelium, these cells diapedese into the tissue and are responsible for the destruction and much of the subsequent tissue damage. MLN-519 attenuates the expression of these cellular proteins, reduces the invasion of leukocytes and hence limits tissue damage. MLN-519 has demonstrated a neuroprotective effect in rat models of middle cerebral artery temporary occlusion. MLN-519 reduces infarct volume, brain edema and increases neurological recovery with a reported therapeutic window of at least 6-hours. These effects are associated with a temporary reduction of circulanting 20S proteasome activity (70-80%), with a reduced leukocyte infiltration and decreased nuclear factor-κB activation. Similar protective results have also been reported in experimental myocardial infarction models in rats and pigs: MLN-519 protects cardiac tissue from ischemia and maintains its functionality as demonstrated by preserved left ventricular developed pressure and contractile function. These data demonstrate substantial clinical value, since many patients are admitted to the hospital hours after the stroke or heart attack has occurred and reperfusion has begun. That inhibition of the proteasome can be of benefit under these clinically-relevant conditions demonstrates their potential in these common life-threatening diseases. An explorative phase I trial has demonstrated that MLN-519 is well tolerated by healthy subjects at levels that are maximally neuroprotective in experimental conditions. It is currently undergoing further evaluation for clinical trials in acute stroke and myocardial infarction.
Keywords: Proteasome inhibitors, NF-κB, Neuroinflammation, Stroke.
ABBREVIATIONS 6-OHDA, 6-hydroxyl dopamine; AD, Alzheimer’s disease; AMC, 7-amino-4-methylcoumarin; BBB, blood-brain barrier; ChTL, chymotrypsin-like; EEG, electroencephalogram; eNOS, endothelial nitric oxide synthase; ER, endoplasmic reticulum, ERAD, ER-associate degradation; hsp, heat shock proteins; HD, Huntington´s disease; HIV, human immunodeficiency virus; IAP, inhibitor of apoptosis proteins; ICAM, intracellular adhesion molecule; IκB, inhibitory-κB; IKK, IκB kinase; IL, interleukin; MCAo, middle cerebral artery occlusion; MM, multiple myeloma; NF-κB, nuclear factor κB; PD, Parkinson’s disease; PGPH, peptidylglutamyl peptide-hydrolyzing; PSI, Cbz-Ile-Glu (O-t-Bu)-Alaleucinal; RT-PCR, real time polymerase chain reaction; TAT, tyrosine amino transferase; TL, trypsin-like; TNF –α, Tumour Necrosis Factor–α, UPS, ubiquitin proteasome system; VCAM-1, Vascular Cell Adhesion Molecole-1.
INTRODUCTION Protein homeostasis is critical to several biological processes and the ubiquitin proteasome system (UPS), which processes more than 80% of all cellular proteins, is the
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1015 principal mechanism for degradation of proteins. It plays an essential role in regulating the intracellular concentration of specific proteins, thereby maintaining homeostasis within the cells. The UPS controls global protein degradation rates and the precisely timed degradation of regulatory proteins, such as cyclins, transcription factors and oncogene products (which are important in cancer and inflammatory disorders) [1-3]. Therefore, targeting the regulation of protein production and destruction has been a major focus of drug research [4-6]. Accordingly, the proteasome has emerged as an attractive target for drug discovery. The 26S proteasome is a large multi-catalytic protease, which has a major role in intracellular proteolysis [7]. It is present within all eukaryotic cells and its function has now been recognised as being crucial for cellular function. The proteasome protects the cell against oxidative stress, and prevents the accumulation of redundant and damaged proteins. Dysfunction of the proteasome has been implicated in the pathogenesis of neurodegenerative conditions like Parkinson’s (PD) and Alzheimer’s Disease (AD) [8]. However, inhibition of the proteasome has also been shown to be beneficial in the treatment of certain cancers and inflammatory conditions [9,10]. Inhibition of the 26S proteasome prevents this targeted proteolysis, which can affect multiple signalling cascades within the cell. Manipulating both the global proteolysis rates and the degradation of particular substrates are both attractive goals for drug design [4,5]. This chapter will review the expanding role of the presently available proteasome inhibitors and their potential clinical uses in the treatment of nervous system diseases.
THE UBIQUITIN-PROTEASOME SYSTEM Current research has focused on the UPS and its role in the pathophysiology of disease processes [11]. Significant advances have been made in the scientific understanding of the fundamental importance of the UPS in processes beyond the proteolytic degradation of damaged, oxidised, or misfolded proteins. In nonclinical studies, targeted degradation of key regulatory proteins by the 26S proteasome has been shown to be involved in controlling the cell cycle, transcription, apoptosis, angiogenesis, cell migration, and metastasis. Several in vitro studies have shown that inhibition of the proteasome affects the temporal stability of various cell-cycle regulatory proteins, especially those that are short lived. Cyclins, cyclindependent kinase inhibitors, and tumour suppressors have all been shown to be substrates for the UPS (Table 1). Inhibiting the degradation of regulators such as p27, p21, and p53 has been implicated as a mechanism by which proteasome inhibition impairs tumour cell growth and survival. The role of the proteasome in cell cycle regulation has provided the opportunity for exploring the therapeutic potential of proteasome inhibition. The UPS regulates the action of transcription factor Nuclear Factor–κB (NF-κB) and this has been an important target for therapeutic drug research [10]. NF-κB is an inducible transcription factor of the Rel family, sequestered in the cytoplasm by the IκB family of proteins. NF-κB exists in several dimeric forms, but the p50/p65 heterodimer is the predominant one [12]. NF-κB, in its inactive form, is normally complexed to the inhibitory protein, inhibitory-κB (IκB) and the complex is sequestered in the cytoplasm, preventing NFκB mediated gene transcription. Phosphorylation of IκB by IκB kinase (IKK) leads to
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activation of NF-κB [13]. Phosphorylated IκB is then ubiquitinated, and subsequently undergoes degradation by the proteasome [14]. In response to stimuli, IκB is phosphorylated, resulting in its ubiquitination and subsequent degradation by the proteasome. NF-κB is thereby released and is rapidly translocated to the nucleus, where it promotes transcription of genes encoding cytokines [e.g. tumour necrosis factor (TNF-α), interleukin-1 (IL-1), IL-6], stress response factors (e.g. cyclooxygenase-2, nitric oxide, 5-lipoxygenase), cell cycle regulators, and cell adhesion molecules (e.g. ICAM-l, VCAM-l, E-selectin). Activation of NF-κB also increases expression of anti-apoptotic proteins such as IAP-l and IAP-2 and the Bcl-2 family [15]. Proteasome inhibition has therefore been an attractive target for drug research in an attempt to reduce NF-κB activation by preventing IκB degradation, thus resulting in a dampening of the inflammatory response [16,17]. Activation of the transcription factor NF-κB can occur when cells are stimulated by cytokines, antigens, oxidative triggers, and cell-cell contact. The role of NF-κB in stimulated cells is to promote transcription of proinflamamtory genes, including cytokines, cell adhesion molecules, and pro-angiogenic molecules. It also suppresses apoptosis in favour of cell growth and migration. Table 1. Proteins targeted by the ubiquitin-proteasome pathway in non-clinical studies Class of Proteins Cyclins and related proteins
Tumour suppressor Oncogenes
Inhibitory proteins Enzymes
Protein Cyclins A, B, D, E
Protein function Cell-cycle progression
Cyclin-dependent (CDK) inhibitors (p27, p21) p53 c-fos c-jun
Regulation of cyclin kinase activity Transcription factor These are two different transcription factors which form a complex; they are also proto-oncogenes Transcription factor Transcription factor Inhibitor of NF-κB Inhibitor of E2F-l Phosphatase Tyrosine metabolism
c-myc N-myc IκB p130 cdc25 phosphatase Tyrosine amino transferase (TAT)
Aberrant NF-κB signalling is an important feature of several neurological disorders like stroke, epilepsy, amyotrophic lateral sclerosis, AD, PD, and Huntington´s diseases (HD). Traumatic brain and spinal cord injuries can also be classified as neurodegenerative. Insult to the brain or spinal cord in these disorders induces a cascade of signalling events that stimulate NF-κB activation in neurons, and injury responsive glial cells [12]. In ischemic brain injury (lack of oxygen in brain leading to neuronal death), which can result in a stroke, the activity of NF-κB is found to be very high. For example, NF-κB (p50) activation has been
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1017 reported to enhance ischemic neuronal death [17]. In the case of epileptic seizures, NF-κB activity has been found to be rapidly increased in hippocampal neurons within 4-16 hours following kainite-induced seizures [18,19]. In rat models of traumatic injury, levels of NF-κB activity are increased in cerebral cortex within hours of the insult [20]. NF-κB activity has been shown to be increased in certain chronic neurodegenerative disorders like AD, PD, HD, and multiple sclerosis [20]. By inducing the production and release of inflammatory cytokines, reactive oxygen molecules and excitotoxins, activation of NF-κB in microglia and astrocytes may contribute to neuronal degeneration. However, activation of NF-κB in neurons can also promote their survival by inducing the expression of genes encoding antiapoptotic proteins such as Bcl-2 and the antioxidant enzyme Mn-superoxide dismutase. From the above information, it is clear that NF-κB is an important transcription factor involved in a range of neurological disorders. Its activation and regulation in different disease states is variable, in a sense that it may be important in the generation of pathogenesis, or it may itself be activated by disease specific proteins. Hence, if the regulation of NF-κB can be selectively controlled, most of these disease processes can be dealt with at a very early stage, when they are more dependent upon cellular factors induced by NF-κB. The degradation of IκB after its phosphorylation is an important event in the activation of NF-κB. Thus, with the knowledge of the molecular mechanisms involving ubiquitination and proteasome dependent degradation, mentioned above, some proteasome inhibitors have been developed in an attempt to modify the NF-κB – proteasome pathway.
THE PROTEASOME AS A THERAPEUTIC DRUG TARGET The proteasome is an abundant multi-enzyme complex that is involved in the degradation of intracellular proteins (see Chapters 6 and 7). Intracellular levels of a vast number of different proteins are regulated by polyubiquitination and subsequent turnover mediated by the proteasome. Interference with proteasome function therefore leads to disturbances in a variety of cellular activities. The 26S proteasome is formed by a core 20S barrel-shaped structure [21]. This is made up of 7 different α and 7 different β sub-units, which are arranged into four stacked heptameric rings. The two outer α rings sandwich the inner core composed of two β rings. The α-subunits provide stability to the proteasome, while the proteolytic sites of the proteasome are located within the individual β-subunits. Each enzymatic site is directed towards the centre of the 20S complex. The regulatory 19S complex (or PA700) caps each end of the 20S core and controls substrate entry into the proteasome. Substrates enter through narrow openings of the outer rings of the 20S proteasome, and must be inserted within the central chamber formed by the β inner core in order to be degraded [7]. This view may be oversimplified, since there are now emerging examples of regulation at the level of proteasome and specific substrate recognition mediated by ubiquitin- and proteasome-binding adaptor proteins such as Rad23 [22]. The substrate protein is initially targeted by the enzyme ubiquitin ligase E3, which ubiquitinates the protein (see Chapter 3 and [23]). Further ubiquitin molecules are subsequently added to create a polyubiquitin chain. The 19S complex contains the ubiquitin recognition sites and this regulates protein entry into the 20S core. The polyubiquitinated
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protein binds to the 19S sub-unit, which cleaves the ubiqutin chain using ATPases, and sends the unfolded protein into the proteasome core for processing. The β-rings have three active sites having chymotrypsin-like (ChTL), trypsin-like (TL), and peptidylglutamyl peptidehydrolyzing (PGPH like or caspase–like) activities. These proteolytic sites degrade the substrate protein into smaller peptide chains. Cleavage of the protein occurs by nucleophilic attack by an N-terminal threonine residue from the β sub-units [24]. Proteasome inhibitors have been designed to target these enzymatic sites, and most interfere with the ChTL activity of the proteasome [25,26]. The proteasome inhibitors comprise five important classes of chemical agents: peptide aldehydes, peptide vinyl sulfones, peptide boronates, peptide epoxyketones, and β-lactones. The role of these drugs have been investigated for potential clinical treatments [10,27]. Several drugs targeting the proteasome are currently in preclinical and clinical testing (Table 2) (see Chapter 40). Table 2. Drugs targeting the proteasome currently in preclinical and clinical testing Reagent Bortezomib (Velcade®, PS-341)
Principle Dipeptide boronic acid
Company/Reference Millennium
MLN519 (MLN-519)
Lactacystin derivative
Millennium
Epoxomicin, Eponemycin Nitrophenyl trileucine vinyl sulfone
Streptomyces epoxyketones Trileucine vinyl sulfone
Meng et al., 1999 [28,29] Bogyo et al., 1997 [30]
Ritonavir
Peptidomimeti c protease inhibitor
Abbott
Experimental Effects Stabilization of cellcycle and proapoptotic proteins, inhibition of antiapoptotic proteins, effects on tumor microenvironment Potent antiinflammatory and neuroprotective effects Cytotoxic effects in various tumor cells Irreversible inhibition of trypsin- and chymotrypsin-like proteasome activities HIV protease inhibitor, also inhibits chymotrypsin-like activity of proteasome
Clinical Trial/Status FDA approved for relapsed and refractory multiple myeloma, ongoing trials for several tumors Phase 1, intended for acute stroke and myocardial infection Preclinical Preclinical
Approved for AIDS, phase 2 studies in tumor patients to be started
Companies: Abbott Laboratories (www.abbott.com), Millennium Pharmaceuticals, Inc(www.mlnm.com).
Due to the importance of the UPS one can foresee that a proteasomal inhibitor would be a toxic agent not suitable for drug development; however different in vitro cell lineages display an astounding variation in the degree of sensitivity to proteasome inhibitors. Moreover, the potent proteasome inhibitor bortezomib (Velcade®) manufactured by Millennium Pharmaceuticals is relatively well tolerated by human patients while it is an effective killer of different cancer cells [31,32] The main focus of research in neurological disease has been on the treatment of neuroinflammation associated with cerebrovascular
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1019 disease [33]. However, targeting the proteasome may not be sufficient to inactivate the NFκB pathway and more specific inhibitors targeting the ubiquitin ligases [E3] are currently being researched [34]. The great advantage of such drugs is their enormous specificity intrinsically related to the ubiquitination mechanism, which relies on the enormous variety of different E3s, many of them specialized only to ubiquitinate just a very limited subset of substrates. For example, MDM2 is the ubiquitin ligase which ubiquitinates itself and the crucial tumor suppressor p53 and therefore may be a target for cancer drug design [35] while inhibition of SCFβTRCP or other E3s which ubiquitinate IkBα may therefore be a suitable target for anti-inflammatory drugs [36]. The design of E3-specific inhibitors pose a great challenge and has not yet gone beyond early laboratory work [34].
NF-ΚB AND CEREBROVASCULAR DISEASE Neuroinflammation has been recognised as an important component in the pathophysiology of acute cerebrovascular disease [37]. Acute stroke results in the activation of an inflammatory cascade, which can lead to increased cerebral infarct size and worsen clinical outcome [38,39]. Various inflammatory mediators have been implicated in this inflammatory process and therapeutic agents are being researched in an attempt to modify this damaging response. Animal models of acute ischemic stroke have shown increased NFκB activity and this has been identified as an important common pathway in neuroinflammation [17,40]. NF-κB triggers the release of interleukins (IL-1, IL-6), TNF-α and cell adhesion molecules (CAM’s) [41]. The formation of reactive oxygen species and TNF-α itself results in further activation of NF-κB [42]. This leads to neutrophil infiltration and these inflammatory mediators are important in the progression of acute stroke [43]. Levels of IL-1 rapidly rise during the acute phase of stroke, and can remain elevated for several days [44]. Studies using IL-1 receptor antagonists have shown a reduction in infarct size in rat models of stroke [45]. TNF-α levels also rise after stroke and can persist for up to 5 days [46]. Overexpression of TNF-α receptors also occurs during acute ischemic stroke [47]. Intracerebral administration of TNF-α prior to middle cerebral artery occlusion (MCAo) significantly enlarges infarct size [48]. However, TNF-α may have beneficial effects during the recovery phase of stroke in cerebral re-modelling. IL-6 levels also rise during the acute period of stroke and again correlate with larger infarct size and poor clinical outcome [39]. IL-6 and TNF-α further trigger the activation of iNOS and COX-2 enzymes which are also implicated in neuronal damage [49]. Leucocyte infiltration of the cerebral tissue also occurs as part of the neuroinflammatory response [50]. This requires the interaction of CAM’s and chemokines. This allows the leucocytes to roll along the endothelium which then undergo diapedesis [51]. Animal models of stroke have shown a strong correlation between CAM’s and cerebral infarct size [52]. Anti-ICAM treatment, so far, has been unsuccessful in patients with acute ischemic stroke. The Enlimomab study used a monoclonal antibody against ICAM-1, which was administered within 6 hours of ischemic stroke onset [53]. The 3-month outcome mortality data and adverse events were worse in the enlimomab group and it appeared that there may have been a pro-inflammatory response.
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In an attempt to modify release of all these inflammatory mediators a central upstream target has been extensively researched. NF-κB inactivation is an attractive therapeutic option and proteasome inhibitors are being tested in the treatment of acute stroke. However, NF-κB action may be beneficial during the recovery phase of stroke and in cerebral re-modelling [12]. Therefore detailed evaluation of any drug would be required.
PRE-CLINICAL STUDIES Most pre-clinical stroke studies have been performed on MLN-519, an analogue of lactacystin [54]. Lactacystin is a bacterial metabolite, which was the initial lead compound found to have proteasomal inhibition activity (a) (Figure 1). Lactacystin acts via a β-lactone ring intermediate called clasto-lactacystin-β-lactone (b). The β-lactone ring reacts with the active site of the proteasome and it mainly inhibits the chymotrypsin-like activity [55]. The β-lactone intermediate irreversibly reacts with the threonine hydroxyl group of the active site [56]. The proteasome inhibitor MLN-519 is also based around the clasto-lactacystin-βlactone strucure; (1R – [1S, 4R, 5S] – 1 – (1 – hydroxy – 2 – methylpropyl) – 4 – propyl – 6 – oxa – 2 – azabicyclo [3.2.1] heptane – 3, 7 – dione) (c). Again, the β-lactone ring is the active part of MLN-519, which forms a covalent bond with the threonine hydroxyl group of the proteasome enzymatic site [26].
Figure 1. Structures of (a)-lactacystin, (b) clasto-lactacystin-β-lactone , and (c) MLN-519.
Pre-clinical studies in rat models of acute ischemic stroke have shown a reduction in cerebral infarct volume after treatment with MLN-519. MCAo rat models were treated with an intravenous infusion of MLN-519, 2 hours after the onset of acute ischemia. MLN-519
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1021 doses ranging from 0.01mg/kg to 0.3 mg/kg were shown to significantly reduce cerebral infarct volumes from 183±42 mm3 to 138±30mm3, respectively. Dose response analysis of infarct volume at 24hrs showed that neuroprotection approached 50-60% with the highest doses (0.1mg/kg to 0.3mg/kg). Evaluation of the rats using electroencephalogram (EEG) and assessment of neurological function showed significant improvements after treatment with MLN-519. Neutrophil infiltration was also significantly decreased in infarcted tissue by up to 70%. Further studies have shown that MLN-519 significantly reduces neutrophil infiltration via inhibition of the NF-κB – proteasome pathway [54]. Analysis of inflammatory mediators showed significant reductions in ICAM and E-selectin levels. Immunohistochemical analysis showed that the drug was most active within the endothelial cells and leucocytes [57]. The use of thrombolytic therapy in the treatment of acute ischemic stroke is being increasingly used in acute stroke units [58]. Patients presenting within three hours of symptom onset, and with no contraindications to treatment, can be treated with recombinant tissue-plasminogen activator (rt-PA). The use of neuroprotective agents in combination with rt-PA is an attractive option in an attempt to minimise cerebral tissue damage. MLN-519 in combination with rt-PA showed significant reductions in cerebral infarct volumes in rat models of MCAo [59]. Combination treatment given up to 6 hours post-ischemia significantly improved neurological recovery and reduced infarct volume. There was no increased incidence of hemorrhagic transformation in the combined treatment groups, compared with controls and MLN-519 alone. This may therefore be an attractive treatment combination to consider for future clinical trials [60]. Rat models of stroke have also shown that NF-κB plays an important role in perilesional inflammation associated with intracerebral hemorrhage [61]. Therefore proteasome inhibition treatment may be beneficial in both acute ischemic and hemorrhagic stroke. The promising results of MLN-519 in pre-clinical stroke animal models has led to clinical trials of this drug.
CLINICAL STUDIES The Phase I study of MLN-519 was performed on healthy male volunteers [62]. This study was a randomised double-blind placebo-controlled trial. It was designed to provide initial safety, tolerability and pharmacodynamic data on MLN-519 in humans. The initial study looked at ascending single bolus doses of MLN-519, in order to establish a therapeutic dose range. This was followed by a multi-dose study, when single bolus doses of MLN-519 were administered over three consecutive days at 24-hour intervals. The primary end-points were the effects of MLN-519 on blood 20S proteasome inhibition and drug toxicity. The aim was to achieve maximal dosing at 80% proteasome inhibition. In pre-clinical drug safety studies MLN-519 toxicity was seen in doses above 8 mg/kg. Gastro-intestinal side effects, weight loss and possible blood pressure lowering was observed in rat studies. These effects were noted when proteasome inhibition was > 95% for extended periods of time (>24hrs). In vitro studies of MLN-519 showed the development of neurotoxicity in spinal neurons after 24hr incubation [63]. Little toxicity was observed at
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80% proteasome inhibition and therefore this level was the target for maximal dosing with MLN-519 [62]. The maximal pharmacodynamic effect of MLN-519 in this phase I study was observed at approximately 1 hour post-dosing. The single dose study investigated doses ranging from 0.012 mg/m2 to 1.6 mg/m2. The maximal dose tested (1.6 mg/m2, ~0.267 mg/kg) reduced the proteasome activity in circulating whole blood cells from 1.31 to 0.60 nmol/AMC/s/mg protein (~54%) [62,64]. The triple-dosing study used doses between 0.5 mg/m2 to 1.6 mg/m2. Male subjects in 17 groups of four were randomly assigned to receive a bolus dose of MLN519 or placebo. Within each group three subjects received the drug and one placebo. Up to 80% blood 20S proteasome inhibition was achieved with the maximal dose of MLN-519, at 1.6 mg/m2 (Figure 2). There were no significant changes in biochemical or hematological parameters observed. ECG and hemodynamic monitoring were normal. There were no drug dose related side effects. Minor side effects were transient and noted only on drug administration – altered test sensation and discomfort in the injection arm. There were no significant differences noted between the treatment and placebo groups, suggesting the symptoms were associated with the diluent and not the drug.
Figure 2. Single dose MLN-519 and 20S proteasome activity with incremental dosing. Maximum proteasome inhibition was achieved at 1-hour post-dosing. Approximately 80% proteasome inhibition was achieved with the highest dosing of 1.6 mg/m2. Proteasome activity returned to within normal limits at 24 hours post-dosing. (Copyright permission: Shah IM et al.: Br J Clin Pharmacol 2002, 54,269-276) [62].
It was not possible to obtain pharmacokinetic data on MLN-519, due to its rapid plasma clearance. Pre-clinical radio-labelled studies of MLN-519 have shown rapid clearance of the drug, within the first 10 minutes of administration. It is mainly taken up by endothelial cells and leukocytes and does not cross the blood brain barrier [54]. The pharmacodynamic data was therefore more important as this provides us with details of proteasomal and drug activity [65].
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1023 Maximal proteasome inhibition was seen at 1-hour post-dosing and activity returned to basal levels within 24 hours. This was also observed in the triple dosing study (Figure 3). Neuroinflammation associated with acute ischemic stroke has been shown to persist for up to 72 hours post-stroke [39]. Hence, the rationale for the triple dosing study in an attempt to mimic a potential neuroprotective treatment regime in acute stroke patients. There was no evidence of accumulation of the pharmacodynamic effect of MLN-519 with repeated dosing. The short action of MLN-519 would be beneficial in acute stroke patients, as NF-κB activity has been associated with cerebral re-modelling [12]. Prolonged treatment may be detrimental in stroke recovery and rehabilitation. Proteasome function has also been shown to decline with ageing [66]. As elderly patients will be the main treatment population, then safety and tolerability data must also be studied in this group. More clinical trials are being planned to further evaluate proteasome inhibitors in the treatment of acute stroke [33,64,67].
Figure 3. Triple dose MLN-519 and 20S proteasome activity (Dose: 1.6 mg/m2). Three consecutive doses of MLN-519 were administered 24 hours apart (arrows). Proteasome activity returned to within normal limits at 24 hours post-dosing and there was no cumulative effect of consecutive dosing.(Copyright permission: Shah IM et al.: Br J Clin Pharmacol 2002, 54:269-76) [62].
BORTEZOMIB The ubiquitin-proteasome pathway plays a significant role in the degradation of regulatory proteins required for cell cycle progression and mitosis [68]. A disruption in the regulation of these cell cycle proteins results in abnormal cell division and tumorogenesis [69]. Proteasome inhibitors selectively target and induce apoptosis in proliferating cancer cells. Therefore these drugs have been an important area of therapeutic research in cancer treatment [70]. Bortezomib (Velcade®; PS-341), a dipeptidyl boronic acid proteasome blocker developed by Millennium Pharmaceuticals (Cambridge, MA) in cooperation with Johnson and Johnson (New Brunswick, NJ), was tested against a panel of 60 tumor cell lines and displayed promising anticancer properties. Bortezomib is the first proteasome inhibitor to be licensed for clinical use [71,72]. It was approved by the FDA in May 2003 for the treatment of patients who have received at least two prior therapies for multiple myeloma (MM), and have
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demonstrated disease progression on the last therapy. Phase 2 studies demonstrated a response to bortezomib in 27% of patients irrespective of prior treatments [72]. It is currently being used in the treatment of the hematological malignancy, MM. It is given as an intravenous injection. Inhibition of NF–κB and reduction in IL-6 levels are postulated mechanisms of action [73]. Bortezomib is cleared rapidly from the circulation and only pharmacodynamic data has been available. Maximal 20S proteasome inhibition with bortezomib has been observed at 1-hour post dosing but its duration of action is longer compared to MLN-519 [32]. Proteasome inhibition returned to baseline at 72 hours postdosing. Early pre-clinical studies showed toxicity with bortezomib was dose dependent and reversible. Bortezomib has also been shown to be effective in drug resistant MM cells and this important property is being further investigated [74]. This is consistent with preclinical findings that the response of MM cells to the drug is independent of their sensitivity to other chemotherapeutics. Nontoxic doses of bortezomib also sensitize several other cancer cells to chemotherapy in vitro and in animal models. It was suggested that the proapoptotic effect of proteasome inhibitors might be due to the observed stabilization of IκB resulting in NF-κB inhibition and the down-regulation of antiapoptotic NF-κB target genes [75]. Another observation was the down-regulation of Bcl-2 in pancreatic cancer cells treated with bortezomib, which would in itself probably be sufficient to induce apoptosis by misbalancing pro- and antiapoptotic proteins [76]. Accumulation of cell cycle inhibitors such as p21 and concomitant cell cycle arrest and apoptosis might also play a role [77]. Bortezomib like other proteasome inhibitors, e.g., lactacystin, also induce p53 accumulation, even though p53 does not seem to be crucial for sensitization of tumor cells [78]. Further clinical trials of bortezomib are ongoing for treatment of other cancers and in combination with other chemotherapeutic agents [79,80]. Peripheral neuropathy, thrombocytopenia and gastrointestinal disturbance have been the most common side effects associated with treatment (see Chapter 43). However, proteasome inhibition of up to 80% was well tolerated and maximal clinical dosing has been aimed to achieve this level of inhibition. There are only few recent preliminary experimental studies on the utility of bortezomib in ischemic stroke and cerebral ischemia (see Chapter 39). A single dose of bortezomib given 1 hr post-MCAo, resulted in a 40% decrease in infarct volume and a 38% decrease in neurological deficit in a rat permanent MCAo model [81]. The functional and histopathologic protection was accompanied by a 67% inhibition of whole blood proteasome activity, a level of inhibition which is commonly achieved in cancer patients in the clinic [81]. The potential neuroprotective effects of bortezomib were also evident in an embolic model of MCAo [82] and in combination with delayed thrombolytic therapy on a rat model of embolic focal cerebral ischemia [83]. Treatment with bortezomib reduced adverse cerebrovascular events including secondary thrombosis, inflammatory responses, and blood-brain barrier (BBB) disruption, and hence reduces infarct volume and neurological functional deficit when administrated within 4 h after stroke onset [83] Combination of bortezomib and rtPA extended the thrombolytic window for stroke to 6 h, which is associated with the improvement of vascular patency and integrity. Real time RT-PCR of endothelial cells isolated by laser-capture microdissection from brain tissue and Western blot analysis showed that bortezomib upregulated endothelial nitric oxide synthase (eNOS) expression and blocks NF-κB activation [83].
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1025
THE NF-ΚB-PROTEASOME PATHWAY AND OTHER INFLAMMATORY-MEDIATED CONDITIONS The effects of proteasome inhibition have also been investigated in other inflammatory conditions [84]. MLN-519 has been tested in an experimental mouse model of autoimmune encephalomyelitis [85]. This was shown to reduce relapses of the disease and a reduction in T-cell activation. This suggests that proteasome inhibitors may be beneficial in other neuroinflammatory conditions like multiple sclerosis. MLN-519 has also shown beneficial effects in a delayed-type hypersensitivity model of asthma [86]. Other common inflammatory conditions, in animal models of psoriasis and arthritis, have also shown significant improvement with proteasome inhibition [87,88]. MLN-519 has also been studied in animal models of myocardial injury and reperfusion [89]. Proteasome inhibition was shown to reduce polymorphonuclear leukocyte infiltration in a ischemia/reperfusion rat heart model. The study showed a reduction in the expression of CAM, P-selectin, and preservation of cardiac function. At 1 mg/kg, MLN-519 treated rats showed a final left ventricular developed pressure of 98±3% in an ischemia reperfusion model, compared to 52±8% in the control group. In a porcine model of myocardial reperfusion injury, MLN-519 was shown to inhibit the activation of NF-κB [90]. There was also a reduction in the release of the myocardial enzymes creatinine kinase MB and troponin I, and a significant reduction in myocardial infarct size. Myocardial function was also preserved. Proteasome inhibitors may therefore have a role in the treatment of vascular reperfusion injury and further research is ongoing [64,91]. The role of NF-κB in the pathophysiology of atherosclerosis is also being further researched [92].
THERAPEUTIC ASPECTS OF PROTEASOME INHIBITION IN NERVOUS SYSTEM DISEASES It is clear that the proteasome represents a central target for the processing and metabolism of multiple proteins whose critical roles in cellular function are being elucidated through the use of selective inhibitors. To avoid eliciting the significant side effects associated with complete inhibition of the proteasome and the possible immunosuppressive effects (with increased risk of infection and cancer) from persistent suppression of NF-κB activation, it is critical that we understand how to partially and temporally attenuate proteasome function to elicit the desired therapeutic effect. Taking into account the central role of the UPS in the biology of eukaryotic cells [2], proteasome inhibitors can be thought of as deadly toxins without any therapeutic value. Unexpectedly, they have been relatively well tolerated drugs because apparently most normal cells tolerate high levels of proteasome inhibition [5,93,94]. The availability of selective proteasome inhibitors such as MG-132 [95] and PSI [96] have boosted research of cellular effects of UPS inhibition. These effects included accumulation of ubiquitinated proteins within the cells [95,96], often in the form of an organized pericentrosomal aggregate called ‘aggresome’ [97,98], as well as blocking different stages of the cell cycle [98,99] and apoptosis [100,101]. While proteasome
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inhibition easily induced apoptosis in various cancer cell lines, primary cells such as diploid human fibroblasts are relatively resistant to the action of proteasome inhibitors, requiring much higher concentrations and longer incubation times [102,103]. Moreover, in terminally differentiated, resting cells low doses of proteasome inhibitors were cytoprotective, e.g. preventing apoptosis induced by nerve growth factor (NGF) deprivation in sympathetic neurons [104], serum deprivation of cerebellar granule cells [105], dehamethasone treatment of thymocytes [106] and IFNγ treatment of lens-derived αTN4-1 cells [107]. Even in some cancer cell lines proteasome inhibition was shown to be cytoprotective [108]. Nevertheless, higher doses of proteasome inhibitors and prolonged treatment inevitably induced cell death [104,106] (see Chapter 21). The effects of proteasome inhibitors can be divided into general, substrate-independent effects and specific, substrate-dependent effects. Specific effects of proteasome inhibition depend on the inhibition of degradation of specific substrates such as cyclins, IκBα, p53 etc. and have been discussed in the previous chapters of this book (see Chapters 7 and 19). Specific effects may be very different depending on the cell type involved: they may be either neutral, beneficial or detrimental to the organism as a whole. General effects of proteasome inhibition do not depend on the cell type and the kind of substrate involved. They result from the accumulation of ubiquitinated proteins, which occurs even after moderate inhibition of the proteasome (see Chapter 12). However, the cell has developed mechanisms to defend against misfolded and aggregated proteins. The first line of defense involves the many molecular chaperones that aid in the normal folding and also refolding of abnormal conformations back into the native state. If this fails, abnormal proteins can be targeted for degradation by covalent attachment of polyubiquitin followed by targeting to the proteasome and degradation [109,110]. The presence of ubiquitin, chaperones and proteasome components in inclusions presumably represents cellular defenses overwhelmed by the excessive aggregation within cells. Even the inclusions themselves are the outcome of an active process by which the cell collects irreversibly aggregated protein, translocates it to an ‘aggresome’ near the nucleus by active transport and attempts to eliminate it, probably by autophagic or other lysosomal-like processes [97,111]. Accumulation of unfolded proteins in the cytosol induces heat shock response characterized by overexpression of cytosolic chaperones such as the heat shock proteins (Hsp), Hsp70 or Hsp90, while accumulation of unfolded proteins in the endoplasmic reticulum secondary to the inhibition of ERAD (ERassociate degradation) induces unfolded protein response [112,113] (see Chapters 11 and 13). Ubiquitinated proteins are not dispersed randomly in the cytosol and nucleus but instead tend to accumulate into discrete subcellular domains, which may correspond to regions of increased protein turnover or ‘proteolytic centers’ [98,115]. In the cytoplasm ubiquitinated proteins coalesce into a single aggregate around the centrosome by a centripete microtubuledependent transport [98]. This central aggregate, sometimes refered to as the ‘aggresome’ recruits proteasomes and other components of the UPS further impairing its function [97,116]. At the same time ubiquitinated proteins in the nuclei coalesce into discrete subnuclear domains called PML bodies [117,118]. It is a matter of debate whether the formation of these cytoplasmic and nuclear inclusions are deleterious to the cells or not [97,119]. However, it is possible to dissociate the proapoptotic effects of proteasome inhibitors from their effects on ‘aggresome’ formation suggesting that those aggregates are
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1027 cytoprotective, recruiting unfolded proteins which may have otherwise wreaked havoc within the cells [120,121]. Moreover, ‘aggresomes’ are often viewed as models of aggregates occurring in human neurons in several neurodegenerative disorders including AD and PD. It is often argued, that neurons which survive do have those aggregates which protected them from cell death, while cells which died probably failed to do so [97,119]. Potential adverse effects of proteasome inhibitors reported from the bedside may stem from either the specific or general effects. Increasing number of patients are receiving treatment with proteasome inhibitors without serious adverse reactions being reported. Most experience is coming from the use of Velcade®. The most frequent adverse effects occurring in ~30% of patients involved weakness, nausea, diarrhoea, decreased appetite, constipation, thrombocytopenia, peripheral neuropathy, pyrexia, vomiting and anemia. These effects were mostly reversible after discontinuation of therapy and/or manageable with secondary treatments. Two cases of death may have been possibly related to the use of proteasome inhibitors [32,122]. The Phase I trial of MLN-519, a different proteasome inhibitor also showed no major adverse effects at all at doses which corresponded to the desired therapeutic values. Minor effects included irritation at the site of injection, altered taste sensation, headache and flu-like syndrome [62]. Another set of clinical data comes from the use of the HIV protease inhibitor Ritonavir in AIDS patients, because Ritonavir is also a bona fide proteasome inhibitor [123]. Compared to Velcade® and MLN-519, Ritonavir is administered continuously over prolonged periods of time eliciting therefore effects of a chronic impairment of proteasome function. The most common adverse effects observed in almost 30% of patients involved nausea and vomiting [124]. Hyperlipidemia induced by chronic Ritonavir administration has been linked to increased hepatic lipoprotein production, caused by the prevention of proteasome-mediated degradation of apoB and activated sterol regulatory element binding proteins in the liver [125]. It is likely that prolonged administration of either Velcade® or MLN-519 will lead to similar effects. All reported adverse effects seem to stem from the specific inhibition of degradation of particular proteins. Prolonged exposure of neural cells in culture to very low concentrations of proteasome inhibitors clearly affects the profile of gene expression therefore a similar situation is very likely to happen in vivo [126]. It is clear, that the current experience with proteasome inhibitors in clinical settings is very encouraging, since they seem to be well tolerated drugs with manageable side effects [127]. Does the accumulation of ubiquitinated proteins around the centrosomes and in the nuclei of cells throughout the entire body somehow affect the function of our cells? The most serious possible complication of inhibiting proteasomes may involve the central nervous system since ‘aggresome’ formation has been postulated as a model for neurodegenerative disorders [97,119]. Neither MLN-519 nor Velcade® efficiently penetrate the brain-blood bareer (BBB) [32,62]. Penetration of Ritonavir through the BBB is considerable, however its levels in the central nervous system are too low to efficiently control HIV infection in AIDS-related dementia [128,129]. Since patients treated with Ritonavir do often develop AIDS-related dementia it is difficult to assess whether the reported adverse neurological effects of Ritonavir are directly related to the drug or to the underlaying disease. However, Ritonavir
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induces significantly more adverse reactions than other HIV protease inhibitors which do not inhibit the proteasome [124]. Other proteasome inhibitors that penetrate more efficiently the BBB − such as PSI or epoxomicin − cause the key features of PD after prolonged exposure [130] although showed protective effects on DA cell death in the rat PD model using 6-hydroxyl dopamine (6OHDA) [131]. This implies that the relationship between proteasome inhibitors, UPS and the pathogenesis of neurodegenerative disorders may be more complex than we thought. (For details, see Chapters 23, 25 and 28). Short courses of treatment with proteasome inhibitors such as the one pursued in stroke patients bears relatively few and mild adverse reactions versus a high possible benefit. Even prolonged treatments such as treatment of HIV and cancer patients is relatively safe, taking into account the seriousness of the primary disease and the life expectancy. The problems of long-term adverse reactions will certainly surface when a population of patients treated with proteasome inhibitors survive for several years. They will require monitoring to detect any possible sign of increased incidence of neurodegenerative disorders or any other effects which may have been caused by the widespread accumulation of ubiquitinated proteins within cells of different tissues. Perhaps focusing on specific aspects of the UPS may provide more promise for neuroprotective efficacy rather than the simpler and less specific proteasome inhibition. The interaction of these events is complex, and the outcome of therapeutic interventions aimed at these elements of cellular injury is uncertain without more rational and specific targeting of these mechanisms and knowledge of the underlying state of the organism with respect to these factors.
CONCLUSION With the recent licensing of Bortezomib, the first proteasome inhibitor in clinical use, the ubiquitin–proteasome pathway has become an important target for therapeutic drug research. Important research areas include inflammatory conditions, vascular disease, HIV and cancer treatments. The main focus of therapeutic targeting in neurological disease has been in the neuroinflammation associated with acute stroke [8,33]. Both, Bortezomib and MLN-519 have shown encouraging results in rat models of acute ischemic stroke, with reduced infarct size and improved neurological outcome. Clinical evaluation of these proteasome inhibitors is ongoing and further safety data are required before these drugs can be licensed for the treatment of acute stroke. In vitro studies of proteasome inhibitors have shown that disruption in the balance of this important regulatory pathway can lead to different pathophysiological disease processes. Chronic dysfunction of the UPS has been associated with neurodegenerative conditions like PD and AD. The UPS also plays an important role in regulation of cell growth and gene expression [26]. The action of NF-κB must also be taken into account as disruption of its function could be detrimental to the body’s immune response [132]. This broad function of the UPS must be thoroughly assessed before the licensing of proteasome inhibitors for clinical use.
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1029 The development of proteasome inhibitors has been an exciting new area of molecular and translational medicine. The elucidation of these molecular pathways has provided us with many new therapeutic drug targets, which will hopefully aid us in the development of specific drugs for the treatment of neurological disease associated with the UPS.
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[79] Hamilton AL, Eder JP, Pavlick AC, Clark JW, Liebes L, Garcia-Carbonero R, Chachoua A, Ryan DP, Soma V, Farrell K, Kinchla N, Boyden J, Yee H, ZeleniuchJacquotte A, Wright J, Elliott P, Adams J, Muggia FM: Proteasome Inhibition With Bortezomib (PS-341): A Phase I Study With Pharmacodynamic End Points Using a Day 1 and Day 4 Schedule in a 14-Day Cycle. J Clin Oncol 2005, 23:6107-6116. [80] Ma MH, Yang HH, Parker K, Manyak S, Friedman JM, Altamirano C, Wu ZQ, Borad MJ, Frantzen M, Roussos E, Neeser J, Mikail A, Adams J, Sjak-Shie N, Vescio RA, Berenson JR: The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents. Clin Cancer Res 2003, 9:1136-1144. [81] Stagliano NE, Ren JM, Riodan W, Finklestein SP: Velcade®, a first-in-class proteasome inhibitor, is efficacious in rat permanent middle cerebral artery occlusion. J Cereb Blood Flow Metab 2005, 25:S38. [82] Henninger N, Sicard KM, Bouley J, Fisher M, Stagliano NE: The proteasome inhibitor VELCADE® reduces infarction in rat models of focal cerebral ischemia. Neurosci Lett 2006, 398:300-305. [83] Zhang L, Zhang ZG, Liu X, Hozeska A, Stagliano N, Riordan W, Lu M, Chopp M: Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator. Thromb Haemost 2006, 95:166-173. [84] Elliott PJ, Ross JS: The proteasome: a new target for novel drug therapies. Am J Clin Pathol 2001, 116:637-646. [85] Vanderlugt CL, Rahbe SM, Elliott PJ, Dal Canto MC, Miller SD: Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519. J Autoimmun 2000, 14:205-211. [86] Elliott PJ, Pien CS, McCormack TA, Chapman ID, Adams J: Proteasome inhibition: A novel mechanism to combat asthma. J Allergy Clin Immunol 1999, 104:294-300. [87] Palombella VJ, Conner EM, Fuseler JW, Destree A, Davis JM, Laroux FS, Wolf RE, Huang J, Brand S, Elliott PJ, Lazarus D, McCormack T, Parent L, Stein R, Adams J, Grisham MB: Role of the proteasome and NF-kappaB in streptococcal cell wallinduced polyarthritis. Proc Natl Acad Sci U S A 1998, 95:15671-15676. [88] Zollner TM, Podda M, Pien C, Elliott PJ, Kaufmann R, Boehncke WH: Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model. J Clin Invest 2002, 109:671-679. [89] Campbell B, Adams J, Shin YK, Lefer AM: Cardioprotective effects of a novel proteasome inhibitor following ischemia and reperfusion in the isolated perfused rat heart. J Mol Cell Cardiol 1999, 31:467-476. [90] Pye J, Ardeshirpour F, McCain A, Bellinger DA, Merricks E, Adams J, Elliott PJ, Pien C, Fischer TH, Baldwin AS, Jr., Nichols TC: Proteasome inhibition ablates activation of NF-kappa B in myocardial reperfusion and reduces reperfusion injury. Am J Physiol Heart Circ Physiol 2003, 284:H919-H926. [91] Meiners S, Laule M, Rother W, Guenther C, Prauka I, Muschick P, Baumann G, Kloetzel PM, Stangl K: Ubiquitin-Proteasome Pathway as a New Target for the Prevention of Restenosis. Circulation 2002, 105:483-489.
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1035 [92] de Winther MP, Kanters E, Kraal G, Hofker MH: Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol 2005, 25:904-914. [93] Golab J: Proteasome inhibitors in the treatment of cancer. Drug Discov Today 2003, 8:575. [94] Adams J: Potential for proteasome inhibition in the treatment of cancer. Drug Discov Today 2003, 8:307-315. [95] Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL: Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 1994, 78:761-71. [96] Figueiredo-Pereira ME, Berg KA, Wilk S: A new inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell. J Neurochem 1994, 63:1578-1581. [97] Kopito RR: Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 2000, 10:524-530. [98] Wojcik C, Schroeter D, Wilk S, Lamprecht J, Paweletz N: Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. Eur J Cell Biol 1996, 71:311-318. [99] Machiels BM, Henfling ME, Gerards WL, Broers JL, Bloemendal H, Ramaekers FC, Schutte B: Detailed analysis of cell cycle kinetics upon proteasome inhibition. Cytometry 1997, 28:243-252. [100] Wojcik C, Stoklosa T, Giermasz A, Golab J, Zagozdzon R, Kawiak J, Wilk S, Komar A, Kaca A, Malejczyk J, Jakobisiak M: Apoptosis induced in L1210 leukaemia cells by an inhibitor of the chymotrypsin-like activity of the proteasome. Apoptosis 1997, 2:455-462. [101] Drexler HC: Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci 1997, 94:855-60. [102] Orlowski RZ, Eswara JR, Lafond-Walker A, Grever MR, Orlowski M, Dang CV: Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor. Cancer Res 1998, 58:4342-8. [103] Pleban E, Bury M, Mlynarczuk I, Wojcik C: Effects of proteasome inhibitor PSI on neoplastic and non-transformed cell lines. Folia Histochem Cytobiol 2001, 39:133-134. [104] Sadoul R, Fernandez PA, Quiquerez AL, Martinou I, Maki M, Schroter M, Becherer JD, Irmler M, Tschopp J, Martinou JC: Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J 1996, 15:384552. [105] Atlante A, Bobba A, Calissano P, Passarella S, Marra E: The apoptosis/necrosis transition in cerebellar granule cells depends on the mutual relationship of the antioxidant and the proteolytic systems which regulate ROS production and cytochrome c release en route to death. J Neurochem 2003, 84:960-971. [106] Grimm LM, Goldberg AL, Poirier GG, Schwartz LM, Osborne BA: Proteasomes play an essential role in thymocyte apoptosis. EMBO J 1996, 15:3835-3844.
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[107] Awasthi N, Wagner BJ: Interferon-gamma induces apoptosis of lens alphaTN4-1 cells and proteasome inhibition has an antiapoptotic effect. Invest Ophthalmol Vis Sci 2004, 45:222-229. [108] Nawrocki ST, Sweeney-Gotsch B, Takamori R, McConkey DJ: The proteasome inhibitor bortezomib enhances the activity of docetaxel in orthotopic human pancreatic tumor xenografts. Mol Cancer Ther 2004, 3:59-70. [109] Ciechanover A, Brundin P: The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 2003, 40:427-446. [110] Goldberg AL: Protein degradation and protection against misfolded or damaged proteins. Nature 2003, 426:895-899. [111] Ravikumar B, Rubinsztein DC: Can autophagy protect against neurodegeneration caused by aggregate-prone proteins? Neuroreport 2004, 15:2443-2445. [112] Lee DH, Goldberg AL: Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Mol Cell Biol 1998, 18:30-38. [113] Kawazoe Y, Nakai A, Tanabe M, Nagata K: Proteasome inhibition leads to the activation of all members of the heat-shock-factor family. Eur J Biochem 1998, 255:356-362. [114] McCracken AA, Brodsky JL: A molecular portrait of the response to unfolded proteins. Genome Biol 2000, 1:REVIEWS1013. [115] Wojcik C: On the spatial organization of ubiquitin-dependent proteolysis in HeLa cells. Folia Histochem Cytobiol 1997, 35:117-118. [116] Bence NF, Sampat RM, Kopito RR: Impairment of the ubiquitin-proteasome system by protein aggregation. Science 2001, 292:1552-1555. [117] Baumann CT, Ma H, Wolford R, Reyes JC, Maruvada P, Lim C, Yen PM, Stallcup MR, Hager GL: The glucocorticoid receptor interacting protein 1 (GRIP1) localizes in discrete nuclear foci that associate with ND10 bodies and are enriched in components of the 26S proteasome. Mol Endocrinol 2001, 15:485-500. [118] Wojcik C, DeMartino GN: Intracellular localization of proteasomes. Int J Biochem Cell Biol 2003, 35:579-589. [119] Sherman MY, Goldberg AL: Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron 2001, 29:15-32. [120] Rideout HJ, Wang Q, Park DS, Stefanis L: Cyclin-dependent kinase activity is required for apoptotic death but not inclusion formation in cortical neurons after proteasomal inhibition. J Neurosci 2003, 23:1237-1245. [121] Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM: Aggresomes Formed by {alpha}-Synuclein and Synphilin-1 Are Cytoprotective. J Biol Chem 2004, 279:4625-4631. [122] Richardson P: Clinical update: proteasome inhibitors in hematologic malignancies. Cancer Treat Rev 2003, 29 Suppl 1:33-39. [123] Andre P, Groettrup M, Klenerman P, de Giuli R, Booth BL, Jr., Cerundolo V, Bonneville M, Jotereau F, Zinkernagel RM, Lotteau V: An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci U S A 1998, 95:13120-13124.
Clinical Experience of Proteasome Inhibitors in Central Nervous System Diseases 1037 [124] Bonfanti P, Valsecchi L, Parazzini F, Carradori S, Pusterla L, Fortuna P, Timillero L, Alessi F, Ghiselli G, Gabbuti A, Di Cintio E, Martinelli C, Faggion I, Landonio S, Quirino T: Incidence of adverse reactions in HIV patients treated with protease inhibitors: a cohort study. Coordinamento Italiano Studio Allergia e Infezione da HIV (CISAI) Group. J Acquir Immune Defic Syndr 2000, 23:236-245. [125] Riddle TM, Schildmeyer NM, Phan C, Fichtenbaum CJ, Hui DY: The HIV protease inhibitor ritonavir increases lipoprotein production and has no effect on lipoprotein clearance in mice. J Lipid Res 2002, 43:1458-1463. [126] Ding Q, Bruce-Keller AJ, Chen Q, Keller JN: Analysis of gene expression in neural cells subject to chronic proteasome inhibition. Free Radic Biol Med 2004, 36:445-455. [127] Adams J: The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004, 4:349-360. [128] Anthonypillai C, Sanderson RN, Gibbs JE, Thomas SA: The distribution of the HIV protease inhibitor, ritonavir, to the brain, cerebrospinal fluid, and choroid plexuses of the guinea pig. J Pharmacol Exp Ther 2004, 308:912-920. [129] van dS, I, Vos CM, Nabulsi L, Blom-Roosemalen MC, Voorwinden HH, de Boer AG, Breimer DD: Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood--brain barrier. AIDS 2001, 15:483-491. [130] McNaught KS, Perl DP, Brownell AL, Olanow CW: Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease. Ann Neurol 2004, 56:149162. [131] Setsuie R, Kabuta T, Wada K: Does proteosome inhibition decrease or accelerate toxininduced dopaminergic neurodegeneration? J Pharmacol Sci 2005, 97:457-460. [132] Karin M, Lin A: NF-kappaB at the crossroads of life and death. Nat Immunol 2002, 3:221-227.
In: The Ubiquitin Proteasome System… ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wójcik, pp. 1039-1056© 2007 Nova Science Publishers, Inc.
Chapter 43
CLINICAL APPLICATION OF PROTEASOME INHIBITOR BORTEZOMIB: CHARACTERIZATION OF NEUROTOXICITY Jens Voortman∗ and Giuseppe Giaccone Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
ABSTRACT Bortezomib (Velcade®) is a dipeptide boronic acid proteasome inhibitor that specifically targets the chymotryptic-like proteolytic activity of the 20S proteasome. It has shown great potential as a novel anti-cancer agent and has been approved for the treatment of multiple myeloma. Therapeutic development as a single agent or in combination with other agents is ongoing in hematological malignancies as well as in various solid tumor types. Furthermore, application of proteasome inhibition therapy in other areas of disease is being explored, such as prevention of reperfusion injury following acute ischemic stroke and management of chronic inflammatory diseases. Bortezomib is generally well tolerated. However, one of the most serious overall as well as dose limiting toxicities has been peripheral neuropathy. Bortezomib-induced peripheral neuropathy constitutes a length-dependent, sensory rather than motor, axonal, small rather than large fiber, polyneuropathy. In agreement with its small fiber neuropathy characteristics, neuropathic pain and symptoms of autonomic dysfunction have also been frequently reported upon bortezomib treatment. Risk factors for bortezomib neurotoxicity include pre-existent neuropathy and prior treatment with neurotoxic (anti-cancer) agents. Additionally, individual susceptibility, and not only cumulative dose, is of great importance. After discontinuing bortezomib therapy, neuropathy resolves in approximately half of the patients. Nevertheless, in severe cases, ∗
Correspondence concerning this article should be addressed to Dr. Jens Voortman, MD; Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: +31 (0)20 4444 300; Fax: +31 (0)20 4444 355; E-mail:
[email protected].
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pharmacologic management of (autonomic) neuropathy and neuropathic pain is required up to several months after discontinuation of bortezomib. The exact biological mechanism of peripheral neuropathy induced by systemic proteasome inhibition therapy still has to be elucidated. Detrimental effects of proteasome inhibition on nerve terminal protein homeostasis as well as myelin production by Schwann cells might explain the high incidence of neurotoxicity in bortezomib-treated patients.
Keywords: bortezomib, neurotoxicity syndromes, peripheral nervous system diseases, proteasome endopeptidase complex, adverse effects, antineoplastic agents,
ABBREVIATIONS APEX, assessment of proteasome inhibition for extending remissions; AUC, area under the curve; Cmax, maximum plasma concentration; CMAP, compound muscle action potential; CMTX1A, X-linked Charcot-Marie-Tooth disease 1A; CNS, central nervous system; CREST, clinical response and efficacy study of bortezomib in the treatment of relapsing multiple myeloma; CTCAE, common terminology criteria for adverse events; d, day; DLT, dose limiting toxicity; FDA, food and drug administration; GR, grade; IENF, intra-epidermal nerve fiber density; MM, multiple myeloma; MTD, maximum tolerated dose; n, number (of patients); NCS, nerve conduction studies; NF-κB, nuclear factor kappa B; PBMC, peripheral blood mononuclear cell; pegLD, pegylated liposomal doxorubicin; 20S PI, 20S proteasome inhibition; PK, pharmacokinetic; PMP22, peripheral myelin protein 22; PN, peripheral neuropathy; SNAP, sensory nerve action potential; q, every; SUMMIT, study of uncontrolled multiple myeloma managed with proteasome inhibition therapy; TNS, total neuropathy score; UPS, ubiquitin-proteasome system.
INTRODUCTION Growing consciousness of the pivotal role of the proteasome in normal cell physiology as well as in human disease propelled the development of proteasome inhibitors for research and therapeutic applications [1-3]. So far two proteasome inhibitors, MLN-519 and bortezomib, have been evaluated clinically [4]. Bortezomib (Velcade®, formerly known as PS-341, LDP-341 and MLN-341) has been dominating the field of proteasome inhibition therapy, with over a hundred clinical studies conducted or ongoing and currently approved for the treatment of multiple myeloma (MM) patients. So far, only one clinical study in healthy volunteers has been published with the lactacystin β-lactone-derived proteasome inhibitor MLN-519 [5]. Boronate proteasome inhibitors, such as bortezomib, are characterized by a pharmacophore containing a functional boronic acid group. They display remarkable selectivity towards the proteasome. Reversible binding to the chymotrypsin-like proteolytic activity, localized within the β5 subunit of the 20S core of the proteasome, results in a temporary inhibition of intracellular proteasome activity [6]. In in vitro studies, bortezomib emerged as a promising novel anti-cancer agent,
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demonstrating unique anti-tumor properties [7]. Bortezomib, and proteasome inhibitors in general, show a striking selectivity in cytotoxicity towards malignant cells compared to normal cells [8-11]. In early clinical experience, exceptional results were obtained in the treatment of patients with relapsing MM, who were refractory to conventional therapies [12]. The Clinical Response and Efficacy Study of Bortezomib in the Treatment of Relapsing Multiple Myeloma (CREST) study, a phase 2 study in heavily pretreated MM patients, showed response rates of 30% and 38% in patients treated with twice-weekly bortezomib dosed at 1.0 and 1.3 mg/m2 respectively [13]. In 2003, on the basis of this and another comparable, larger phase 2 study, the Study of Uncontrolled Multiple Myeloma Managed with Proteasome Inhibition Therapy (SUMMIT), bortezomib received fast-track FDA approval for treatment of patients with relapsed refractory MM [14]. Additional indications for treatment with bortezomib are likely to follow considering the observed efficacy in other hematological malignancies such as mantle cell lymphoma and Waldenström macroglobulinemia as well as in several solid tumor types [15-17]. Preclinical studies attribute a critical part of bortezomib’s activity in hematological malignancies to the inhibitory effect of proteasome inhibition on activation of transcription factor nuclear factor kappa B (NF-κB) [8,18,19]. It is therefore not a surprising development that systemic proteasome inhibition therapy is expanding to other areas of disease characterized by pathologic activation of NF-κB mediated signaling, such as chronic inflammatory diseases and reperfusion injury following acute ischemic stroke [20-22].
BORTEZOMIB PHARMACOLOGY Pharmacokinetic Profile Bortezomib is administered as a short intravenous injection. The bortezomib plasma profile is best described by a two-compartment pharmacokinetic (PK) model with a rapid initial distribution half-life (t1/2α: 0.22 to 0.46 hours), followed by a longer terminal elimination phase (t1/2β: > 10 days) [23]. Though in general plasma PK profiles are consistent among patients, maximum plasma concentration (Cmax) values did not show an apparent relationship to dose and varied significantly among individuals [23]. Animal studies, using radioactively labeled bortezomib, showed rapid distribution into most tissues with the exception of brain, testis and some parts of eye and optic nerve [24]. In humans, over 30 metabolites, none pharmacologically active, were identified [25].
Pharmacodynamic Profile As detection of bortezomib in serum proved to be difficult by its rapid removal from the vascular compartment, early-on in the clinical development of bortezomib a pharmacodynamic assay was developed to monitor the degree of 20S proteasome inhibition (20S PI) in peripheral blood mononuclear cells (PBMCs) [26]. It was demonstrated that 20S
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PI in peripheral PBMCs was related both to the dose of bortezomib in milligrams per square meter, and to the total dose of bortezomib in milligrams. However, at higher doses of bortezomib, 20S PI is not an ideal surrogate for the bortezomib serum level as it reaches a plateau at around 60-75% [23]. Peak proteasome inhibition is reached one hour post injection, and proteasome activity in PBMCs typically restores to baseline levels 72 hours after injection of bortezomib. Administration of bortezomib is therefore bound to a maximum frequency of twice per week, allowing proteasome activity to restore before the next dose is administered [12]. It must be noted that proteasome expression levels and subtype distribution differ among cell types, and even within single cells the relative proportion of proteasome-subtypes, such as immunoproteasomes, is subject to change [27,28]. Furthermore, proteasome activity is different in normal cells compared to malignant cells [11]. Thus far comparative analysis of the level of proteasome inhibition in PBMCs and simultaneously obtained tumor samples were conducted in only a handful of patients. A comparable outcome for the degree of 20S PI in PBMCs and tumor samples was found [23,29]. However, 20S PI in the bone marrow was shown to be around half of that observed in the matched whole blood sample. There are no indications that tolerance or tachyphylaxis develop upon repeated doses of bortezomib [23,29].
INCIDENCE OF BORTEZOMIB-INDUCED NEUROPATHY Phase 1 Studies Four different dosing schedules of bortezomib were assessed in seven phase 1 studies, which served to determine safety, and the maximum tolerated dose of bortezomib in each schedule. Six studies were conducted in adult patients and one in pediatric patients (see Table 1). The first study in humans evaluated a weekly schedule of bortezomib on days 1, 8, 15, and 22 of a 35-day cycle. In following phase 1 studies, more dose-intense twice-weekly schedules were assessed: days 1 and 4 of a 14 day cycle; days 1, 4, 8 and 11 of a 21 day cycle; days 1, 4, 8, 11, 15, 18, 22 and 25 of a 42 day cycle [12,30-33]. Patients receiving twice-weekly bortezomib had their therapy most commonly interrupted during the third week of treatment, the major toxicity being malaise and fatigue [12,32]. Therefore twice-weekly bortezomib for two weeks followed by the third week off, days 1, 4, 8 and 11 of a 21-day cycle, became the standard regimen. Sensory neuropathy was among the most commonly observed dose limiting toxicities, next to thrombocytopenia, diarrhea, fatigue and (orthostatic) hypotension. Occurrence of severe peripheral neuropathy did not allow further dose-escalation of bortezomib in three out of seven dose finding studies [29,30,32].
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Table 1. Incidence of peripheral neuropathy in phase 1 dose finding studies with bortezomib DLT (mg/m2)
Neurotoxicity
2.0 diarrhea hypotension 1.75 + 1.90 sensory neuropathy fatigue diarrhea 1.56 sensory neuropathy diarrhea I: 1.50 + 1.70 trombocytopenia sensory neuropathy
Overall, any grade GR3: 0.13-1.8 mg/m2 2.0 mg/m2 Overall, any grade: GR3: 0.25-1.45 mg/m2 1.75 mg/m2 1.90 mg/m2
8% 0% 20% 28% 0% 8% 29%
Overall, any grade: GR3: 0.13-1.08 mg/m2 1.30 mg/m2 1.56 mg/m2 I: Overall, any grade GR3: 0.50-1.25 mg/m2 1.50-1.70 mg/m2
12% 0% 33% 17% 18% 0% 6%
II:1.50
I: 1.60 trombocytopenia
II: Overall, any grade: GR3:
19% 0%
1.04
1.20 + 1.38 trombocytopenia fatigue and malaise hyponatriemia hypokalemia 1.50 orth. hypotension nausea diarrhea hypokalemia fluid retention 1.60 trombocytopenia
Overall, any grade: GR3:
19% 0%
Overall, any grade: GR3:
13% 0%
Grade 1 >GR1
7% 0%
Studies
No.
Malignancy
Schema
Papandreou (2004)
53
advanced solid tumors
Hamilton (2005)
40
advanced solid tumors/ lymphomas
d1, 8, 15, 22 q35d d1, 4 q14d
Aghajanian (2002)
42
advanced solid tumors
d1, 4, 8, 11 q21d
1.56
Dy (2005)
I:28
advanced solid tumors/ lymphomas/ multiple myeloma
I: d1, 4, 8, 11, 15, 18, 22, 25 q42d
I: 1.50
II: d1, 4, 8, 11 q21d d1, 4, 8, 11, 15, 18, 22, 25 q42d
II:16
MTD (mg/m2) 1.60
1.75
Orlowski (2002)
27
refractory hematologic malignancies
Cortes (2004)
refractory/ relapsed acute leukemias
d1, 4, 8, 11, 15, 18, 22, 25 q42d
1.25
15
recurrent/ refractory solid tumors (pediatric patients)
d1, 4, 8, 11 q21d
1.20
Blaney (2004)
15
n indicates number of patients; d, day; q, every; MTD, maximum tolerated dose; DLT, dose limiting toxicity; GR, grade.
In clinical studies, sensory or motor neuropathy is graded according to the Common Terminology Criteria for Adverse Events (CTCAE) (see Table 2) [34]. Grade 3 neuropathy, which is generally regarded as dose limiting, implies function is affected, e.g. of the hand or foot, however there is no interference with ‘activities of daily living’ (self-care). Overall incidence of drug-related neuropathy, all grades, in phase 1 studies in adult patients varied from 8% to 28% with a clear rise in incidence and severity at higher dose and higher dose-
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intensity of bortezomib [12,23,29,30,32,33]. In the weekly schedule overall incidence of peripheral neuropathy was relatively low at 8% compared to 12 to 28% in the more doseintense twice-weekly schedules of bortezomib. It was furthermore observed pre-existing neuropathy and prior neurotoxic treatment predisposes to bortezomib-induced neurotoxicity [30]. In a phase 1 study in pediatric patients a twice-weekly schedule of bortezomib was evaluated, at 1.2 or 1.6 mg/m2 per dose [31]. Levels of 20S PI were comparable to those found in adults. No dose-limiting peripheral neuropathy was observed in pediatric patients and hardly any neurotoxicity was observed in this small study: only 1 out of 15 patients experienced a grade 1 neuropathy. An explanation for this could be that more than 80% of patients received just one cycle of therapy. Furthermore 10 out of 15 patients were treated at 1.2 mg/m2, which is lower than the adult MTD. Age-related differences in proteasome activity in children compared to adults might also play a role [35,36]. Table 2. Grading of neuropathy and neuropathic pain according to the Common Terminology Criteria for Adverse Events (CTCAE)
Adverse Event
0
neuropathymotor
normal
neuropathysensory
normal
neuropathic pain
none
Grade 2 NEUROLOGY asymptomatic, symptomatic weakness on exam/ weakness testing only interfering with function, but not interfering with activities of daily living asymptomatic; loss sensory loss of deep tendon alteration or reflexes or paresthesia paresthesia (including tingling), (including tingling) interfering with but not interfering function, but not with function interfering with activities of daily living PAIN mild pain not moderate pain: pain interfering with or analgesics function interfering with function, but not interfering with activities of daily living 1
3
4
weakness interfering with activities of daily living; bracing or assistance to walk
life-threatening; disabling e.g. paralysis
sensory alteration or paresthesia interfering with activities of daily living
disabling
severe pain: pain or analgesics severely interfering with activities of daily living
disabling
Phase 2 and 3 Studies In phase 2 and 3 studies, which served to assess efficacy of bortezomib treatment in MM patients as well as several other hematological malignancies and solid tumor types, peripheral
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neuropathy was among the most frequently observed toxicities necessitating dose reductions or discontinuation of treatment. In the previously mentioned SUMMIT and CREST phase 2 studies, a total number of 256 patients with relapsed and/ or refractory MM were treated with 1.0 or 1.3 mg/m2 bortezomib twice-weekly. Peripheral neuropathy was reported in 35% of patients whereby the number of events per 100 patient doses steadily increased through the first 5 cycles, peaking at 5.3% at cycle 5, and steadily decreased thereafter. In the SUMMIT study (202 patients), cumulative dose-related peripheral neuropathy was considered ‘the most clinically significant adverse event’. Dose reductions for peripheral neuropathy were necessary in 12% of patients and 5% of patients discontinued treatment due to peripheral neuropathy. Especially patients with pre-existent neuropathy and/ or prior exposure to neurotoxic anti-cancer agents, such as taxanes and platinum compounds, are more prone to develop high-grade neuropathy [35]. The incidence of treatment-emergent grade 3 neuropathy was 16% in patients who had peripheral neuropathy prior to bortezomib treatment compared to 3% in patients without prior peripheral neuropathy [13,14,37]. In the largest study with bortezomib conducted to date, the Assessment of Proteasome Inhibition for Extending Remissions (APEX) phase 3 study, 669 patients with relapsed MM were randomized to receive either high-dose dexamethasone (336 patients) or twice-weekly bortezomib (333 patients) at 1.3 mg/m2 [38]. Regarding neurotoxicity, incidence was comparable to that observed in the SUMMIT and CREST studies. In the bortezomib arm, overall incidence of peripheral neuropathy was 36%. Peripheral neuropathy necessitated early discontinuation of treatment in 8% of patients, making it the predominant reason for treatment discontinuation. Incidence of grade 3 and 4 neuropathy was 7% and 1% respectively. In the dexamethasone arm overall incidence of peripheral neuropathy was 9%, grade 3 and 4 incidence was below 1%. Furthermore, in the bortezomib arm paresthesias were reported in 21% of patients, grade 3 in 2% of patients, compared to 8% and 0% respectively in the dexamethasone arm. In an extension study of the SUMMIT and CREST studies, allowing patients who benefited from the treatment to continue bortezomib treatment beyond the initial eight threeweek cycles, only in 14% of patients new or worsening of peripheral neuropathy was observed, compared to 30% when these same patients participated in the original SUMMIT or CREST studies [39]. An important consequence of this observation could be that maintenance therapy with bortezomib might be possible in certain patients not susceptible for its neurotoxic side effects. It is important to realize in this regard that bortezomib is not only being developed for treatment of malignant neoplastic diseases but also in chronic inflammatory diseases [20]. In phase 1 studies, a higher maximum tolerated dose was established in solid tumor patients compared to patients with hematological malignancies, 1.5 mg/m2 and 1.3 mg/m2 respectively. In part this was due to a greater bone marrow reserve in solid tumor patients, resulting in less pronounced bortezomib-induced thrombocytopenia. So far, the sizes of published studies in solid tumor patients were relatively small, varying from 16 to 37 patients. Striking is the great variability in incidence of peripheral neuropathy between different studies in solid tumor patients. A study in patients with metastatic neuro-endocrine tumors
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who did not have any prior chemotherapy, showed the highest overall and grade 3 incidence at 63% and 37% respectively. Other studies with a similar or higher dose-intensity report overall incidences of 21% to 48% and grade 3 incidences of 3% to 15% [40-46]. Larger studies in solid tumor patients will have to show whether or not overall and treatment-emergent neurotoxicity is comparable to what has been observed in the large studies conducted in MM patients. A comparison will remain difficult, as most hematological patients included in published studies with bortezomib were generally more exposed to prior neurotoxic treatments than solid tumor patients.
Combination Studies As inhibition of proteasome activity is increasingly considered as a rational target for chemosensitization, bortezomib is being combined with other chemotherapeutic agents in many studies [47]. However, an undesirable effect of combining proteasome inhibition and chemotherapy could be potentiation of neurotoxicity, especially when bortezomib is combined with other neurotoxic agents. A randomized phase 2 study in advanced non-small cell lung cancer patients, who had received one prior chemotherapy regimen, compared bortezomib alone to the combination of docetaxel and bortezomib. More grade 3 neuropathy was observed in the bortezomib alone arm than in the combination arm, 15% vs. 5% respectively. It must be noted that bortezomib was dosed higher at 1.5 mg/m2 in the bortezomib-alone arm vs. 1.3 mg/m2 in the combination arm [48]. In a dose finding study combining bortezomib with pegylated liposomal doxorubicin (pegLD) in 42 patients with hematological malignancies, the overall incidence of neuropathy was 55% [49]. Grade 3 incidence was 6%, 17% and 33% at doses of 1.30 mg/m2, 1.40 mg/m2 and 1.50 mg/m2, respectively. The dose of pegLD was kept constant at 30 mg/m2 in all cohorts. Neuropathy as observed in this study was related only to bortezomib dose and comparable to, or slightly higher than incidences in single agent bortezomib studies at similar dose-intensity. It must be noted that patients in this study were heavily pretreated with a median number of five prior, often neurotoxic, chemotherapy regimens, which is a predisposing factor for bortezomib-induced neuropathy. In another small dose finding study bortezomib was combined with a fixed dose of carboplatin (area under the curve [AUC] 5). Fifteen patients with recurrent ovarian or primary peritoneal cancer refractory to at least one platinum-based chemotherapeutic regimen, were treated. Even though patients had received prior neurotoxic treatment and bortezomib was combined with a relatively neurotoxic chemotherapy, overall incidence of sensory neuropathy remained rather low at 27%. Grade 3 dose limiting sensory neuropathy was observed only in one patient treated at the highest dose level of 1.5 mg/m2 bortezomib [50]. Preliminary results of a dose finding study combining bortezomib with gemcitabine and cisplatin in 34 chemonaive patients with advanced solid tumors indicate there was no potentiation of neurotoxicity [51]. Furthermore, a patient has been described who developed
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grade 3 peripheral neuropathy following bortezomib treatment, which improved to grade 1 despite subsequent treatment with cisplatin-based chemotherapy [43]. Overall, preliminary results indicate that when bortezomib is administered concurrently with other chemotherapeutic drugs such as pegylated liposomal doxorubicin, somewhat neurotoxic drugs such as docetaxel and carboplatin and a known neurotoxic chemotherapeutic agent such as cisplatin, no potentiation of neurotoxicity occurs. Future and currently running combination studies will have to show whether or not neurotoxicity is manageable, especially in combination with other reputedly neurotoxic agents such as thalidomide, paclitaxel and vincristine. It might be that prior damage to peripheral nerves due to prior neurotoxic treatment or other illnesses predisposes more to bortezomib-induced neurotoxicity than concurrent combinations of bortezomib and other (neurotoxic) agents in chemonaive or second line treated patients.
CLINICAL MANIFESTATIONS Peripheral Neuropathy In animal studies with bortezomib, the nervous system had already been determined as a target organ of toxicity. Neurotoxicity of repeat dose bortezomib in animals included axonal swelling and degeneration in peripheral nerves, dorsal spinal roots, and tracts of the spinal cord as well as multifocal hemorrhage and necrosis in the brain [24,52]. Clinical signs of bortezomib-induced neuropathy in patients included tingling, pain, diminished pinprick, vibratory and temperature sense. Motor symptoms are uncommon. Typically paresthesias were more intense in the distal lower limbs compared to the hands, increasing in severity with every dose of bortezomib. Neuropathic pain was also mainly confined to the legs and feet [30,43,50,53]. Nerve conduction studies (NCS) showed reduced or absent sensory nerve action potential (SNAP) amplitude and reduced peroneal compound muscle action potential (CMAP) amplitude and which is consistent with an axonal polyneuropathy. Skin-biopsies showed decreased intra-epidermal nerve fiber (IENF) density, which is typical for ‘dying back’ axonopathy [54]. A strong correlation was found between Total Neuropathy Score (TNS), a validated measure of peripheral nerve function, SNAP amplitude values and cumulative bortezomib dose [53,55]. Bortezomib-induced neuropathy has been characterized by NCS and quantitative sensory testing as a length-dependent, sensory, axonal polyneuropathy with predominantly small fiber (A-δ myelinated afferent; nociceptive C unmyelinated afferent) involvement [43,53,37]. Small fiber neuropathies are generally characterized by peripheral pain [57]. Concordantly, bortezomib-induced neuropathy can also be extremely painful, occasionally requiring narcotic analgesia and other drugs such as gabapentin and amitriptyline for pain management. This can be necessary for many months following discontinuation of bortezomib [23,30,50]. Apart from several described cases of severe neuropathic pain associated with bortezomib treatment, overall incidence of neuropathic pain is somewhat unclear when reviewing the published studies. In one study, all patients with neuropathy were reported to experience neuropathic pain and this was clearly specified in the adverse events
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listing, in other studies, a pain component has not been specified [43]. Though not clearly designated as neuropathic pain, ‘pain in limb’ was reported in the SUMMIT study in 13% of patients, grade 3 in 7% of patients. In the APEX study pain in limb was reported in 15% of patients in the bortezomib arm vs. 7% in the dexamethasone treated arm. Grade 3 pain in limb was solely observed in the bortezomib arm with an incidence of 2% [38]. In the phase 1 dose finding study evaluating weekly bortezomib, pain in limb was seen in 3% of patients at the lower seven dose levels, compared to 30% of patients at the highest three dose levels of bortezomib (1.6 - 2.0 mg/m2) [23]. It is therefore likely the frequently reported ‘pain in limb’ reported in studies with bortezomib, is related to a small fiber like neuropathy induced by bortezomib. In the SUMMIT study complete resolution or improvement of PN was observed ‘in the majority of patients’ [13,14,37,39]. In the APEX study patients treated with bortezomib who developed grade 2 or higher peripheral neuropathy had in 46% complete resolution (return to baseline) and in 5% improvement of symptoms at last assessment. Median time to resolution was 3.5 months [38]. De facto in 49% of patients with grade 2 or higher peripheral neuropathy, symptoms did not improve after discontinuation of bortezomib. Furthermore, worsening or onset of neuropathy after discontinuation of bortezomib has been described in a few patients [43]. Generally speaking, bortezomib-induced neuropathy is dose-dependent. However, it can manifest itself after a single dose of bortezomib while some patients will not develop neuropathy, even upon prolonged exposure. The median cumulative dose at the onset of neuropathy was determined in one 16-patient study at 13.3 mg/m2 ranging from 1.5 to 28.5 mg/m2 [43]. This suggests individual susceptibility varies greatly.
Autonomic Instability Proposed as a small fiber neuropathy based on clinical findings and diagnostic testing, it is expected that autonomic functions be affected as well. Small A-δ fibers and C fibers carry autonomic functions such as bowel movements and blood pressure in addition to temperature and pain sensation [56]. In phase 1 studies, a dose-dependent increase in incidence of hypotension was observed and in two studies (orthostatic) hypotension was one of the dose-limiting toxicities [23,33]. Hypotension did not appear to be related to cardiac failure nor adrenal dysfunction. At higher dose levels, ‘autonomic instability’, characterized by postural hypotension and syncope were partly attributed to bortezomib therapy and reported to resolve upon discontinuation of bortezomib treatment [12,23]. In a phase 2 study evaluating bortezomib treatment in sixteen patients, six out of ten patients with grade 2 to 3 peripheral neuropathy also showed grade 2 to 3 symptoms ‘possibly related to autonomic neuropathy’ such as orthostatic hypotension, syncope, dizziness, ileus and abdominal cramps. With the exception of abdominal cramps, symptoms indicative for autonomic dysfunction occurred at cumulative doses equal or greater to those at onset of peripheral sensory neuropathy. Only in one patient symptoms ‘possibly related to autonomic neuropathy’ (grade 3 ileus) were observed without symptoms of peripheral
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sensory neuropathy. In contrast, in four out of ten patients with peripheral sensory neuropathy, there were no symptoms indicating autonomic neuropathy [43]. In the APEX phase 3 study, adverse events implicated in autonomic dysfunction such as constipation, abdominal pain and headache were reported more also frequently in the bortezomib arm compared to the dexamethasone arm with overall incidences of 42% vs. 15%, 16% vs. 4% and 26% vs. 13% respectively [38]. It is likely autonomic neuropathy represents a later event in bortezomib-induced neurotoxicity, following or coinciding with, but generally not preceding peripheral sensory neuropathy.
Ototoxicity In one case report severe irreversible bilateral hearing loss after bortezomib therapy has been described in a MM patient with a minor prior hearing impairment [57]. Typical about this case was that not only medium and high frequencies were affected, as in cisplatininduced ototoxicity, but all frequencies. Furthermore deterioration of hearing loss in the low frequency range continued for twelve months. This may have been an idiosyncratic effect in this one patient, as no other reports of bortezomib-induced ototoxicity have been published, even though many patients treated with bortezomib had been exposed to known ototoxic agents such as cisplatin. Nevertheless, for the time being, caution is warranted for ototoxic effects of bortezomib treatment, especially in patients with prior hearing loss, or when combining bortezomib with ototoxic drugs such as cisplatin.
Central Nervous System Effects So far there are no indications that bortezomib therapy is associated with effects on the CNS. In 6% of patients of the extended phase 2 CREST/ SUMMIT studies, with treatment durations up to eleven months, several CNS events, such as memory impairment and mental state changes, were reported. However, they were not clearly attributed to bortezomib, but rather to disease progression and confounding illnesses [39].
BORTEZOMIB-INDUCED PERIPHERAL NEUROPATHY Pathogenesis Pathologic states associated with the ubiquitin-proteasome system (UPS) can be the result of either a loss of function or a gain of function of this system. In the first scenario, proteins are stabilized, in the latter proteins are degraded in an abnormal or accelerated fashion. In the case of blunt and abrupt inhibition of proteasome function by a chemical inhibitor such as bortezomib, loss of function of the UPS occurs resulting in stabilization of proteasome-degraded proteins.
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Ubiquitination plays a role among others in neuronal survival, synaptogenesis and axon function [58-60; and Chapters 15-18]. The marked neurotoxicity occurring upon systemic proteasome inhibition is therefore not surprising. Furthermore, neurodegenerative disorders are strongly associated with aberrations in the UPS, either as a primary cause or as a secondary consequence [61]. Nerve conduction studies and quantitative sensory testing indicate that bortezomib causes a length dependent axonal sensory neuropathy with predominantly small fiber involvement [37]. Considering the role of the UPS in the peripheral nerve system, it is very important to distinguish differences in effects of proteasome inhibition on the neuron cell body compared to the distal axon. Neuronal differentiation and neurite outgrowth is known to be promoted by proteasome inhibition [62-64]. In fact the first proteasome inhibitor, lactacystin, was discovered using a neurite outgrowth assay [65]. In contrast to these neuritogenic effects on the neuron cell body, proteasome inhibition has been shown to have detrimental effects on growth and long-term maintenance of mature axons. In sympathetic and sensory explant cultures from mice, proteasome inhibition led to ‘dying-back’ degeneration of nerve terminals [66]. A proposed explanation for this finding was the intense and continuous anterograde and retrograde protein transport occurring in the axon terminal. Proteins delivered to the axon terminals by anterograde axonal transport must either be transported back to the cell body by retrograde axonal transport, or be degraded in situ. Inhibition of proteasomal degradation could simply disturb the balance between delivery and degradation of axonal proteins, causing them to accumulate to toxic levels in nerve terminals resulting in degenerative axonopathy [66]. In addition to generally reported degenerative axonopathy, demyelinating neuropathy has also been associated with bortezomib treatment [23,67]. The role of the UPS in demyelinating neuropathies such as X-linked Charcot-Marie-Tooth disease 1A (CMTX1A) has been studied quite extensively (see Chapters 17 and 26). Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein mainly expressed by Schwann cells. Correct expression levels of PMP22 are essential for normal peripheral nerve function. Duplication of the gene and concomitant high expression levels are associated with peripheral demyelinating and axonal neuropathies [68,69]. Levels of PMP22, a protein with a very short half-life, are regulated by the UPS. Preclinical studies have shown that, when the proteasome is inhibited, PMP22 accumulates in perinuclear aggresomes. The exact mechanism by which PMP22 accumulation and aggresome formation might contribute to cellular alterations and demyelination needs yet to be elucidated. A possible mechanism is that intracellular retention will reduce the amount of protein that is incorporated into myelin resulting in peripheral nerve demyelination and dysfunction of Schwann cells [70]. CNS side effects have not been clearly associated with bortezomib treatment. Aside from headache, no marked increase in incidence of memory loss or altered motor function have been related to bortezomib treatment. This could be a result of a limited crossing of the blood brain barrier by bortezomib. Potentially, for CNS effects to occur, they might require a more sustained level of inhibition over a longer period of time.
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CONCLUSIONS Peripheral sensory neuropathy has been reported as an important and common toxicity of bortezomib treatment, occurring in about one third of patients. Neuropathy induced by bortezomib is predominantly cumulative though individual susceptibility varies greatly. Risk factors for bortezomib-induced neurotoxicity are prior exposure to neurotoxic agents as well as pre-existent neuropathy. Features of bortezomib neuropathy are characteristic for a small fiber neuropathy, characterized by a more sensory than motor neuropathy, neuropathic pain and autonomic dysfunction. Treatment is symptomatic with analgesics in case of neuropathic pain. Symptoms resolve in over half of patients after discontinuation of bortezomib therapy. Detrimental effects of proteasome inhibition on nerve terminal protein homeostasis as well as myelin production by Schwann cells might explain the high incidence of neurotoxicity in bortezomib-treated patients.
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[58] Yi JJ, Ehlers MD: Ubiquitin and protein turnover in synapse function. Neuron 2005, 47:629-632. [59] Allen E, Ding J, Wang W, Pramanik S, Chou J, Yau V, Yang Y: Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival. Nature 2005, 438:224-228. [60] Korhonen L, Lindholm D: The ubiquitin proteasome system in synaptic and axonal degeneration: a new twist to an old cycle. J Cell Biol 2004, 165:27-30. [61] Ciechanover A, Brundin P: The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 2003, 40:427-446. [62] Fenteany G, Standaert R, Reichard G, Corey E, Schreiber S: A {beta}-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line. PNAS 1994, 91:3358-3362. [63] Ohtani-Kaneko R, Takada K, Iigo M, Hara M, Yokosawa H, Kawashima S, Ohkawa K, Hirata K: Proteasome inhibitors which induce neurite outgrowth from PC12h cells cause different subcellular accumulations of multi-ubiquitin chains. Neurochem Res 1998, 23:1435-1443. [64] Obin M, Mesco E, Gong X, Haas AL, Joseph J, Taylor A: Neurite outgrowth in PC12 Cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis. J Biol Chem 1999, 274:11789-11795. [65] Omura S, Fujimoto T, Otoguro K, Matsuzaki K, Moriguchi R, Tanaka H, Sasaki Y: Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J Antibiot (Tokyo) 1991, 44:113-116. [66] Laser H, Mack TG, Wagner D, Coleman MP: Proteasome inhibition arrests neurite outgrowth and causes "dying-back" degeneration in primary culture. J Neurosci Res 2003, 74:906-16. [67] Umapathi T, Chaudhry V: Toxic neuropathy. Curr Opin Neurol 2005, 18:574-80. [68] Chance PF, Abbas N, Lensch MW, Pentao L, Roa BB, Patel PI, Lupski JR: Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17. Hum Mol Genet 1994, 3:223-228. [69] Ryan MC, Shooter EM, Notterpek L: Aggresome Formation in Neuropathy Models Based on Peripheral Myelin Protein 22 Mutations. Neurobiol Dis 2002, 10:109-118. [70] Notterpek L, Ryan MC, Tobler AR, Shooter EM: PMP22 accumulation in aggresomes: implications for CMT1A pathology. Neurobiol Dis 1999, 6:450-60.
BOOK GLOSSARY 11 S cap 19S cap
20S proteasome
26S proteasome
See: PA28. Also known as PA700 (proteasome activator of 700 kDa). A subcomplex of the 26S proteasome that acts as a receptor of ubiquitinated proteins, deubiquitinates proteins, unfolds them, opens the entrance to the 20S proteasome and translocates the polypeptide chains into it. Subdivided into lid and base complexes linked by Rpn10 (S5a). Different nomenclatures of the subunits exist, but the most widely accepted is the Rpn/Rpt nomenclature, where Rpt designate proteasomal ATP-ases (Rpt1-6) and Rpn designate non-proteasomal subunits (Rpn1-3 and Rpn5-12). Few additional components lack the Rpn names (e.g. UCH37). Multisubunit proteolytic core of the 26S proteasome; formerly known as multicatalytic proteinase complex (MPC); is a member of the N-terminal nucleophile hydrolases family; is organized in four stacked 7-membered rings composed of 2 outer α and 2 inner β rings arranged in a cylinder-like shape, with a central catalytic chamber and two antechambers. The entrance to the antechambers is occluded in inactive (latent) 20S proteasomes by N-terminal extensions of several α subunits. Activation of the 20S proteasome can be achieved by physical or chemical means or by binding to activators such as PA28 and PA700. It is characterized by the presence of three main hydrolytic activities, referred to as chymotrypsin-like activity (ChTL), trypsin-like activity (TL) and post-glutamyl peptide-hydrolyzing (PGPH) activity (also known as caspase-like). 20S proteasomes can degrade only short peptides and some denatured proteins, they can not recognize or bind polyubiquitinated proteins. Different nomenclature of the 20S subunits exist, but the most widely accepted is the α/β nomenclature, which distinguishes 7 α subunits (α1-α7) and 10 β subunits (β1-β7 and β1i, β2i, β5i). Composed of the core 20S proteasome associated with one or two 19S caps (PA700). The 26S proteasome are the active form of proteasomes, engaged in the degradation of ubiquitinated proteins. The 19S cap provides the capacity to bind polyubiquitinated proteins, deubiquitinate them, unfold them and translocate them to the central 20S proteasome, which provides the proteolytic sites.
1058 AAA (atpases associated with various cellular activities) proteins Adhesion molecule
After eight Aggresome
Akt/Protein kinase B
Alfa(α)-synuclein
Algogen: Allodynia: Allosteric effect Alzheimer’s disease (AD)
Ampa Amygdale
Amyloid fibrils
Mario DiNapoli and Cezary Wójcik A superfamily of enzymatic machines that posses a structurally conserved ATPase domain and diverse cellular functions. Includes the proteasomal ATP-ases (Rpt1-6) and VCP. Specialized cell surface molecules that are involved in interactions between different cells. Include different families of proteins such as cadherins, integrins, selectins, ICAM, etc. A zebrafish homologue of the Drosophila delta gene. Inclusion body, which is assembled around centrosomes in the area of the proteolytic center of the cell through centripete microtubulemediated transport; it is enriched in ubiquinated proteins, chaperones and proteasomes, and surrounded by a cage of intermediate filaments. Arise as a consequence of proteasome inhibition or/and massive overexpression of misfolded proteins. Aggresomes are often considers in vitro models of inclusion bodies found in neurodegenerative disorders. A serine/threonine kinase that has a wide range of substrates; it acts downstream of PI3K to regulate many biological processes, such as proliferation, apoptosis and growth and is involved in tumorigenesis. One in a family of structurally related proteins that are prominently expressed in the central nervous system. Aggregated α-synuclein proteins form brain lesions that are hallmarks of some neurodegenerative diseases (synucleinopathies). The gene for αsynuclein, which is called SNCA, is on chromosome 4q21. One form of hereditary Parkinson’s disease is due to mutations in SNCA. Another form of hereditary Parkinson disease is due to a triplication of SNCA. See also: Parkinson’s disease. A substance that produces pain e.g. capsaicin, mustard oil. The perception of normally innocuous stimuli such as light touch as painful. This is a frequent symptom of neuropathic pain. Coupling of conformational change between two widely separated binding sites. A degenerative disease of the brain associated with the development of protein deposits in the cerebral cortex and characterized by confusion, disorientation, memory failure, speech disturbances. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acic. Agonist for AMPA receptor, mimics the effects of glutamate. (Latin, corpus amygdaloideum) an almond-shaped set of neurons located deep in the brain's medial temporal lobe. Shown to play a key role in the processing of emotions, the amygdala is a part of the limbic system. Structures formed by many disease-causing proteins when they
Book Glossary
Amyotrophic lateral sclerosis / parkinsonism dementia complex of guam (als/pdc)
Anaphase promoting complex (apc)
Angiogenesis Antigen Anti-nuclear antibodies Antisense
Aplysia Apoptosis
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aggregate.Amyloid fibrils share common biochemical characteristics such as detergent insolubility, high β-sheet content and a cross β structure, protease resistance and the ability to bind lipophilic dyes, such as congo red. ALS is a progressive neurodegenerative disorder involving primarily the motor neurons of the cerebral cortex (upper motor neurons, umn), brain stem and spinal cord (lower motor neurons, lmn). Guamanian als/pdc amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam (als/pdc) is a chronic neurodegenerative disorder highly prevalent in the native chamorro population of guam island in the western pacific. The etiopathogenesis of this disorder is not yet elucidated, although it has been hypothesized that environmental factors such as aluminium or neurotoxins might be involved. Neuropathologically, guamanian als/pdc shows a severe cortical atrophy and neuronal loss. The neuropathological hallmark of als/pdc is the widespread nft formation, especially in the isocortex and hippocampal formation. A large multi-protein complex consisting of at least 11 subunits with E3 ubiquitin ligase activity, implicated in mediating proteolysis during cell-cycle progression. Recently, roles for the APC in neural development have been described. Also known as the cyclosome. The formation of blood vessels, such as occurs during embryogenesis, tissue repair or tumorigenesis. A substance that stimulates an immune response, especially the production of antibodies. Autoantibodies to nuclear antigens found in different systemic autoimmune diseases, in particular systemic lupus erythematosus and diseases affecting the connective tissue. Oligonucleotides with a sequence that is complementary to the mRNA of a given molecule can be used to block its translation. The subsequent temporary elimination of the protein of interest often provides useful information on its biological function. Sea slug that belongs to the family Aplysiidae and is a genus of sea hares. A process of programmed cell death which can be initiated (initiation phase) through an extrinsic (TNF, FAS ligand) or intrinsic pathway (DNA damage, oxidative stress), leading to activation of caspases, in particular caspase 3 (execution phase). Activation of caspases brings upon cleavage of multiple cellular proteins, including some proteasomal subunits. Subsequent activation of endonucleases induces internucleosomal DNA cleavage. Morphological features of apoptosis include loss of cell
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Arborisation
Ariadne
Armadillo superfamily
Ataxia Ataxin
Autophagy
Axon
Axonal dystrophy
Mario DiNapoli and Cezary Wójcik adhesion, peripheral chromatin condensation, and cell blebbing, ultimately leading to the division of the cell into multiple apoptotic bodies, which ar ephagocytosed by surrounding cells. In distinction to necrosis, apoptosis does not elicit an inflammatory response. The process by which an axonal or dendritic growth cone changes its morphology upon reaching its target by the extension and retraction of branch tips to form an elaborate stucture resembling a tree or arbor. A Drosophila gene encoding a RING finger domain-containing protein. Null mutants are lethal and exibit a large number of abnormalities such as in neural development including axon pathfinding. The armadillo repeat is an approximately 40 amino acids long tandemly repeated sequence motif first identified in the Drosophila segment polarity gene product armadillo, a protein that mediates cell adhesion. Similar repeats were later found in the mammalian armadillo homolog β-catenin, the junctional plaque protein plakoglobin, the adenomatous polyposis coli (APC) tumor suppressor protein, and a number of other proteins. These proteins exert several functions through interactions of their tandem armadillo repeats domain with diverse binding partners. The proteins combine structural roles as cell-contact and cytoskeletonassociated proteins and signaling functions by generating and transducing signals affecting gene expression. Impairment of the ability to perform smoothly coordinated voluntary movements. Protein associated with spinocerebellar ataxia, e.g. ataxin-1 is the protein associated with SCA-1. The ataxin proteins contain a polyglutamine tract. A process by which areas of cytoplasm including entire organelles are surrounded by membranes, likely derived from the endoplasmic reticulum. Once the entire area of cytoplasm is secluded it fuses with primary lysosomes, leading to the degradation of enclosed cytoplasmic structures. Autophagy is controlled by a complex cascade of enzymatic reactions including the covalent conjugation of two different ubiquitin-like proteins of the Atg family. Autophagy is triggered in situations of starvation providing to the cell a steady source of nutrients, however excessive autophagy leads to autophagic cell death. Also called nerve fiber, is a long slender projection of a nerve cell, or neuron, that conducts electrical impulses away from the neuron's cell body or soma. Generic term for mis-shapen axons in pathology, encompassing both larger spheroids and smaller varicosities.
Book Glossary Axonal pruning Axonal spheroid
Axonal transport
baculovirus inhibitor of apoptosis protein repeat (BIR)
Bag-1
Bang-sensitive paralytic Base of the 19s cap (pa700)
Basic helix-loop-
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The cell-autonomous, programmed removal of superfluous axonal branches that arise during development Focal swelling of an axon, usually in the CNS, to many times its usual diameter, typically 10-50 µm. Spheroids are often filled with disorganised cytoskeleton and organelles, and many stain positively for APP. The flow of proteins, organelles and other axonal components along the axon. Axonal transport is bidirectional, with anterograde transport moving away from the cell body and retrograde transport moving towards it. Components also move with different speeds, classified as slow and fast axonal transport. BIR is a domain of tandem repeats separated by a variable length linker that seems to confer cell death-preventing activity. The BIR domains characterize the Inhibitor of Apoptosis (IAP) family of proteins (MEROPS proteinase inhibitor family I32, clan IV) that suppress apoptosis by interacting with and inhibiting the enzymatic activity of both initiator and effector caspases (MEROPS peptidase family C14). Several distinct mammalian IAPs including XIAP, cIAP1, c-IAP2, and ML-IAP, have been identified, and they all exhibit antiapoptotic activity in cell culture. The functional unit in each IAP protein is the baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. Most mammalian IAPs have more than one BIR domain, with the different BIR domains performing distinct functions. For example, in XIAP, the third BIR domain (BIR3) potently inhibits the catalytic activity of caspase-9, whereas the linker sequences immediately preceding the second BIR domain (BIR2) selectively targets caspase-3 or -7. BAG-1 (also known as RAP46; BCL2-associated athanogene) is an anti-apoptotic protein, which has been shown previously to interact with molecular chaperones of the Hsp70/Hsc70 family, characterized by an N-terminal ubiquitin-like domain, which binds to the 26S proteasome, providing a link between molecular chaperones and the UPS. A class of fly mutant that exhibits hyperactive seizure behavior followed by temporary paralysis in response to mechanical stimulation, such as a “bang” of the culture vial. A subdivision of the 19S cap (PA700) formed by a hexameric ring of proteasomal ATP-ases (Rpt1-6) as well as two largest, nonATP-ase subunits of the PA700 (Rpn1 and 2).The base attaches to the α ring of the 20S proteasome on one side and to the lid of the PA700 on the other, through the Rpn10 subunit, which forms the hinge region. A family of positive and negative regulators of transcription i.e.
1062 helix (bhlh)
Basic helix–loop– helix (bhlh)
Bcl2 Bcl-2 family (b-cell lymphoma-2 family). Bendless
Beta (β)-Amyloid
Beta (β)-Sheet structures
Bir motif
Blm3p
Bone morphogenetic
Mario DiNapoli and Cezary Wójcik transcription factors characterised by a basic (positively charged) helix-loop-helix motif mediating sequence specific DNA recognition. A structural motif present in many transcription factors that is characterized by two α-helices separated by a loop.The helices mediate dimerization, and the adjacent basic region is required for DNA binding. A protein that promotes the survival of neurons by stabilizing mitochondrial membranes and decreasing oxidative stress. These are proteins with a structural similarity to Bcl-2, the prototypical inhibitor of apoptosis. The Bcl-2 family comprises proteins that both block and enhance apoptosis. A Drosophila gene which when mutated leads to defects in the giant fibre axon pathfiding and escape-jump response. Bendless encodes a ubiquitin-conjugating enzyme and was the first mutation in a component of the UPS which exhibited axon pathfinding defects. An amyloid derived from a larger precursor protein (APP: Amyloid precursor protein) and is a component of the neurofibrillary tangles and plaques characteristic of Alzheimer's disease. The β sheet (also β-pleated sheet or β strand) is a commonly occurring form of regular secondary structure in proteins. It consists of a stretch of amino acids whose peptide backbones are almost fully extended, resulting in an elongated pleatlike structure in which the peptide carbonyls point in alternating directions relative to the plane of the sheet. A typical strand is about five to ten amino acids long. In the most common usage, β strand refers to a single continuous stretch of amino acids adopting an extended conformation and involved in hydrogen bonds; by contrast, a β sheet refers to an assembly of such strands that are hydrogenbonded to each other. However, the term ‘β sheet’ is also sometimes used as a synonym of ‘βstrand’, i.e., for a single segment of extended, hydrogen-bonded amino acids. A ~70 amino-acid zinc-finger motif called the baculoviral inhibitor of apoptosis repeat. The number of BIR domains in a given IAP varies from one to three, but they are invariably present at the amino-terminus of the protein, and mediate the interaction with caspases. Initially identified as an extragenic suppressor of the blm3-1 mutation in a genetic screen to detect genes controlling sensitivity to bleomycin, a drug that induces DNA double strand breaks. Yeast ortholog of the mammalian 20S proteasome activator PA200. It is now designated as Blm10p. Members of the transforming growth factor βfamily of molecules
Book Glossary protein (bmp) Bortezomib (a dipeptidyl boronic acid, ps-341)
Braap Brain derived neurotrophic factor Brain stem Calcium/calmodulin protein kinase ii (camkii)
Calpain/calpastatin
Capsaicin
Carney syndrome
Casein kinase i and ii (chi/ii)
1063
having multiple roles in development, including synaptogenesis. The first proteasome inhibitor that has progressed to clinical trails for the treatment of multiple myeloma (MM) and other cancers. Received the US Food and Drug Administration approval (VelcadeTM, Millenium Pharmaceuticals, Cambridge, MA) for the treatment of patients with relapsed and refractory MM. The mode of action depends largely on the inhibition of anti-apoptotic and anti-inflammatory NF-κB pathway both in the MM cells and in bone marrow stromal cells. Proteasomal activity cleaving after branched-chain amino acids. A neurotrophin playing roles in proliferation, differentiation and survival of neurons during development, as well as in the synaptic activity and plasticity in many groups of mature neurones. The lower part of the brain, adjoining and structurally continuous with the spinal cord. Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) is a serine/threonine kinase. It is a Ca2+ /calmodulin-dependent, truncated monomer (1-325 amino acid residues) of the subunit. Autophosphorylation of threonine 286 in the presence of Ca2+ and calmodulin activates CaMKII and produces substantial Ca2+ /calmodulin-independent activity. Calpain is a calcium-activated proteinase of eukaryotic cells that itself activates several cellular enzymes. In erythrocytes, it affects several proteins important for the determination of cellular shape and deformability. This protease is also involves in apoptosis, cytoskeletal reorganization and muscle protein degradation. Calpain exists as a heterodimer composed of a small regulatory subunit and one of three large catalytic subunits, designated calpain1, 2 and 3. Calpastatin regulates calpain by inhibiting both the proteolytic activity of calpain and its binding to membranes. Calpastatin exists in two types, tissue type (100-120 kDa) and erythrocyte type (70 kDa), resulting from both alternative splicing and proteolytic processing. Irritant chemical responsible for the burning sensation of chilli peppers, activates the ion channel TRPV1 (transient receptor potential V1). Multiple Neoplasia Syndrome characterized by spotty skin pigmentation, cardiac and soft tissue (skin, mucous membrane) myxomas, psammomatous melanotic schwannomas and endocrine tumors including Cushing’s syndrome from nodular adrenocortical hyperplasia, pituitary adenomas (acromegaly or prolactinoma), Sertoli cell tumors and Leydig cell tumors. Casein Kinases (CKI and II) are serine/threonine protein kinases. Numerous isoforms have been described, most with monomeric
1064
Caspases
Cbcvhl complex
Cdk inhibitor p21
Cdk inhibitor p27kip1
Central sensitisation
Centrosome
CFTR Chaperones
Mario DiNapoli and Cezary Wójcik structure. Caspases are members of the cysteine-aspartic acid protease (caspase) family and are generated by a unique gene. They exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme Their sequential activation plays a central role in the execution-phase of cell apoptosis. Caspase 3 cleaves and activates caspases 6, 7 and 9 (executor caspases), and the protein itself is processed by caspases 4, 8, 9 and 10 (initiator caspases). An SCF-related complex of elongin B, elongin C, cullin-2 and the RING-finger protein Rbx1/Roc-1.The substrate recognizing subunit pVHL binds to the elongin B/C complex through a motif known as the Socs box. It is believed that the von Hippel–Lindau cancer syndrome is a direct consequence of a loss of cellular CBCVHL-mediated ubiquitylation activity. It binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at G1. The expression of the coding gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. This protein can interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. It binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at G1. The degradation of this protein, which is triggered by its CDK dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state. The phenomenon of synaptic facilitation in the dorsal horn of the spinal cord, which occurs following tissue damage or nerve injury, whereby activation of spinal cord neurons can be elicited by a weaker sensory signal than in normal animals. This sensitisation has some characteristics of synaptic plasticity associated with learning and memory (for example, hippocampal long-term potentiation). A structure adjacent to the nucleus formed by a pair of centrioles surrounded by amorphous pericentriolar material, serving as a microtubule-organizing center. See: Cystic fibrosis transmembrane conductance regulator. Proteins whose function is to assist other proteins in achieving proper folding. Many chaperones are heat shock proteins, that is,
Book Glossary
Chaperonin
Charcot-MarieTooth disease Chemical synapse Chemokines Chip (carboxyl terminus of hsp70interacting protein): Cht-l Cks1 (cdc2associated protein) Clpap (clpxp)
Cluster of differentiation (cd)
Combinatorial chemistry
Conformational diseases
1065
proteins expressed in response to elevated temperatures or other cellular stresses. Other chaperones are involved in folding newly made proteins as they are extruded from the ribosome. Although most newly synthesized proteins can fold in absence of chaperones, a minority strictly requires them. A subclass of chaperones, forming cylindrical stacked complexes with an inner cavity, which assist the folding of nascent, non-native polypeptides into their native, functional state. A group of peripheral neuropathies either affecting primarily the axons (type II) or the Schwann cells (type I). Specialized junction through which neurons signal to one another and to non-neuronal cells such as muscles or glands. Class of pro-inflammatory cytokines that have the ability to attract and activate leukocytes. A co-chaperone that negatively regulates the ATPase and chaperone activities of Hsc70. It has U box-dependent ubiquitin ligase activity that targets chaperone substrates for proteasomedependent degradation. Chymotrypsin-like activity; proteasomal activity cleaving after hydrophobic residues and associated to the β5 subunit. Cofactor for Skp2; induces allosteric alterations in Skp2 molecule, allowing it to bind phosphorylated substrate A major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATPhydrolyzing, chaperone component, ClpA(X). Molecules are cell surface molecules recognized by specific sets of antibodies. The CD nomenclature is established during International Workshops and Conferences on Human Leukocyte Differentiation Antigens (HLDA). This system was meant to classify many monoclonal antibodies, generated by different laboratories, against various cell surface molecules on leukocytes. Around 300 CD molecules have been identified. A technology for synthesizing and characterizing collections of chemical compounds (libraries) and the screening of those libraries for compounds with useful properties; the modern approach to drug discovery. Conformational diseases are diseases where cellular functions are compromised because of misfolded proteins. The conceptional framework of conformational diseases is found in the cellular protein quality control systems which in the normal and young cell eliminate misfolded proteins. Misfolding may occur in proteins with an intrinsic ability to aggregate and in oxidatively damaged proteins, which accumulate by ageing. If the protein quality control systems are not sufficiently efficient cell toxic protein complexes
1066
Consanguineous Constitutive proteasome Corticobasal degeneration (cbd)
Covalently bound inhibitors of the 20s proteasome
Cowden syndrome
Creb (camp response element-binding protein)
Cyclins
Cystic fibrosis transmembrane conductance regulator (Cftr) Cytokine
Mario DiNapoli and Cezary Wójcik may accumulate. This pathogenesis is a major contributing factor in the development of late onset neurodegenerative disorders. Related by blood, usually referring to marriage between close relatives. 20S proteasome whose active subunits are β1, β5 and β2, in contrast to the immunoproteasome. A slowly progressive disorder characterized by neurodegenerative changes of certain brain regions, including the cerebral cortex (particularly the frontal and parietal lobes) and parts of the basal ganglia. Most patients initially develop symptoms in their 60s or 70s. Primary findings may include stiffness (rigidity); slowness of movement (bradykinesia); loss of the ability to coordinate and execute certain purposeful movements of the arms or legs (limb apraxia); the sensation that a limb is not one's own (‘alien limb phenomenon’); and other sensory abnormalities. Affected individuals may also develop slurred, labored speech (dysarthria); dystonia; and irregular, involuntary, ‘shock-like’ contractions of certain muscle groups, particularly of the hands and forearms, that may be provoked or aggravated by voluntary movement and certain external stimuli (action and reflex myoclonus). Peptide or non-peptide – based small molecules bearing diverse functional groups that bind covalently to the hydroxyl group of the N-terminal threonine-1 residue of each catalytic β-subunit; inhibit the activity either reversibly or irreversibly; commonly used to explore the role for the proteasome in physiology and pathology; exhibit anti-cancer and anti-inflammatory activities. A hereditary predisposition to tumors: hamartomas of the skin, mucous membranes, breast and thyroid that is caused by PTEN mutations CREB binds to cAMP-responsive gene promoters that have in common an 8-base enhancer known as the cAMP-response element (CRE). Cyclic AMP (cAMP) second messenger pathways provide a chief means by which cellular growth, differentiation, and function can be influenced by extracellular signals. These function as positive regulatory subunits of cyclindependent kinases (CDKs). Cyclin–CDK complexes are usually activated at specific points during the cell cycle and have a specific set of substrates. A multispanning transmembrane chloride ion channel and regulator of other transporters; mutations cause cystic fibrosis.
Small proteins or biological factors that are released by cells and have specific effects on cell-cell interaction, communication, and
Book Glossary
Deadly seven Degradasome
Degron
Deleted in colorectal carcinoma Delta
Deltex
Demyelinating neuropathy Demyelination Dendrites
Dentatorubral Pallidoluysian Atrophy
Deubiquitinating enzymes (dubs)
Dislocation
1067
behavior of other cells. A zebrafish homologue of the Drosophila notch gene. A coupled multiprotein system that physically links the 26S proteasome and one or more ubiquitin receptors with components of the ubiquitin conjugation system and other ancillary factors. Amino acid sequence, conformational determinant or chemically modified protein structure that confers metabolic instability to proteins and acts as a degradation signal. Examples of degrons include the cyclin destruction box and destabilizing N-terminal amino acids. One of the receptors for the axon guidance cue Netrin-1 identified for its potential role as a tumour suppressor gene. A neurogenic gene first indentified in Drosophila which when mutated leads to an excess of neurones differentiating. Delta is a transmembrane protein which interacts with the Notch protein medeating the process of lateral inhibition. Identified in Drosophila as a positive regulator of the Notch signalling pathway. Deltex contains a RING-H2 domain at the cterminus and two copies of a WWE protein-protein interaction domain indicating it may be an E3 ubiquitin ligase. Peripheral nerve disease associated with the loss or destruction of myelin from Schwann cells. Loss of the myelin sheath surrounding myelinated axons, which occurs as a result of disease or damage. Projections of a neuron (usually branched) that act to conduct the electrical stimulation received from other cells to and from the cell body (from Greek dendron - tree). This is a rare neurodegenerative disease reported mostly in Japan. It is characterised by epilepsy, chorea and ataxia. It is caused by the expansion of a CAG nucleotide repeat in a gene on chromosome 12. Like Huntington's disease the onset of the symptoms are earlier, and the disorder more severe, if the defective gene is inherited paternally. Early onset and severe symptoms are more marked the longer the CAG repeat. Multiple cellular enzymes able to cleave the peptide and isopeptide (isopeptidases) bonds formed between ubiquitin molecules or between ubiquitin and the substrate protein. DUBs perform main three functions: 1) they are necessary components of the 26S proteasome, removing polyubiquitin chain before the substrate is degraded; 2) they are required for the generation of free ubiquitin from the products of the ubiquitin-fusion genes; 3) they edit and rescue ubiquitinated substrates, opposing the action of the E1-E2E3 cascade. See: retrotranslocation.
1068 Dlk-1
Dopamine:
Dorsal horn Dysarthria E1
E2
E3
E4
Ecm29
Ectopic neurite outgrowth Electroencephalogra
Mario DiNapoli and Cezary Wójcik A mitogen activated kinase kinase kinase which in C. elegans is regulated via ubiquitination and regulates synaptogenesis downstream of RPM-1, the C elegans homologue of the Drosophila highwire gene. An endogenous catecholamine and major transmitter in the extrapyramidal system of the brain important in regulating movement. In the synthesis of catecholamines from tyrosine, it is the immediate precursor to norepinephrine and epinephrine. The area of the spinal cord where the majority of sensory afferents terminate, comprising the laminae I-VI. Speech disorder resulting from the inability to properly control the muscles of the mouth. Ubiquitin-activating enzyme (UBA); an enzyme that activates ubiquitin by forming a ubiquitin–E1 thiol ester bond, first step in the ubiquitination cascade. There are two isoforms of the E1 in humans, E1A and E1B. Ubiquitin conjugating enzyme (UBC); an enzyme that conjugates ubiquitin by transferring the activated ubiquitin from an E1 and forming an ubiquitin–E2 thiol ester bond; it interacts with specific E3 enzymes. There are ~20 known E2s in the human genome. Ubiquitin ligases; enzymes whcih bring upon specificity to the UPS, recognizing the substrate to be ubiquitinated. There are over 700 different E3s in the human genome, which contain either the HECT (Homologous to the E6-AP Carboxyl Terminus) domain or the RING (really interesting new gene) domain (or the closely related U-box domain). Ubiquitination can occur in two ways: Directly from E2, catalysed by RING domain E3s and via a thiol linkage to the E3 enzyme, catalysed by HECT domain E3s. E3s containing the HECT domain are monomeric, while enzymes containing the RING domain form multisubunit complexes, such as the APC or the SCF. Ubiquitin chain elongation factor; A specialized ubiquitin ligase that is capable of elongating oligoubiquitinated substartes. Known human E4s include Ufd2A and B. ~200-kDa HEAT-repeat protein that associates with the 26S proteasome. Many species are present in mouse brain ranging from 55 kDa to greater than 250 kDa and are likely to arise by alternative splicing and/or proteolytic processing. Ecm29 has been proposed to function as an adaptor to link the 26S proteasome to endocytic, secretory, transport and protein quality control pathways. A class of mutations identified in C. elegans with defects in the axon outgrowth of specific neurone types. A recording of the summated electrical potentials of neurons in the
Book Glossary m (eeg) Embolus Endbulbs Endocytosis Endothelium Ephb2
Epigenetic inheritance
Epilepsy Erad (endoplasmic reticulum-associated degradation)
Erythropoietin Esrom
Etiology Familial cylindromatosis familial
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cerebral cortex using scalp electrodes. A clot formed by platelets or leukocytes that blocks a blood vessel. Large swellings, up to 50 µm diameter, that develop terminally on both proximal and distal axon stumps after transection. The process by which eukaryotic cells take up material from the outside by invagination of the plasma membrane. The layer of epithelial cells that lines the blood and lymph vessels of the body. A member of the erythropoietin producing hepatocellular family of tyrosine kinase receptors playing roles in tunourigenesis and axon guidance. Transmission of phenotypic changes without alteration of the genetic code; includes traits trasmited through the pattern of DNA methylation and through the pattern of posttranslational modification of histones. A brain disease characterized by the presence of recurrent and spontaneous, unprovoked seizures The process by which lumenal and transmembrane proteins present in the endoplasmic reticulum are degraded; not all ERAD depends on the UPS, and not all ERAD involves retrotranslocation from the ER to the cytosol, however the term ERAD is often used to describe the retrotranslocation of substrates from the ER followed by their ubiquitination and UPS-dependent degradation. ERAD is a quality control mechanism, which prevents the accumulation of misfolded proteins in the ER or their secretion to the extracellular space, it therefore counteracts ER stress and complements the unfolded protein response. Depending on whether the misfolded domain is localized within the lumen, the membrane or the cytosol, the ERAD pathway can be subdivided into ERAD-L, ERAD-M and ERAD-C, with different sets of factors required for each pathway. All subdivisions of ERAD converge on the cytosolic site of the ER membrane, where substrates are delivered to the 26S proteasome, often with the assistance of the VCP-Ufd1-Npl4 complex. A renal hormone that is induced by anaemia and that activates haemoglobin synthesis by bone marrow red-cell precursors. The zebrafish homologue of the Drosophila highwire gene. Esrom functions as an E3 ubiquitin ligase and is required for the topographic mapping of zebrafish RGCs in the optic tectum. The cause or origin of disease. See. Turban tumor syndrome. An autosomal dominantly inherited dementia, histologically
1070 encephalopathy with neuroserpin inclusion bodies (fenib), Fat facets Frazzled Frontotemporal dementia with parkinsonism linked to chromosome 17 (ftdp-17)
Gamma γ-Interferon Ganglion mother cell
Genomic imprinting Giant axonal neuropathy (GAN)
Gigaxonin
Glass bottomed boat Glioblastoma
Glucocorticoid
Mario DiNapoli and Cezary Wójcik characterized by unique neuronal inclusion bodies, and biochemically by polymers of the neuron-specific serpin, neuroserpin. A member of the Drosophila deubiquitinating enzyme family which may antagonize ubiquitin-dependent mechanisms. The Drosophila homologue of the netrin receptor DCC. Hereditary Frontotemporal Dementia with Parkinsonism-17 (FTDP-17) is a progressive dementia that can present with a variety of clinical features, including behavioral and cognitive changes, psychiatric symptoms, language disturbances, and/or motor dysfunction. Onset of these symptoms typically occurs between 40 and 60 years of age. In all these diseases, the symptoms observed were related to mutation in the tau gene. Immunomodulatory cytokine. During neurogenesis in insects neuroblats undergo eight waves of mitosis giving rise to progeny known as the ganglion mother cell. Each ganglion mother cell performs one equal cell division yielding two neurones. A phenomenon of epigenetic inheritance in which a gene’s expression pattern is dependent on the parent-of-origin. GAN is a rare hereditary motor and sensory neuropathy (HSMN) that severely affects the central nervous system. The first symptoms appear in early childhood. This disorder is characterized by abnormalities in the peripheral and central nervous systems including low muscle tone (hypotonia), muscle weakness, decreased reflexes, impaired muscle coordination (ataxia), seizures and mental retardation. Pale, tightly curled hair is frequently seen in those affected. Giant axonal neuropathy follows autosomal recessive genetic inheritance. Gigaxonin controls protein degradation, and is essential for neuronal function and survival. Gigaxonin is now known as a ubiquitin scaffolding protein that controls MAP1B-LC degradation. Mutations in the GAN gene, which encodes the ubiquitously expressed protein gigaxonin, results in a sensory and motor neuropathy called Giant Axonal Neuropathy (GAN). Features of GAN include axonal degeneration. See Giant axonal neuropathy. The Drosophila homologue of bone morphogenetic protein. It is the most frequent astrocytic gliomas and the most malignant of neuroepithelial tumors. It can be primary, arising as such since the beginning, or secondary by malignant transformation of a previous astrocytoma. A steroid hormone synthesized and secreted by specialized cells in the adrenal cortex that exerts wide-ranging effects on nearly every
Book Glossary
Glucocorticoid receptor Growth cone
Gsk3 α and β glycogen synthase kinase3 alpha and beta)
Guidance cue
Hcdc4/fbw/archipela go/ago. Hdj-1/Hdj-2
HEAT repeat
Heat shock protein
Hect Hereditary inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB) and
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tissue in the body including the brain. The receptor for glucocorticoid hormones (e.g. cortisol) that is a member of the nuclear receptor superfamily. The tip of a developing axon or dendrite with long thin filopodia and lamellipodia responsible for sensing the environment and guiding the axon or dendrite to its targeted during development or regeneration. GSK-3 is a serine/threonine kinase, which is involved in many cell functions, including, insulin pathway, growth factor and nutrient signaling, cell division, apoptosis, modulation of transcription factors AP-1 and CREB, and specification of cell fate1. Phosphorylation of GSK-3β on serine 9 results in its inactivation. GSK-3β has also been shown to phosphorylate tau, the major component of neurofibrillary lesions of Alzheimer’s disease A signal which guides the growth cones of axons or denrites to their correct target during neural development. Examples of guidance cues include the netrin and semaphorin families. F-box protein with 7 tandem tryptophan-aspartic acid repeats. It binds directly to cyclin E and is thought to target it for ubiquitinmediated degradation. Hdj-1 and Hdj-2 are members of the Hsp40 family of cochaperones that utilize a conserved J-domain to regulate the ATPase activity of Hsp70. Named after huntingtin, eukaryotic elongation factor 3, the PR65/A subunit of protein phosphatase 2A, and the target of rapamycin (TOR) lipid kinase, HEAT repeats are α-helical domains composed of roughly 50 amino acid residues which pack together to form elongated superhelices or solenoids. Canonical HEAT repeats consist of two helices, which form helical hairpins that stack upon one another into a single domain with a continuous hydrophobic core. These domains are found in a wide variety of proteins of differing activities that function as scaffold, anchoring or adaptor proteins. Heat shock proteins (Hsps) are a group of molecular chaperones, which are normally up-regulated when a cell undergoes various type of environmental stresses such as heat, cold or oxygen deprivation. A protein domain homologous to the E6-assopciated protein (E6AP) C terminus characteristic of this family of ubiquitin ligases. IBMPFD is a rare, complex and ultimately lethal, autosomal dominant disorder (MIM 605382). IBMPFD features adult-onset proximal and distal muscle weakness (clinically resembling limb girdle muscular dystrophy), early-onset PDB in most cases, and premature FTD. Mutations in the valosin-containing protein (VCP)
1072 frontotemporal dementia (FTD) (IBMPFD) High throughput screening (hts)
Highwire Hippocampus
Hrs (hepatocyte growth factorregulated tyrosine kinase substrate) Hsp100
Hsp40
Hsp60
Mario DiNapoli and Cezary Wójcik on chromosome 9p13-p12 were recently found to be associated with IBMPFD. The process in which thousands of compounds are screened against a known or unknown target and the ones exhibiting the biggest positive effect are taken on for more detailed analysis; can be performed using microwell-or cell array-based assays or using pooled libraries. A potential Drosophila RING-H2 E3 ubiquitin ligase which negatively regulates synaptogenesis. A part of the brain located inside the temporal lobe, forming a part of the limbic system and plays a part in memory and spatial navigation. The name derives from its curved shape in coronal sections of the brain, which to some resembles a seahorse (Greek: hippokampos). Hrs is localised to early endosomes in a manner that requires phosphoinositide 3-kinase (PI 3-kinase) activity.
Heat shock protein 100 (Hsp100) chaperones are members of the AAA+ protein family (adenosine triphosphatases with diverse activities) that share a common ATPase domain and form large ring-shaped structures. In yeast, Hsp104, the best-characterized Hsp100, regulates protein aggregation, disaggregation and thermotolerance, but no mammalian homologue has been identified so far. Hsp40 co-chaperones bind Hsp70 through a conserved J-domain and stimulate ATP hydrolysis, resulting in a conformational switch that closes the substrate-binding pocket of Hsp70 and facilitates the capture of non-native protein substrates. Hsp40s also bind protein substrates and target these substrates to Hsp70, enhancing the efficiency of the Hsp70/Hsp40 refolding cycle. Higher eukaryotes have many Hsp40 family members,whose differential expression or localization might regulate the substrate specificity of conserved Hsp70 family members. Hsp60 chaperones are heptameric complexes of identical subunits stacked back to back in a double-ring structure that contains a large central cavity in which protein folding is thought to occur. In eukaryotes, Hsp60 family members (also called Group I chaperonins) are found in the mitochondria, and cooperate with a cofactor of the Hsp10 family. A second class of chaperonins (Group II chaperonins) is found in the eukaryotic cytosol but has no HSP10 cofactor. The best-characterized Group II chaperonin is TRiC, which comprises eight subunits per ring encoded by
Book Glossary
Hsp70
Hsp90
Huntingtin (htt) Huntington’s disease (hd)
Hybrid proteasomes Hyperalgesia:
HypothalamicPituitary-Adrenal (HPA) axis
Iap (inhibitor of apoptosis proteins)
Icer (inducible camp
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different genes. TRiC is thought to be crucial for the folding of actin and tubulin in the eukaryotic cytosol. Hsp70 chaperones (with HSP40s, their co-chaperones) assist in the stabilization and folding of many substrates and are found in most cellular compartments. In humans, 11 genes that encode Hsp70 family members have been identified, including the constitutive cytosolic member heat shock cognate 70 (HSC70), the stressinduced cytosolic HSP70, the endoplasmic reticulum-localized glucose-regulated protein 78 (GRP78) and the mitochondrial GRP75. All Hsp70 proteins have a conserved amino-terminal ATPase domain that binds and hydrolyses ATP, and a carboxy (C)terminal substrate-binding domain. Hsp90 chaperones are an essential component of the eukaryotic cytosol, where they stabilize misfolded proteins and regulate the activity of various signalling proteins, including steroid hormone receptors, tyrosine kinases, nitric oxide synthase and calcineurin. The polyglutamine containing protein associated with Huntington’s disease. An autosomal, dominantly inherited disorder characterized by the onset of progressive chorea (involuntary, forcible, rapid, jerky movements), dementia, and ataxia. Huntington’s disease is a polyglutamine tract disorder. Proteasomes composed of a 20S proteasome capped on one end by a 19S cap (PA700) and on the other end by 11S cap (PA28). Heightened sensitivity to noxious stimuli which can be a short-term effect following tissue damage, or can be chronic, as occurs in neuropathic pain. The hierarchy of stress hormones that serve to ultimately regulate the synthesis and secretion of glucocorticoids from cells in the adrenal cortex. For example, neuropeptides (e.g. corticotrophin releasing factor or CRF) secreted from specialized neuroendocrine cells of the hypothalamus stimulate the secretion of hormones from specialized cells of the anterior pituitary (e.g. adrenocorticotropic hormone or ACTH), which ultimate regulate hormone (e.g. cortisol) synthesis and secretion from cells in the adrenal cortex. A negative feedback loop operates at all levels of this axis driven by the secreted hormones. All contain one or more baculoviral IAP repeat motifs involved in mediating protein-protein interactions. Many IAPs also possess a RING domain which enables recruitment of ubiquuitin-conjugating enzymes and catalyses the transfer of ubiquitin onto target proteins. IAP protein levels can themselves be regulated by ubiquitinmediated proteolysis. A splice variant of CREM (multiexonic gene that encodes both
1074 early repressor)
Idiopathic epilepsy Immediate-early gene Immunoproteasome
Inclusion bodies
Infarct Inflammation
Inflammatory pain: Interictal Intrathecal Ionophoresis, ionophoretic
Ischemia Isoelectric focusing
isopeptidases Iκb-kinase (ikk)
J2 prostaglandins
Mario DiNapoli and Cezary Wójcik activators and antagonists of cAMP-inducible transcription) that is induced by activation of the adenylyl cyclase signal transduction pathway. ICER serves as a dominant-negative repressor of cAMPinduced transcription. Epilepsy with no known cause. A gene that is transcribed rapidly and transiently in response to cellular stimulation. 20S proteasome assembling the three catalytically-active subunits β1i, β5i and β2i which replace their constitutive homologues under the influence of γ-interferon. Cellular structures found inside neurons that are composed of aggregated proteins, including amyloid fibrils, molecular chaperones and components of the UPS. Recent studies indicate that the formation of inclusion bodies correlates with neuronal survival and is a protective response. An area of tissue death due to a local lack of oxygen. A localized protective reaction of tissues to irritation, injury or infection haracterized by heat, pain, redness, swelling and sometimes loss of function. Pain arising from tissue damage, or release of inflammatory agents, which can be short-term or chronic. The interval between seizures. The fluid-filled space between the spinal cord and surrounding dural membrane. In electrophysiological studies, a method of drug application close to the recording site in nervous tissue. The process involves ejection of an ionised pharmacological agent from the tip of a glass electrode using a small electric current arranged in opposite polarity. A low oxygen state usually due to obstruction of the arterial blood supply or inadequate blood flow leading to hypoxia in the tissue. An immunoblotting technique used for the detection of immunoglobuline synthesis inside the central nervous system (intrathecal antibody synthesis). See: deubiquitinating enzymes. The 700–900-kDa IκB-kinase (IKK) complex includes the catalytic subunits IKKα and IKKβ and the regulatory subunit IKKγNEMO. Both catalytic substrates are involved in the activation of NF-κB transcription factors, but they do so by distinct mechanisms and substrates.As shown by genetic studies, IKKβ is essential for inducible IκB phosphorylation and degradation. A group of potent hormone-like lipid compounds that are derived from arachidonic acid, contain 20 carbon atoms including a five-
Book Glossary
Jamm Jnk
Josephin domain
Kennedy Disease Ki antigen Lactacystin
Lateral inhibition Leukocyte Leukodystrophy
Lewy bodies:
Lid of the 19S cap (PA700)
Ligand gated ion channel
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carbon ring, and modulate inflammation. Jab1/MPN/Mov34 proteases are metallo-enzymes that have JAMM or MPN+ metal-binding domain for deubiquitinating activity. The c-Jun amino-terminal kinase belongs to the group of mitogenactivated protein kinases (MAPKs) and is activated in mammalian cells by environmental stress, pro-inflammatory cytokines and mitogenic stimuli. JNK regulates the activities of many transcription factors, and is required for the regulation of inflammatory responses, cell proliferation and apoptosis. The Josephin domain is an eukaryotic protein module of about 180 residues, which occurs in stand-alone form in Josephin-like proteins, and as an amino-terminal domain associated with two or three copies of the ubiquitin-interacting motif (UIM) in ataxin 3like proteins. It is a mainly α helical cysteine-protease domain predicted to be active against ubiquitin chains or related substrates. The Josephin domain contains two conserved histidines and one cysteine that is required for the ubiquitin protease activity. See: (X-linked) Spinal and Bulbar Muscular Atrophy. Old name for PA28γ Lactacystin is a microbial metabolite originally isolated from Streptomyces that is now widely used as a selective inhibitor of the 20S proteasome The process during neurogenesis by which neuroblasts inhibit their neighbouring cells from becoming neurones. white corpuscles in the blood involved with host defenses. A disorder of the white matter of the brain, the part of the brain that contains myelinated nerve fibers. The white matter is white because it is the color of myelin, the insulation covering the nerve fibers. (The white matter is as opposed to the gray matter, the cortex of the brain which contains the nerve cell bodies). The white matter is involved in the conduction of nerve impulses in the brain intracytoplasmic, eosinophilic, round α Synuclein (αS)-positive inclusions found in neurons. The presence of Lewy bodies is a histological hallmark of Parkinson’s disease (PD). They are found typically in the substantia nigra and locus coeruleus but are also seen in the neocortex. The lid is a subdivision of the 19S cap (PA700) distal to the 20S proteasome, which attaches to the base through a hinge formed by Rpn10 and is formed by all the subunits of the PA700 with the exception of proteasomal ATP-ases (Rpt1-6), Rpn1 and Rpn2. Transmembrane ion channel that will open and close to allow transport of ions in response to binding of neurotransmitter (or other chemical signal). For example, in neurons, calcium channels open in response to specific stimuli and this entry of ionic calcium
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Ligand of numbprotein X Lipid peroxidation
lipid rafts
Liquid facets Long-term depression Long-term potentiation (ltp).
Lysosome
Machado-Joseph Disease
Macrophage
Major histocompatibility molecules (MHC)
Mario DiNapoli and Cezary Wójcik is important for regulation of many events in neurons. A Drosophila RING finger containing E3 ubiqitin ligase which ubiqutinates and targets numb for ubiqitin-mediated proteolysis regulating Notch signalling. An autocatalytic process in which free radicals attack double bonds in membrane lipids, resulting in structural damage to membranes and the liberation of toxic aldehydes such as 4-hydroxynonenal. Membrane microdomains, formed by high concentrations of sphingolipids and cholesterol immersed in a phospholipidrich environment, that are involved in specialized pathways of protein/lipid transport and signalling The Drosophila homologue of the vertebrate endocytic protein epsin and is a target of the deubiquitinating enzeme fat facets The theory that down-regulation of a post-synaptic receptor will lead to loss of responsiveness to neurotransmitter stimulation, resulting in a depressed response to stimuli in the future An enduring increase in the amplitude of excitatory postsynaptic potentials as a result of high-frequencyb (tetanic) stimulation of afferent pathways. It is measured both as the amplitude of excitatory postsynaptic potentials and as the magnitude of the postsynaptic cell population spike. LTP is most frequently studied in the hippocampus and is often considered to be the cellular basis of learning and memory in vertebrates. A membrane-bound organelle characterized by a low pH that contains high concentrations of hydrolytic enzymes including multiple proteases, mainly of the cathepsin family. also called spinocerebellar ataxia type III, is a rare, inherited, ataxia (lack of muscular control) affecting the central nervous system and characterized by the slow degeneration of particular areas of the brain called the hindbrain. Patients with MJD may eventually become crippled and/or paralyzed but their intellect remains intact. The onset of symptoms of MJD varies from early teens to late adulthood. Three forms of Machado-Joseph Disease are recognized: Types MJD-I, MJD-II, and MJD-III. The differences in the types of MJD relate to the age of onset and severity. Earlier onset usually produces more severe symptoms. phagocytic cell of mammalian tissues that become activated in response to foreign materials or tissue injury and play an important role in killing foreign cells, release of pro-inflammatory substances, and antigen presentation. are cell surface proteins found on most cells of the body. There are three classes of MHC molecules. Class I and II molecules participate in presentation of antigens to T cells. MHC class I molecules typically interact with the cell surface receptor of a type
Book Glossary
Maps Mdm2 gene
Medea MEN-1 (multiple endocrine neoplasia type 1) Mendelian disorder
Menin Mesencephalon Metabotropic glutamate receptors
Microarray
Microtubule binding domain of tau protein Microtubule-affinity regulating kinase
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of lymphocytes known as killer or cytotoxic T cells, while MHC class II molecules present antigens to helper T cells. Microtubule associated proteins. Proteins involved in the polymerisation and the stability of microtubules. encodes for E3-like ubiquitin-protein ligase, a nuclear phosphoprotein that binds and inhibits transactivation by tumor protein p53, as part of an autoregulatory negative feedback loop. Overexpression of this gene can result in excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. This protein has E3 ubiquitin ligase activity, which targets tumor protein p53 for proteasomal degradation. A Drosophila SMAD mediating intracellular signalling downstream of BMP receptors. autosomal dominant condition that describes the association of the occurrence of tumors involving two or more endocrine glands: parathyroid hyperplasia, pancreatic endocrine tumors, pituitary adenomas a disease that adheres to single gene inheritance patterns, such as autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive gene located on 11q13 encodes menin, a tumor suppressor gene that is mutated in MEN-1. (or midbrain) is the middle of three vesicles that arise from the neural tube of developing brain. A group of cell surface receptors that bind the excitatory neurotransmitter glutamate ( while glutamate is also an amino acid used for making proteins it can also act as neurotransmitter in the nervous system. Generally, these receptors are seven-pass transmembrane G-protein coupled receptors, and the binding of ligand to the receptor results in activation of a biochemical signalling pathway inside the neuron. DNA Microarrays, commonly known as gene chips, are small, solid supports (glass slides or silicon chips) onto which the sequences from thousands of genes are immobilized at fixed locations. Microarrays may be used to assay the gene expression of thousand of genes simultaneously within a single sample or to compare gene expression in two different tissue samples or cell types. The Microtubule binding domain was given to the C-terminal part of tau protein. This side of the molecule is involved in the binding and the stability of microtubules. It will differ by the incorporation or not of the exon 10 of the tau gene. kinase with an apparent molecular mass of 110 kDa that phosphorylates the neuronal MAPs tau and MAP2 and the
1078 (mark)
Mind bomb
Mineralocorticoid receptor Mjd (Machado Joseph disease proteases) Molecular chaperones Morula Mtoc (microtubule organizing center)
Mts (microtubules)
Multivesicular bodies (mbvs) Mushroom body Myelin: Nedd8 (neddylation)
Mario DiNapoli and Cezary Wójcik ubiquitous MAP4 on their homologous KXGS motifs. The kinase caused rapid detachment of all three MAPs from microtubules, resulting in high dynamic instability, and was therefore termed MARK (MAP/microtubule affinity-regulating kinase). A zebrafish RNG E3 ubiqutin ligase which regulates Notch-Delta signalling via promoting the ubiquitination and internalization of Delta. The receptor for mineralocorticoid hormones (e.g. aldosterone) that is a member of the nuclear receptor superfamily Machado Joseph disease proteases are cysteine proteases that have Josephin domain for deubiquitinating activity. See: chaperones. The Drosophila orthologoue of the anaphase promoting complex subunit 2 gene. Cellular structure from which the microtubular cytoskeleton radiates towards cell periphery; usually a synonym for the centrosome plus the associated pericentrosomal material, however e.g. in human oocytes MTOC are actually acentrosomal, made only of the pericentrosomal material. A type of filamentous protein polymer found in the cytoplasm of eukaryotic cells, polymer of α and β tubulin arranged into a 13 protofilaments forming an empty tubule of ~24 nm of diamteter and varying length from several micrometers to possible millimeters in axons of nerve cells. MTs occurs singly or in pairs, triplets, or bundles. Microtubules is one of the main components of the cytoskeleton, they emanate from the MTOC located close to the nucleus and project to the periphery. In axons, MTs are involved in retrograde and anterograde transport.MTs also form the centrosomes, basal bodies, cilia and flagella as well as the spindle during mitosis and meiosis. Late endosomal organelles that form by invagination of the endosomal membrane to form intraluminal vesicles with subsequent fusion of the MVBs with lysosomes. The olfactory learning and memory centre in insects. Multilayed, lipid-rich membrane that wraps nerves to increase the efficiency of signal propagation along axons. Nedd8 is a ubiquitin-like small protein modifier. The Nedd8 conjugation process, called neddylation, is similar to ubiquitination. Neddylation utilizes the E1 activating-enzyme complex composed of two subunits, APP-BP1 and UBA3, and the E2 conjugating-enzyme, UBC12. The only known substrates of neddylation are Cullin family proteins (Cul1, Cul2, Cul3, Cul4A,
Book Glossary
N-end rule
Neocortex
Nerve growth factor: (NGF)
Netrin-1
Neuralized Neuralized homology repeat Neurodegeneration
Neurofibrillary degeneration (NFD)
Neurofibrillary tangles (nfts)
Neurofibromatosis
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Cul4B, and Cul5) which have been shown to be modified by Nedd8 in mammalian cells. The rule which determines that protein stability depends on specific amino acids present at the N-terminus. While for example Met is characteristic of long lived proteins, N-terminal Arg induces quick ubiquitination and proteasomal degradation of proteins. Destabilizing amino acids at the N-terminus are the “degrons”. a part of the brain of mammals (also birds and reptiles); the top layer of the cerebral hemispheres. Other names neopallium and isocortex. A naturally occurring molecule in the body which stimulates the growth and differentiation of the sympathetic and certain sensory nerves. NGF is a protein that consists of 3 types of polypeptide chains -- alpha, beta and gamma -- that interact to form the protein. The NGF β chain (NGF β) is solely responsible for the nerve growth stimulating activity of NGF. The NGF β gene is in chromosome band 1p22. A secreted protein with homology to the extracellular matrix molecule laminin with attractive and repulsive effects on growing axons. A Drosohila E3 ubiquitin ligase which regulates the Notch-Delta signalling pathway via the ubiqutination of Delta. A novel protein domain of unknown function identified in the neuralized gene. Progressive damage or death of neurons causing a gradual decline of bodily functions regulated by the affected parts of the nervous system neurofibrillary degeneration is the formation of coarse, argentophilic, intracytoplasmic fibres, often in complex tangles within intracranial nerve cells that are undergoing aging. NFD corresponds to the intracellular accumulation of pathological fibrils in the cytosol of neurons. In Alzheimer’s disease, NFD may be caused by the abnormal aggregation of tau proteins. Accumulation of twisted protein fragments inside neurons. Neurofibrillary tangles are one of the characteristics structural abnormalities found in the brains of patients with Alzheimer's disease patients. Upon autopsy, the presence of amyloid plaques and neurofibrillary tangles is used to positively diagnose Alzheimer's disease. In Alzheimer’s disease and other tauopathies, tangles are mainly formed by abnormally modified tau protein. Autosomal dominant disorders associated with deregulated Schwann cell proliferation and are classified as type 1 or type 2. The hallmark of NF1 is neurofibromas, while NF2 is associated with schwannomas.
1080 Neurogenesis Neuropathic pain Neuropathy Neuroserpin Neutrophil
NF-κB
NF-κb essential modulator (NEMO)
Nissl’s bodies
Nmda
Nob1p (Nin One Binding Protein) Nociception, nociceptive: Noncovalent inhibitors of the 20S proteasome
Nondisjunction
Mario DiNapoli and Cezary Wójcik The stage of development during which neuronal precursors cells proliferate to generate neurones. Chronic pain arising from damage to the nervous system, either to the peripheral nerves or to the central nervous system. Any disorder affecting any segment of the peripheral nervous system. In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. a white blood cell with conspicuous cytoplasmic granules (granulocyte) involved in host defense. nuclear factor-κB; a ubiquitous prosurvival and proinflammatory transcription factor composed of two different subunits of the Rel family. Inactive NF-κB is bound to an inhibitory protein IκB in the cytoplasm, which masks its nuclear localization signal. Upon activation of specific receptors (for example TNF R1), IκBα is phosphorylated, ubiquitinated and degraded by the proteasome releasing active NF-κB, which translocates to the nucleus and induces the expression of specific genes. is a regulatory subunit of the inhibitor of IκB kinase (IKK) complex. It contains multiple coiled-coil motifs and a zinc finger at the COOH-terminal end. NEMO is required for the assembly and activation of the IKK complex in response to a wide range of NFκB-stimulating signals, including tumor necrosis factor (TNF). Chromophil substance in the form of granules found in the cell bodies and dendrites of neurons, but is absent from axons. They consist principally of the ribose type of nucleic acid and nucleoprotein and stain strongly with basic aniline dyes. They are concerned with protein synthesis and metabolism; their condition varies with physiological and pathological conditions. N-methyl-D-aspartic acid. Agonist for the NMDA receptor, mimics the effects of glutamate. A synthetic amino acid derivative that is useful in neurochemical research to distinguish between different glutamate receptor subtypes. Nin One Binding Protein involved in the maturation of 20S proteasome by Ump1 The sensory detection of noxious stimuli, which may result in a sensation of pain. small molecules that bind reversibly to the substrate binding sites in the active sites located on the β-subunits without modifying the catalytic active N-terminal threonine; generated by combinatorial chemistry or identified by high throughput screening ; exhibit low cytotoxicity against normal cells. failure of chromosomes to segregate during meiosis
Book Glossary Non-dopaminergic neurons:
Normoxic N-terminal nucleophile hydrolase (Ntn hydrolases) Ntn-hydrolases Nuclear export signal (NE)
nuclear factor-κb (nf-κb)
Nuclear receptor
Nucleation
NZB domain
Oligoclonal bands
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neurons that use neurotransmitters other than dopamine. Nondopaminergic neurons affected by PD include the noradrenergic neurons in the locus coeruleus, serotonergic cells in the dorsal raphe, cholinergic cells in the nucleus basalis of Meynert and pyramidal neurons in parts of the hippocampal formation At or containing a normal level of oxygen. superfamily of three known enzymes that use the side chain of the amino-terminal residue as the nucleophile in the catalytic attack at the carbonyl carbon. The nucleophile (protor donor) is threonine in the 20S proteasome, serine in penicillin acylase and cysteine in glutamine PRPP amidotransferase. class of enzymes which perform their catalytic activities relaying on the N-terminal aminoacid residue as nucleophile is an amino acid sequence used to localize the protein to the cell nucleus through the nuclear pore complex. Usually, this signal consists of a few short sequences of positively charged lysines or arginines. A family of transcription factors important for pro-inflammatory and antiapoptotic responses. They are activated by the phosphorylation and subsequent ubiquitindependent proteolytic degradation of their respective inhibitors, known as inhibitor of κB (IκB). Phosphorylation of IκB occurs through tissuespecific kinases, IκB kinase 1 (IKK1) and IKK2. A specific family of hormone, vitamin or small metabolite receptors that share a common structural and functional organization including a zinc-finger DNA-binding domain and carboxyl-terminal ligand-binding domain. These receptors are generally localized predominantly in the nucleus but can also be found in the cytoplasm. A process by which the addition of a small amount of preaggregated protein to a monomeric preparation of the same protein robustly accelerates the assembly of amyloid fibrils. Also known as NZF. Putative zinc finger domain found at the Cterminus of Npl4, a cofactor of valosin-containing protein, that binds polyubiquitylated substrates. Similar domains are also found in TAK1-binding protein (TAB2), Vps36 and RBCK2. NZB domains appear to bind both Lys-48- and Lys-63-linked ubiquitin chains. Immunoglobulins visualized as discrete bands by isoelectric focusing. If the bands are present in the cerebrospinal fluid only and not in the corresponding serum, this is interpreted as a sign of intrathecal immunoglobuline synthesis. Oligoclonal bands in the cerebrospinal fluid are found in different inflammatory diseases affecting the central nervous system, in particular in multiple
1082
Omuralide
Open gate proteasome
Out (Ovarian tumor proteases)
Oxidative stress P element
Pa200
Pa28
PAC1-PAC2 complex Pael-R Pam
Mario DiNapoli and Cezary Wójcik sclerosis the name for the highly selective proteasome inhibitor β-clastolactacystin proposed by Corey group in honor of discovery of the lactacystin by Omura research group; its synthetic analog, MLN519, is currently under clinical evaluation for the treatment of acute stroke and myocardial infarction. The 20S core particle of the proteasome is characterized by a central axial channel, which is gated at both ends. Regulatory subunits, such as the 19S particle, or the presence of substrates can modify the size of the gates. The proteasome therefore can assume dynamically both an open and close gate conformation. Mutant proteasomes with deletion of the α3 N-terminal chain are open gate proteasomes, which are able to degrade substrates at a faster rate than the wilde type. A Drosophila protein involved in oocyte morphogenesis. OUT are deubiquitinating enzymes that have homology to viral cysteine proteases in its catalytic domain sequence. As the putative catalytic cysteine is replaced by a serine it is not clear whether OTU is an active protease or in inactivated protease homologue. A build-up of free radicals and H2O2 resulting in cell dmage and disease. a small segment of DNA called a transposable element that is capable of moving from one genomic location to another in Drosophila The mammalian ortholog of blm3p (now called blm10p), a ~200kDa protein composed of numerous HEAT repeats, that binds either one or both ends of the 20S proteasome and activates peptide hydrolysis by the 20S particle in vitro. PA200 is a nuclear protein proposed to link proteasomes to repair mechanisms at DNA double strand breaks. proteasome activator formed by different members of the family of small 28 kDa proteins termed α, β or γ that share significant sequence homology with one another and function as ATPindependent homo- (α/α, γ/γ) or hetero- (α/β) heptameric rings that cap one or both ends of the 20S catalytic core particle. Binding of PA28 oligomers to the 20S proteasome causes gating and activation of the proteasome’s peptidase activity. Also known as 11S cap. PA28 γ known formerly as Ki antigen. associates with the α-subunits before α-rings are complete; functios as a scaffold for α-ring assembly A putative G protein-coupled transmembrane polypeptide identified as an intracellular substrate of Parkin. Protein associated with Myc, originally identified from a human cDNA library by its interaction with the transcriptional activating
Book Glossary
PAN (proteasomeactivating nucleotidase) Paraneoplastic cerebellar degeneration Paraneoplastic encephalomyelitis Parkin
Parkinson’s disease (PD)
Parvalbumin immunoreactive neurons
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domain of the c-terminus of Myc. PAM is the human homolgoue of the Drosophila highwire and C.elegans RPN-1 genes. The ATPase complex from archaebacteria that is highly homologous to the ATPases of the eukaryotic 19S proteasomeregulatory complex. Quality control: A system for ‘proof-reading’ that distinguishes native from non-native protein conformations. Cerebellar dysfunction characterised by loss of balance and coordination, with a subacute onset, that occurs in association with cancer Encephalomyelitis, in particular affecting the brainstem and the limbic system, that is associated with different malignancies Parkin is an E3 ligase in the ubiquitin-proteasome system. Many mutations in parkin have been associated with a familial form of Parkinson's disease termed autosomal recessive juvenile parkinsonism. How loss of function of parkin leads to dopaminergic cell death in this disease is unclear. The prevailing hypothesis is that parkin helps degrade one or more proteins toxic to dopaminergic neurons. Putative substrates of parkin include synphilin-1, CDC-rel1, CDC-rel2, cyclinE, p38 tRNA synthase, Pael-R, synaptotagmin XI, synphilin-1, sp22 and parkin itself. See also Ubiquitin ligase. is a neurodegenerative movement disorder, clinically characterized by a resting tremor, rigidity, hypokinesia and postural instability. The neuropathological hallmarks are intraneuronal Lewy bodies and dystrophic neurites (Lewy neurites), which both contain aggregated proteins, such as a-synuclein, ubiquitinated proteins, parkin and Pael-R (a parkin substrate). The loss of dopaminergic neurons in the substantia nigra pars compacta is the major cause of the clinical movement problems but it has been shown that more widespread neuropathology is present in the brains of PD patients, including degeneration of noradrenergic, serotonergic, peptidergic and cholinergic systems, before degeneration of the substantia nigra occurs. The autosomal dominant and recessive forms of PD are caused by mutations in the genes encoding a-synuclein, two ubiquitination enzymes (parkin and UCH-L1), the molecular chaperone DJ-1 and the signaling molecule leucine-rich repeat kinase 2. Moreover, overexpression of a-synuclein by duplication or triplication of the gene can also lead to PD. Similar to other neurodegenerative diseases, PD is primarily a sporadic disorder with a complex etiology. Parvalbumin is a calcium binding protein that belongs to the so called ‘EF-hand’ family of calcium binding proteins. Other notable members are calbindin-D28K and calretinin.These three proteins have been used as markers for distinct subpopulations of cortical
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Pathognomonic Paw withdrawal latency: Paw withdrawal threshold: : PC domain
PelizaeusMerzbacher disease:
Penetrance Pgph
Pharmacology
Mario DiNapoli and Cezary Wójcik interneurons. distinctively characteristic for a particular disease to the point of aiding diagnosis Behavioural measurement – recording of the latency for an animal to withdraw its paw from a noxious thermal stimulus. Behavioural measurement – recording of the mechanical force or pressure required for an animal to withdraw its paw. Repeat of 35-40 residues found in subunits S1 and S2 of the 26S proteasome and subunit Apc1 of the anaphase-promoting complex or cyclosome. The most highly conserved feature in these repeats is an alternating pattern of large aliphatic residues and glycine or alanine. The variable part of the repeats contains a pattern of hydrophobic and hydrophilic residues with a periodicity of 3.6 typical of amphipathic helices. These repeats are proposed to fold into structures resembling α-helical toroids. A disorder of the central nervous system (CNS) in which there is loss of myelin, the sheath around the nerves. The disease is clinically characterized by nystagmus (rhythmical oscillation of the eyes), impaired motor development, tremor, progressive spasticity (increased muscle tone), ataxia (wobbliness), choreoathetotic movements, and dysartria (difficulty speaking). PelizaeusMerzbacher disease (PMD) in its classical form manifests in infancy or early childhood and progresses to severe spasticity and ataxia. The lifespan may be shortened.PMD is due to mutation in the gene PLP1. This gene is located on the X chromosome in band Xq22. The disease describes an X-linked pattern of inheritance with boys who have the mutation affected with the disease while females with the mutation are carriers. The PLP1 gene encodes proteolipid protein (PLP), the most abundant protein of the myelin sheath in the CNS. The mutation in PLP1 in PMD results in loss of myelin and that, in turn, causes the neurological abnormalities. The severity of myelin loss is dependent on the particular PLP1 mutation and can range from early lethal forms of PMD to a mild disorder known as spastic paraplegia type 2 (SPG2). Among the mutations in the PLP1 gene locus that can cause PMD is a duplication of PLP1 in which the duplicated region may be far away from the original PLP locus in chromosome region Xq22. The PLP1 duplication is almost always present in the mothers of affected boys and usually can be traced to the maternal grandfather. the frequency of expression of a phenotype for a given genotype Peptidylglutamyl-peptide hydrolysing activity; proteasomal activity cleaving after acidic residues and associated to the β1 subunit Also called ‘caspase-like’ activity or “post-acidic” activity. the study of the drugs with respect to their origin, nature,
Book Glossary
Phfs: Paired helical filaments
Phosphorylation / Hyperphosphorylati on of tau protein
Phr-1 PI3K (phosphatidylinosito l-3-kinase) Pick's Disease (pid)
Piriform cortex
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properties, and mechanisms of actions and their effects on living tissues and organisms. Filaments found in degenerating neurons in Alzheimer’s disease. These filaments are mostly composed of abnormal tau proteins. These filaments are also characteristic of other neuropathological disorders called “tauopathies”. The phosphorylation is the addition of a phosphate group to a compound by an enzyme (e.g., thymidine kinase, tyrosine kinase…). Phosphorylation is an essential step in many cellular processes. Phosphorylation may cause conformational changes in proteins or activate particular enzymes. Concerning tau proteins, its phosphorylation will change its activity in neurons and mostly its capacity to bind and polymerase Microtubules. The hyperphosphorylation of tau is an increase of its normal phosphorylation and an appearance of new phosphorylation sites on tau molecule. The hyperphosphorylation of tau is thought to be involved in its intraneuronal aggregation as described in Alzheimer’s disease and other tauopathies. The mouse homologue of the Drosophila highwire gene, a potential E3 ubiquitin ligase. large family of enzymes that catalyse the phosphorylation of inositol-containing lipids, thus transmitting signals from tyrosine kinases and G-protein coupled receptors. PI3K pathway regulates proliferation, growth, apoptosis and cytoskeletal rearrangement. Pick's disease is a dementing illness associated with deterioration of the frontal and temporal lobes of the brain. Symptoms may include a decline in social behavior (including disinhibition, tactlessness, and breaches of interpersonal etiquette), emotional blunting, apathy, changes in eating habits (including increased appetite, weight gain, and increased preference for sweets), attention problems, decreased insight, speech and language problems (including reduced speech ability, repetition of phrases heard, reduced use of nouns, difficulty naming objects, loss of word meaning, diminished writing ability, and mutism), and difficulty recognizing faces. Though Alzheimer's disease and other forms of dementia can sometimes cause similar symptoms, Pick's disease is more likely to cause certain deficits in behavior and speech (such as disinhibition or loss of nouns), while memory and visuospatial function (which are frequently affected by Alzheimer's Disease) tend to be relatively spared. Also, the onset of Pick's Disease (usually between the ages of 45 and 65) is earlier than is normally seen in Alzheimer's disease. part of paleopallium that together with olfactory cortex relates to the perception of smells; present in amphibians, reptiles, birds and
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Polyglutamine (polyq) diseases
Polyglutamine tract Postsynaptic density
Preselenins
Prevalence Primary afferents: Prion
Prion diseases
Prion protein
Mario DiNapoli and Cezary Wójcik mammals Group of neurodegenerative diseases that includes Huntington’s, Spinal Bulbar Muscular Atrophy, Dentatorubral Pallidoluysian Atrophy and a number of Spinocerebellar Ataxias (1, 2, 3, 6, 7, 17). These diseases are caused by mutation within the coding regions of several unrelated proteins resulting in the expansion of polyglutamine tracts within these proteins. A primary hallmark of polyQ diseases is the presence of intracellular, often nuclear, polyQ inclusion bodies deposited within the diseased brains of polyQ patients. PolyQ inclusions contained the polyQ-expanded protein along with chaperones and components of the ubiquitinproteasome system. A repeated sequence of glutamine residues within a protein. The intracellular area immediately beneath the postsynaptic membrane of a synapse, which contains a high density of specialised proteins, particularly receptor binding proteins associated with synaptic function. Presenilins are essential components of γ-secretase, a protease complex catalyzing intramembrane proteolysis of various type I membrane proteins, including the amyloid precursor protein and the Notch receptor. Important in the pathogenesis of Alzheimer’s disease and in normal development and expressed in many tissues, presenilins (PS1 and PS2) are proteins with multiple transmembrane domains and are processed into N-terminal and Cterminal fragments (NTF and CTF). Both proteins are predominantly located within the endoplasmic reticulum (ER) and early Golgi apparatus. The exact functions associated with PS proteins have not been fully characterized yet. the total number of cases of a disease in a given population at a given time. Peripheral sensory neurons, which are activated by stimuli to the periphery and synapse in the dorsal horn of the spinal cord. This term is an abbreviation for “proteinaceous infectious particle”, the putative infectious agent of prion diseases according to the prion hypothesis. A group of neurodegenerative diseases that affect humans and animals, also known as transmissible spongiform encephalopathies. An example of a human prion disease is Creutzfeldt-Jakob disease, examples of animal prion diseases are scrapie in sheep and bovine spongiform encephalopathy in cattle. A protein that plays a key role in prion diseases. The normal form of the prion protein is called PrPC (C: cellular). In diseased tissue PrPC has been post-translationally modified into a disease associated form, often called PrPSc (Sc: scrapie), which is thought
Book Glossary
PRKAR1A (protein kinase A regulatory subunit 1(α)
Progressive supranuclear palsy (PSP)
Projection domain of tau protein Proline directed protein kinases (PDPK) Proteasome activator
Proteasome cleavage prediction algorithm
Proteasome inhibitors
Proteasomes Protein activator Protein aggregate
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to be part of the infectious unit of prion diseases, the prion. 381-amino acid protein. The holoenzyme of PKA, a tetramer consisting of 2 regulatory and 2 catalytic subunits, is inactive in the absence of cAMP. Activation occurs when 2 cAMP molecules bind to each regulatory subunit, eliciting a reversible conformational change that releases active catalytic subunits. Germline mutations in PRKAR1A, an apparent tumor-suppressor gene, are responsible for the Carney complex phenotype. Progressive supranuclear palsy (PSP) (or the Steele-RichardsonOlszewski syndrome, after the Canadian physicians who described it in 1963) is a rare degenerative disorder involving the gradual deterioration and death of selected neurons in the brain. Typical effects are problems with control of gait and balance, and an inability to aim the eyes properly, especially in the vertical directions (downward gaze palsy). Other symptoms may be alterations of mood and behavior, depression and apathy as well as mild dementia. There is currently no effective treatment for the disease. The projection domain was given to the N-terminal part of Tau proteins. The PDPK are kinases, which phosphorylate serine and threonine only if these amino acids are followed by a praline. Group of molecules that include the 19S subunit of the 26S proteasome, PA28 and PA200 (blm10p) which bind one or both ends of the 20S proteasome and activate its catalytic activity in an ATP-dependent (19S subunit) or ATP-independent manner (PA28 and PA200). Bioinformatics tools based on experimental data on proteasome degradation and on mathematical models. These algorithms are useful to predict the position of proteasome cleavage (also known as cleavage site) in a given amino acid sequence. These algorithms are used in immunology to predict intracellularly generated antigenic peptides presented on MHC class I molecules in the context of antigen presentation. These are classified into four groups: lactacystin and β-lactone derivates, vinyl sulfones, peptide aldehydes and peptide boronates. The aldehyde and boronate inhibitors are reversible and more amenable to clinical use. A term used to describe either 20S proteasomes (constitutive or immunoproteasomes ) or 26S proteasomes. a binding protein that regulates positively another protein An abnormal protein assembly that results from the cohesion of two or more misfolded monomeric proteins. Protein aggregates that
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Protein aggregation
Protein Kinase A,B,C and N (PKA/B/C and N)
Mario DiNapoli and Cezary Wójcik form amyloid fibrils are often resistant to solubilization with ionic detergents after boiling. the clumping of proteins, in particular proteins which are misfolded or which contain multiple β-sheets. Protein aggregation depends mostly on hydrophobic interactions. PKA: In cell biology, cAMP-dependent protein kinase (cAPK), also known as protein kinase A (PKA, EC 2.7.1.37), refers to a family of enzymes whose activity is dependent on the level of cyclic AMP (cAMP) in the cell. Each PKA is a holoenzyme that consists of two regulatory and two catalytic subunits. Under low levels of cAMP, the holoenzyme remains intact and is catalytically inactive. When the concentration of cAMP rises (e.g. activation of adenylate cyclases by certain G protein-coupled receptors, inhibition of phosphodiesterases which degrade cAMP), cAMP binds to the two binding sites on the regulatory subunits, which then undergo a conformational change that releases the catalytic subunits. The free catalytic subunits can then catalyze the transfer of ATP terminal phosphates to protein substrates at serine, or threonine residues. This phosphorylation usually results in a change in activity of the substrate. Since PKAs are present in a variety of cells and act on different substrates, PKA and cAMP regulation are involved in many different pathways. In addition, the effects of PKA phosphylation are generally transient because protein phosphatases quickly dephosphorylate PKA targets. PKB: Akt1, also known as "Akt" or protein kinase B (PKB) is an important molecule in mammalian cellular signaling. There are three genes in the "Akt family": Akt1, Akt2, and Akt3. Akt1 is involved in cellular survival pathways, by inhibiting apoptotic processes. Akt1 is also able to induce protein synthesis pathways, and is therefore a key signaling protein in the cellular pathways that lead to skeletal muscle hypertrophy, and general tissue growth. Since it can block apoptosis, and thereby promote cell survival, Akt1 has been implicated as a major factor in many types of cancer. Akt2 is an important signaling molecule in the Insulin signaling pathway. It's required to induce glucose transport. The role of Akt3 is less clear, though it appears to be predominantly expressed in brain. PKC: Protein kinase C ('PKC', EC 2.7.1.37) is actually a family of protein kinases consisting of ~10 isozymes. They are divided into three subfamilies: conventional (or classical), novel, and atypical based on their second messenger requirements. Conventional (c) PKCs contain the isoforms I, II, and III. These require Ca2+, diacylglycerol (DAG), and a phospholipid such as phosphatidylcholine for activation. Novel (n)PKCs isoforms require DAG, but do not require Ca2+ for activation. Thus,
Book Glossary
Protein Phosphatase 1, 2A and 2B (PP1/2A and 2B)
Proteolysis inducers
Proteolytic center of the cell
Proto-oncogene protein c-myctranscription factor
PTEN (protein and tensin homolog
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conventional and novel PKCs are activated through the same signal transduction pathway as phospholipase C. On the other hand, Atypical (a)PKCs require neither Ca2+ nor diacylglycerol for activation. Protein phosphatases are enzymes that remove phosphate groups that have been attached to amino acid residues of proteins by protein kinases. Whereas a kinase enzymatically adds a phosphate to a protein, a phosphatase's purpose is phosphate removal. It should be noted that phosphate addition and removal do not necessarily correspond to enzyme activation or inhibition, and that several enzymes have separate phosphorylation sites for activating or inhibiting functional regulation. CDK, for example can be either activated or deactivated depending on the speicific amino acid residue being phosphorylated. The phosphates are important in signal transduction by regulating the proteins they are attached to. To reverse the regulatory effect, the phosphate has to be removed. This occurs on its own by hydrolysis or is mediated by protein phosphatases. Serine and threonine phosphates are stable under physiological conditions, so a phosphatase has to remove the phosphate to reverse the regulation. There are four known groups: PP1, PP2A, PP2B (AKA calcineurin) and PP2C. The first three have sequence homology in the catalytic domain, but differ in substrate specifity. Ser/Thr-specific protein phosphatases are regulated by their location within the cell and by specific inhibitor proteins. small molecules designated to recruit a disease-promoting protein for ubiquitination and degradation by the 20S proteasome; include proteolysis-targeting chimeric molecules (protacs), and the small molecule proteolysis inducers (SMPI); currently being tested in preclinical settings for therapeutic potential in selected types of cancer. An area around the centrosome, enriched in proteasomes and other components of the UPS; multiple substrates of the UPS arrive to the p.c. by microtubule mediated transport, where they are ubiquitinated and degraded. When the UPS is overwhelmed, its degradative capacity is diminished leading to the accumulation of proteins forming a structured aggregate, or aggresome. is the product of v-myc myelocytomatosis viral oncogene homolog, and is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. It functions as a transcription factor that regulates transcription of specific target genes. encodes a protein-and lipid phosphatase that controls PI3K (phosphatidylinositol-3-kinase) cascade and also intervenes in cell
1090 deleted on chromosome 10)/ MMAC1 (mutated in multiple advanced cancers-1) PTTG (pituitary tumor transforming gene) Purkinje cells
Reactive oxygen species (ROS). Reperfusion Retrotranslocation
RING-FINGER
Ring-finger proteins
RNA interference
Roundabout Scf ubiquitin-ligase complex
Mario DiNapoli and Cezary Wójcik cycle regulation, DNA repair, apoptosis, senescence, and inhibition of angiogenesis
located on chromosome 5q33, encodes human securin that blocks sister chromatid separation until the beginning of anaphase. Its over-expression can cause aneuploidy. Inhibitory neurons in the cerebellum that use GABA γaminobutyric acid) as their neurotransmitter.Their cell bodies are situated beneath the molecular layer, and their dendrites branch extensively in this layer. Their axons project into the underlying white matter, and they provide the only output from the cerebellar cortex. Highly reactive oxygenbased molecules with an unpaired electron in their outer orbital that are capable of damaging proteins, lipids and nucleic acids. Examples include hydrogen peroxide and hydroxyl radicals. the restoration of blood flow to an organ or tissue. also called dislocation. A process by which proteins destined for degradation by cytoplasmic 26S proteasomes (ERAD) are extracted from the ER in a direction opposite to their insertion during protein synthesis (translocation). Retrotranslocation occurs either through the same Sec61 channel used for translocation, or through a specialized channel composed of derlins, Doa10 and other proteins. The RING (really interesting new gene) consensus sequence is: CX2CX(9–39)CX(1–3)HX(2–3)C/HX2CX(4–8)CX2C. The cysteines and histidines represent metal binding sites. The first, second, fifth and sixth of these bind one zinc ion and the third, fourth, seventh and eighth bind the second zinc ion. A family of proteins that are structurally defined by the presence of the zinc-binding RING-finger motif. Many RING-finger proteins are ubiquitin ligases or subunits thereof. A process by which small interfering RNAs induce cleavage of specific mRNAs within cells inducing a functional konockdown of a specific gene product. While RNAi is an ancient mechanism which evolved as a response against certain viruses and retrotransposomes, it can be exploited for experimental purposes or for therapy. A transmembrane receptor for the slit family of ligands involved in mediating axon guidance. A multisubunit E3 ubiquitin ligase, which is composed of Skp1, cullin-1 protein, F-box protein, and Rbx1/Roc-1 RINGfinger protein. The F-box protein is the substrate recruiting factor.
Book Glossary Schwann cell: Semaphorin 1A
Semaphorin 3A
Sequestosome 1
SKP2 (S-phase kinase interacting protein 2 / CDK2/Cyclin Aassociated protein p45) SMAD Small mothers against decapentaplegic Smad ubiquitin regulatory factor Small heat shock proteins
Small inhibitory rna
Snaap Somatosensory: Spherical and annular oligomers
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Glial cells of the peripheral nervous system, responsible for myelin formation and other supportive functions. A transmembrane member of the semaphorin family of molecules which may have an attractive effect on growing axons during development. A secreted member of the semaphorin family of molecules implicated in having attractive and repulsive effect on growing axons during development. a protein with seven structural motifs: an SH2 domain that binds the tyrosine kinase p56lck, an acidic interaction domain (AID) that binds the atypical PKC ζ, a ZZ type ZINC finger that binds the receptor interactive protein (RIP) involved in TNFα-induced apoptosis, a binding site for the RING-finger protein tumor necrosis factor receptor-associated factor 6 (TRAF6), two PEST sequences and a UBA domain that binds polyubiquitin chains F box protein, gene located on chromosome 5p13, recognizes protein substrates (e.g.p27) for ubiquitination by SCF complex/ ubiquitin ligase.
Intracellular mediators of the BMP signalling pathway.
Regulate BMP singalling pathway by targeting SMADs for ubiquitin-mediated proteolysis. sHsps have a molecular mass of less than 40 kDa and assemble into large, oligomeric structures that resemble a hollow ball.All sHSPs contain a conserved, C-terminal α-crystallin domain of about 100 residues that mediates oligomeric assembly. Similar to HSP90 chaperones, sHSPs transiently interact with and stabilize misfolded substrates, conceivably until the HSP70/HSP40 system can actively refold them. A small RNA molecule that interferes with normal RNA processing, causing rapid degradation of the endogenous RNA and thereby precluding translation. This provides a simple way of studying the effects of the absence of a gene product in simple organisms and in cells. proteasomal activity cleaving after small neutral amino acids The perception of sensory (such as mechanical, thermal and chemical) stimuli. Metastable structures observed in many amyloid-forming proteins that might be on a pathway to fibril formation. These structures have been proposed to be the principal toxic entities that mediate
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Spinobulbar muscular atrophy (SMBA): spinocerebellar ataxia, type III Spinocerebellar Ataxias (scas):
STAM (signaltransducing adaptor molecule) Straight filaments (sfs)
Stress activated Protein Kinases (SAPK)
Substantia nigra:
Sumo (Sumoylation)
Mario DiNapoli and Cezary Wójcik neuronal dysfunction. A polyglutamine tract disorder that manifests as a neuromuscular disease due to an expanded polyglutamine tract in the androgen receptor. See: Machado-Joseph Disease. A group of dominantly inherited diseases linked by the presence of a polyglutamine repeat in the relevant protein. They are of predominantly late-onset and may be subdivided based on clinical features and genetic mapping. STAM interacts with Hrs and may therefore be involved in endocytosis/vesicular transport. straight filaments are found in Alzheimer’s disease and other tauopathies such as the corticobasal degeneration and the progressive supranuclear palsy. They correspond to a specific aggregation of tau protein (4R tau). Protein kinases are enzymes that modify other proteins by adding phosphate groups to them (phosphorylation), changing their function radically. About thirty percent of proteins can be modified by kinases. Disregulated kinase activity is the root cause of many diseases, especially cancers, as kinases regulate many aspects of cell growth, movement, and apoptosis. Kinase-inhibiting drugs are being developed to treat several diseases. Kinases are stressactivated when specific events such as DNA damage or an overload of Ca2+ ions is detected. There are also kinases that are activated only by stress, referred to as c-Jun N-terminal kinases, or JNKs. These stress activated protein kinases respond to stress stimuli like cytokines, ultrafiolet radiation, heat schock, and osmotic shock. They're also involved in cell differentiation and apoptosis. a dark band of gray matter deep within the brain where pigmented cells manufacture the neurotransmitter dopamine for movement control. Degeneration of cells in this region lead to the neurologic movement disorder PD. Small Ubiquitin-related Modifier or SUMO proteins are a family of small proteins, most are around 100 amino acids in length and 12 kDa in mass, that are covalently attached to and detached from other proteins in cells to modify their function, using a mechanism analogous to, but distinct from, ubiquitin. The exact length and mass varies between SUMO family members and depends on which organism the protein comes from. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO
Book Glossary
Suppressor of deltex Suppressors of cytokine signaling (SOCS) Suspended paw elevation time: Symptomatic epilepsy Synapse
Tau tubulin kinase (ttk) Tauopathies
T-l (trypsin-like activity) Tnf-receptor family
Tnf-receptorassociated factors (trafs)
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is produced when the last four amino acids of the C-terminus have been cleaved off. Sumoylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. A Drosophila E3 ubiquitin ligase that regulates the Notch signalling pathway. proteins comprise a family of cytoplasmic negative feedback regulators of cytokine signaling. These proteins inhibit JAK kinases activated by numerous cytokine receptors. Behavioural measurement – test for cold allodynia in neuropathic animals – recording suspension of the injured paw from 4ºC water over several seconds following immersion of the paws. epilepsy with an identifiable cause, such as a brain tumor, brain trauma, or some other neurological disorder. Region of contact between the axon of the pre-synaptic neuron and the dendrite of the post-synaptic neuron. Synapses are specialized structures that create a microdomain between cells. This allows for the localized release of neurotransmitter from the pre-synaptic axon, resulting in the interaction of neurotransmitter with the neurotransmitter receptors present on the surface of the dendrite. TTK can phosphorylate serine, threonine and tyrosine hydroxyamino acids. This kinase is associated with cell proliferation and was shown to phosphorylate tau protein. Neurodegenerative disorders involving deposition of abnormal tau protein isoforms in neurons and glial cells in the brain. Pathological aggregations of tau proteins are sometimes associated with mutation of the tau gene on chromosome 17 in patients with FTDP-17. Proteasomal activity cleaving after basic residues and associated to the β2 subunit Members of this family function as trimers and multimers of trimers, and can trigger proliferation, survival, differentiation or death. A subfamily that comprises the death receptors Fas/CD95 and TNF-R1, as well as some other members of this family, contains a cytoplasmic region — the death domain — which is essential for inducing apoptosis. However, at the same tiem TNF-R1 can also elicit a prosurvival and proinflammatory response, activating NFκB. These are adaptor proteins for various cell-surface receptors.Most TRAFs encode a RING-finger motif at their amino-terminus; in the case of TRAF2 and TRAF5, the RING-finger is required for NFκB activation.
1094 TPR (tetratricorepeat motif)
Trail (tnf-related apoptosis inducing ligand) Transport mechanism of substrate
Transporters associated with antigen processing (TAP proteins) Tric/CCT Tubulin
Tubulin associated unit (tau)
Tumor suppressor
Mario DiNapoli and Cezary Wójcik The TPR domain is a 34-residue helix-turn-helix motif that facilitates protein–protein interactions. The TPRs of HOP, the cyclophillins and CHIP bind their cognate chaperones at the same C-terminal EEVD motif. Hip's TPRs enable it to bind the ATPase domain of Hsp70, but not the EEVD site. This induces apoptosis preferentially in transformed cells. In contrast to other death-inducing ligands, TRAIL is expressed in a wide range of tissues. Mechanisms responsible for the transport of substrate molecules within the 20S proteolytic chamber of the proteasome. Substrates enter the proteasome core particle in a partially unfolded or ubiquitinated state. The substrate composition influences the interaction with the external alpha ring of the 20S and/or with the regulatory caps subunits (19S, PA28). The rate at which substrates are degraded within the 20S can strongly be influenced by the transport mechanism. The forces defining the transport mechanism are complex and still largely unknown. are proteins of the endoplasmic reticulum responsible for the transport of cytosolic peptides to the lumen of ER. This process uses energy from ATP degradations and is necessary for the loading of peptide antigens into grooves of MHC class I molecules. Group II chaperonins, such as TCP-1 ring complex in the eukaryote is the protein which makes up microtubules. Microtubules are assembled from dimers of α- and β-tubulin. Each of these subunits has three domains. γ-tubulin is important in the nucleation and polar orientation of microtubule. Tubulin binds GTP and assembles onto the (+) ends of microtubules in the GTP-bound state. Once assembled into microtubules, it hydrolyzes GTP into GDP. The GDP-bound form of tubulin will disassemble from the tip of a microtubule, though it will not spontaneously fall out of the middle. This GTP cycle is essential for the dynamic instability of the microtubule. Tubulin was long thought to be specific to eukaryotes. Recently, however, the prokaryotic cell division protein FtsZ was shown to be evolutionarily related to tubulin. Delta and epsilon tubulin have been found to localize at centrioles and may play a role in forming the mitotic spindle during mitosis. Alpha and Beta Tubulins are proteins that have a molecular weight of approximately 55 kiloDaltons (kDa) each. Tau proteins are mainly expressed in neurons (6 isoforms in the central nervous system) where they act on the polymerisation and stability of Microtubules. These proteins belong to the family of Microtubule associated proteins (MAPs). regulates cell cycle, specifically the transition from G0 to G1. It
Book Glossary protein p53
Tumour-necrosis factor-α (tnf-α) Turban tumor syndrome.
Ubb+1
Ubiquitin
Ubiquitin activating enzyme (UBA) Ubiquitin and proteasomedependent proteolytic system (UPS) Ubiquitin conjugating enzyme (UBC)
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has low levels in normal cells and high levels in tumor cells. It contains DNA-binding, oligomerization and transcription activation domains. It binds as a tetramer to a p53-binding site and activates downstream genes inhibiting growth and/or invasion, functioning as a tumor suppressor. Mutants of p53, frequent in many human cancers, fail to bind the consensus DNA binding site, and hence cause loss of tumor suppressor activity. Alterations of the TP53 gene occur not only as somatic mutations in human malignancies, but also as germline mutations. A prototypic member of a family of cytokines that interact with several receptors, among them receptors that are responsible for eliciting apoptosis. Also called Familial cylindromatosis. A genetic syndrome in which numerous benign tumors of skin adnexa (such as the sweat glands) develop, principally on the head and neck. This disorder is inherited in an autosomal manner and is caused by mutation of the CYLD gene on chromosome 16q12-q13. Mutation of CYLD has been likened to having faulty brakes on a car. Instead of a pileup of cars, a pileup of cells results. Topical application of aspirin, another type of brake on cell proliferation, may possibly be useful. the turban tumor syndrome. Mutant ubiquitin. UBB+1 is a mutant form of ubiquitin that lacks the C-terminal Gly of wild type ubiquitin and instead has a 19 amino-acids extension. This mutant can itself be ubiquitinated but is unable to bind other proteins. UBB+1 is a powerful inhibitor of the proteasome activity when its intracellular concentration reaches a certain threshold. a heat stable low molecular mass (~8 kDa) protein formed by 76 amino acids, largely preserved during the phylogenesis, which is covalently attached through isopeptide bonds to substrate proteins (ubiquitination), often forming multiple adducts in form of polyubiquitin chains, serving in form of oligomers and in an ATP dependent manner binds the protein destined to destruction in the ubiquitin proteolytic system. See: E1. is the major nuclear and cytoplasmic proteolytic system which involves the degradation of proteins by 20S and 26S proteasomes, usually after previous ubiquitination. It requires energy in form of ATP for both ubiquitination and degradation by the 26S proteasomes. See: E2.
1096 Ubiquitin C-terminal hydrolases Uch Ubiquitin fusion degradation (UFD) Ubiquitin ligase Ubiquitin receptors
Ubiquitin-associated (UBA) domain
Ubiquitination (ubiquitylation)
Ubiquitinconjugating domain (ubc) Ubiquitininteracting motif (UIM)
Mario DiNapoli and Cezary Wójcik Ubiquitin C-terminal hydrolases are cysteine proteases that generate free ubiquitin mainly from ubiquitin adducts and ubiquitin precursors. a subdivision of the UPS in the cytosol, where uncleavable ubiquitin fused to other proteins destabilizes these proteins and induces their ubiquitination and proteasomal degradation. See: E3. Class of proteins that contain ubiquitin-binding domains that are known to associate directly with either mono- or polyubiquitin signals in partner proteins or proteasomal substrates rather than participate in ubiquitylation reactions. Known ubiquitin-binding domains include the ubiquitin-interacting motif (UIM), the ubiquitin-associated (UBA) domain, the ubiquitin-conjugating enzyme variant (UEV), VHS (Vps 27, Hrs, STAM), NZF (Npl4 zinc finger), and the polyubiquitin-associated zinc finger (PAZ) among others. Small domain of about 40-55 residues whose three-dimensional structure is a compact three-helix bundle of low sequence conservation. UBA domains have a relatively high affinity for Lys48-linked polyubiquitin chains and UBA domain proteins have been implicated in the ubiquitin fusion degradation pathway involving Lys-29-linked ubiquitin chains. Affinity of UBA domain proteins for monoubiquitin is ~10-500 μM whereas affinity towards polyubiquitin chains is between 2 and 4 orders of magnitude higher. The process of covalent attachement of ubiquitin to other proteins, which is achieved either through an isopeptide bond between an εamino group of Lys of the substrate and the C-terminal Gly of ubiquitin or through a peptide bond between N-terminal amino group of the substrate and the C-terminal Gly of ubiquitin. Monoubiquitination is the attachement of a single ubiquitin moiety to the substrate, multiubiquitination is the attachement of multiple ubiquitin moieties to different sites on the substrate, while polyubiquitination is the assembly of polyubiquitin chains, where one ubiquitin is conjugated to another through isopeptide linkages involving one of the 6 Lys present in the ubiquitin molecule. Ubiquitination is carried on by the E1-E2-E3 cascade of enzymes. The ~16-kDa ubiquitin conjugating domain of E2s harbours the active-site cysteine residue that is required for the formation of a thioester-linked E2-ubiquitin complex. Composed of approximately 20 amino acids, UIMs are characterized by “LALAL” motifs within the sequence of the ubiquitin-binding sites of subunit S5a of the 26S proteasome. UIMs are present in diverse protein families, including proteins
Book Glossary
Ubiquitin-like (UBL) domain
Ubiquitin-specific proteases (usp) UBX domain
Ump1 Unfolded protein response (UPR)
UT3 domain
1097
involved in ubiquitylation and ubiquitin metabolism, proteolysis, and endocytosis. They bind either mono- or polyubiquitin with affinities ranging between 100 and 400 μM. This domain is found at or near the N-terminus of proteins and is defined by a stretch of 45-80 residues with significant sequence homology and very similar three-dimensional structure to ubiquitin. UBL domains bind to the 26S proteasome, where they may dock on the S1, S2 or S5a subunits of the 19S regulatory complex. Most UBL-containing proteins have functions related to the ubiquitin-proteasome system and may promote the assembly of proteasomal supercomplexes or degradasomes. See: deubiquitinating enzymes. The UBX domain comprises ~80-residue C-terminal modules structurally related to ubiquitin in spite of low sequence conservation with the latter. UBX domain proteins can be grouped into five evolutionarily conserved families represented by the human cofactor p47, and the Y33K, FAF-1, UBXD1 and Rep-8 proteins. The UBX domain has been proposed to function as a general binding module for valosin-containing protein/cdc48, an hexameric segregase that dissociates protein complexes. short-lived protein identified as a chaperone necessary for a correct proteasome assembly and maturation A concerted cellular reaction to the presence of misfolded proteins in the ER. It involves three main branches, depending on the activity of three ER transmembrane proteins: PEK/PERK, IRE1 and ATF6. The initial response involves activation of PEK/PERK, which phosphorylates cytsosolic eIF2α, leading to translational attenuation of most gene products accompanied by specific translational activation of a specialized array of gene products characterized by the presence of IRES or 5’ upstream alternative ORFs. Subsequently activation of the IRE1 endonuclease leads to the alternative cytosolic splicing of the transcription factor XBP1, while transit to the Golgi of ATF6 leads to the proteolytic cleavage of its transactivation domain. XBP1 and ATF6 induce the transcription of an array of genes which include ER chaperones, proteins involved in ERAD, and other aspects of tehs ecretory pathway. UPR has therefore a cytoprotective function, however when overactive, it can lead to apoptosis through a pthway involving activation of caspase 12 in mice or its functional homolog in humans, caspase 4. Region corresponding to amino acids 1-211 of the sequence of Ufd1. Structurally, it contains a double-ψ β-barrel fold and a αβ roll that resemble the N-terminal region of valosin-containing
1098
VCP (valosincontaining protein)
VHS domain
Wallerian degeneration
WD40 repeat
Mario DiNapoli and Cezary Wójcik protein (VCP). The UT3 domain of Ufd1 contains two nonoverlapping ubiquitin-binding regions located in the N-terminal double-ψ β-barrel domain: One that binds monoubiquitin with low affinity (Kd of 1-2 mM), and a region that binds polyubiquitin in Lys-48 linkage. An essential, ubiquitous and abundant ATPase of the AAA family of 97 kDa, forming a ring-shaped homohexamer of a 97 kDa . VCP is able to bind polyubiquitin chains and misfolded proteins. It associates with over 30 different proteins forming complexes involved in diverse cellular activities, including membrane fusion, mitosis, apoptosis, nucleotide repair and ubiquitin-dependent degradation of proteins. VCP functions in the UPS often in association with the Ufd1-Npl4 heterodimer. In particular, VCPUfd1-Npl4 is believed to participate in the retrotranslocation of proteins from the ER and their delivery to the 26S proteasome (ERAD). Also known as p97, Ter94 (in Drosophila) and Cdc48 (in yeast). The VHS (Vps-27, Hrs and STAM) domain is a ~150-residue long domain that contains eight α-helices (α1 through α8) and a Cterminal extension, and are often found at the N-terminus of proteins involved in membrane targeting/cargo recognition along the endocytic and secretory pathways. The eight α-helices fold into a curved double-layer superhelical structure with concave and convex surfaces. The first two helical hairpins (α1-α2 and α3-α4) within the VHS domain resemble HEAT repeats, whereas the third repeat, consisting of helices α5, α6 and α7, is reminiscent of the three-helix ARM repeat. VHS domains interact with sorting receptors and sorting signals within the cargo such as ubiquitin. The degeneration of an axon distal to a site of injury, which begins to occur at about 1.5 days after a lesion. Wallerian degeneration is delayed approximately tenfold in rats or mice that carry the dominantly acting slow Wallerian degeneration (WldS) gene. Minimally conserved domain of approximately 40-60 amino acid residues characterized by a glycine-histidine (GH) dipeptide 11 to 24 residues from its N-terminus, and separated approximately 40 amino acids from the end tryptophan-aspartic acid (WD) dipeptide. WD40 proteins are speculated to form a circularized β propeller structure that functions as a scaffold to which proteins bind through coordination with residues on the top and bottom surfaces of the propeller to constitute multiprotein complex assemblies. Most WD40-repeat proteins contain a cluster of at least 4 repeats and participate in many essential biological functions ranging from signal transduction, RNA synthesis and processing, vesicular trafficking, and cytoskeletal assembly to cell cycle regulation and
Book Glossary
Wishful thinking X-linked Spinal Bulbar Muscular Atrophy
XPC binding domain
1099
apoptosis. A Drosophila type II BMP receptor involved in retrograde signalling during synaptic development. Also called Kennedy Disease is a rare, slowly progressive muscular disorder that affects males only and is inherited as an X-linked genetic trait. Uncontrollable twitching (fasciculations) followed by weakness and wasting of the muscles becomes apparent some time after the age of fifteen. The muscles of the face, lips, tongue, mouth, throat, vocal chords, trunk and limbs may be affected. Very large calves may also be found in some patients with this disorder. Kennedy disease is caused by a mutation in the androgen receptor (AR) gene. Androgen insensitivity leads to abnormal swelling of the breasts (gynecomastia), small testes and infertility. Region in Rad23 that binds to Rad4, the yeast homolog of Xeroderma pigmentosum group C complementing protein (XPC) promoting the assembly of a multiprotein nucleotide excision repair (NER) complex at the site of DNA lesions. The XPC domain of Rad23 also binds cytosolic peptide:N-glycanase allowing the formation of a degradasome for the turning over of N-linked glycoproteins dislocated from the endoplasmic reticulum.
Appendix I. Nomenclature of proteasome subunits [Reprinted with the permission of BIOMOL International LP (www.biomol.com)] Nomenclature Baumeister , et al. [1]
Gene 'Old' human
1º Acc. # (human)
Seq. length (amino acids)
MW
Coux et al. [2]
Groll et al. [3]
Miscellaneou s
UniP rotK B [4]
Human
Yeast (S.c.)
20S α-type subunits α1 iota
Pro-α6
α1_sc
α6
PSMA6
PRS2
P60900
246
27399
α2
C3
Pro-α2
α2_sc
α2
PSMA2
PRS4
P25787
233
25767
α3
C9
Pro-α4
α3_sc
α4
PSMA4
PRS5
P25789
261
29484
α4
C6
Pro-α3
α4_sc
α7
PSMA7
248
27887
zeta
Pro-α1
α5_sc
α5
PSMA5
P28066
241
26411
α6
C2
Pro-α5
α6_sc
α1
PSMA1
P25786
263
29556
α7
C8
Pro-α7
α7_sc
nu, Pros30, p30k, Pre5 Pre10, Prs1, C1, Prc1
α3
PSMA3
PRE 6 PUP 2 PRE 5 PRS1
O14818
α5
Pros27, p27k, C7, Prs2, Y8, Prc2, Scl1 Pre8, Prs4, Y7 Pre9, Prs5, Y13 XAPC-7, Pre6 Pup2, Doa5
P25788
254
28302
20S β-type subunits β1 Y
Pro-β3
β1_sc
β6
PSMB6
Lmp2
Pro-β3
β9
PSMB9
PRE 3 -
P28072
β1i
delta, Lmp9, Pre3 Ring12
β2
Z
Pro-β2
β7
PSMB7
Pro-β2
β10
β3
MECL1 C10
Pro-β6
β3_sc
theta, Pup3
β3
PSMB1 0 PSMB3
PUP 1 -
Q99436
β2i
Lmp19, MC14, Pup1 Lmp10
P49720
25358/2190 4 23264/2127 6 29965/2521 8 28936/2464 8 22949
β4
C7
Pro-β4
β4_sc
Pre1, C11
β2
PSMB2
P49721
201
22836
β5
X
Pro-β1
β5_sc
β5
PSMB5
P28074
208/20 4
22897/2245 8
β5i
Lmp7
Pro-β1
β8
PSMB8
-
P28062
β6
C5
Pro-β5
β6_sc
β1
PSMB1
PRS3
P20618
276/20 4 241
30354/2266 0 26489
β7
N3
Pro-β7
β7_sc
epsilon, Lmp17, MB1, Pre2, Doa3, Prg1 Ring10, Y2, C13 gamma, Pre7, Prs3, C5, Pts1 beta, Pros26, Pre4
PUP 3 PRE 1 PRE 2
239/20 5 219/19 9 277/23 4 273/23 4 205
β4
PSMB4
PRE 4
P28070
264/21 9
29192/2438 0
β2_sc
P28065
P40306
(Da)
Mario DiNapoli and Cezary Wojcik
1102
Appendix I. Nomenclature of proteasome subunits (cont.d) 19S Regulator (19S cap, PA700) Nomenclature
Gene
Finley, Dubiel Miscellaneous et al. et al. [5] [6] 19S (PA700) regulator ATPase subunits Rpt1 S7 p48, Mss1, Yta3, Cim5 Rpt2 S4 p56, Yhs4, Yta5, Mts2 Rpt3 S6b p48, Tbp7, Yta2, Ynt1, MS73 Rpt4 S10b p42, Sug2, Pcs1, Crl13, CADp44 Rpt5 S6a p50, Tbp1, Yta1 Rpt6 S8 p45, Trip1, Sug1, Cim3, Crl3, Tby1, Tbp10, m56 19S (PA700) regulator non-ATPase subunits Rpn1 S2 p97, Trap2, Nas1, Hrd2, Rpd1, Mts4 Rpn2 S1 p112, Sen3 Rpn3 S3 p58, Sun2 Rpn4 Son1, Ufd5 Rpn5 p55, Nas5 Rpn6 S9 p44.5, Nas4/6? Rpn7 S10a p44, HUMORF07 Rpn8 S12 p40, Mov-34h, Nas3 Rpn9 S11 p40.5, Les1, Nas7 Rpn10 S5a p54, ASF1, Sun1, Mcb1, Mbp1 Rpn11 S13 Poh1, Mpr1, Pad1h Rpn12 S14 p31, Nin1, Mts3 Rpn13 YLR421C S5b p50.5 S15 p27-L p28, Gankyrin, Nas6
Ma, et al. [8]
11Sα 11Sβ 11Sγ
PA28α PA28β PA28γ
Realini, et al. [9] REGα REGβ REGγ
MW
Human
Yeast (S.c.)
(human)
Subunit 7 Subunit 4 Subunit 6b Subunit 10b Subunit 6a Subunit 8
PSMC2 PSMC1 PSMC4 PSMC6
CIM5 YTA5 YTA2 SUG2
P35998 P62191 P43686 P62333
432 440 418 389
48503 49185 47336 44173
PSMC3 PSMC5
YTA1 SUG1
P17980 P62195
439 406
49204 45626
Subunit 2
PSMD2
HRD2
Q13200
908
100200
Subunit 1 Subunit 3
PSMD1 PSMD3
Subunit 12 Subunit 11 Subunit 6 Subunit 7 Subunit 13 Subunit 4
PSMD12 PSMD11 PSMD6 PSMD7 PSMD13 PSMD4
SEN3 SUN2 RPN4 YDL147W YDL097C
Q99460 O43242 Q03465(Sc) O00232 O00231 Q15008 P51665 Q9UNM6 P55036
953 534 531 455 421 389 324 376 377
105836 60978 60153 52773 47333 45531 37025 42918 40736
Subunit 14 Subunit 8
PSMD14 PSMD8
Subunit 5 Subunit 9 Subunit 10
PSMD5 PSMD9 PSMD10
O00487 P48556 O13563(Sc) Q16401 O00233 O75832
310 257 156 503 223 226
34577 30005 17902 56065 24654 24428
Kandil, et al. [10]
Ki antigen
Seq. length (amino acids)
UniProtK B [4]
11S Activator (11S cap, PA28) Nomenclature Dubiel, et al. [7]
1º Acc #
YOR261C SUN1 MPR1 NIN1 RPN13 NAS2
Gene
1º Acc #
UniProtKB [4]
(human)
(human)
Subunit 1 Subunit 2 Subunit 3
PSME1 PSME2 PSME3
Q06323 Q9UL46 P61289
Seq. length (amino acids) 249 238 254
(Da)
MW (Da)
28723 27230 29506
REFERENCES [1] Baumeister W, Walz J, Zuhl F, Seemuller E: The proteasome: paradigm of a selfcompartmentalizing protease. Cell 1998, 92:367-380. [2] Coux O, Nothwang HG, Silva P, I, Recillas TF, Bey F, Scherrer K: Phylogenic relationships of the amino acid sequences of prosome (proteasome, MCP) subunits. Mol Gen Genet 1994, 245:769-780.
Appendix
1103
[3] Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R: Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 1997, 386:463-471. [4] Apweiler R, Bairoch A, Wu C. The Universal Protein Resource. UniProtKB 2006 September 15 [cited 2006 Sep 15];Available from: URL: http://www.ebi.uniprot.org/index.shtml [5] Finley D, Tanaka K, Mann C, Feldmann H, Hochstrasser M, Vierstra R, Johnston S, Hampton R, Haber J, Mccusker J, Silver P, Frontali L, Thorsness P, Varshavsky A, Byers B, Madura K, Reed SI, Wolf D, Jentsch S, Sommer T, Baumeister W, Goldberg A, Fried V, Rubin DM, Toh-e A,.: Unified nomenclature for subunits of the Saccharomyces cerevisiae proteasome regulatory particle. Trends Biochem Sci 1998, 23:244-245. [6] Dubiel W, Ferrell K, Rechsteiner M: Subunits of the regulatory complex of the 26S protease. Mol Biol Rep 1995, 21:27-34. [7] Dubiel W, Pratt G, Ferrell K, Rechsteiner M: Purification of an 11 S regulator of the multicatalytic protease. J Biol Chem 1992, 267:22369-22377. [8] Ma CP, Slaughter CA, DeMartino GN: Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain). J Biol Chem 1992, 267:10515-10523. [9] Realini C, Jensen CC, Zhang Z, Johnston SC, Knowlton JR, Hill CP, Rechsteiner M: Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators. J Biol Chem 1997, 272:25483-25492. [10] Kandil E, Kohda K, Ishibashi T, Tanaka K, Kasahara M: PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes. Immunogenetics 1997, 46:337-344.
INDEX
6 6-OHDA, 600, 609, 611, 613, 763, 1012, 1026
A access, xii, xiii, 50, 58, 78, 117, 125, 142, 146, 169, 181, 185, 208, 211, 212, 213, 217, 218, 249, 252, 309, 310, 319, 340, 430, 446, 509, 684, 837, 879 accessibility, 217, 244, 395, 565, 902 accounting, 332, 591, 685, 877, 880 acetylcholine, 335, 412, 416, 436, 698, 699, 898, 912 achievement, 414 acid, ix, x, xii, xxxiv, xxxvi, 1, 3, 4, 10, 11, 22, 23, 28, 36, 38, 41, 42, 44, 53, 64, 72, 73, 74, 78, 79, 81, 86, 90, 98, 99, 117, 118, 126, 132, 133, 140, 152, 177, 179, 181, 186, 214, 239, 243, 244, 254, 286, 292, 297, 308, 309, 312, 313, 314, 316, 320, 321, 323, 326, 329, 331, 332, 333, 344, 352, 374, 378, 379, 401, 412, 431, 436, 442, 449, 459, 524, 555, 560, 589, 590, 634, 663, 665, 667, 682, 693, 754, 772, 788, 800, 840, 868, 885, 889, 899, 902, 903, 905, 923, 940, 946, 961, 962, 966, 967, 969, 981, 992, 1016, 1021, 1037, 1038, 1060, 1061, 1062, 1065, 1069, 1087, 1088, 1096 acidosis, 53, 930 acromegaly, 877, 883, 888, 1061 ACTH, 1071 action potential, 433, 653, 1038, 1045 activated receptors, 434, 729
activation, xix, xx, xxi, xxiv, xxvi, xxxi, xxxiv, xxxv, 28, 31, 33, 59, 62, 68, 78, 81, 82, 86, 90, 91, 105, 123, 126, 127, 128, 132, 134, 145, 146, 155, 157, 167, 173, 176, 181, 183, 185, 194, 195, 198, 248, 250, 263, 267, 278, 284, 285, 286, 288, 299, 300, 301, 302, 322, 333, 337, 340, 350, 354, 355, 358, 360, 364, 368, 379, 381, 385, 391, 399, 418, 420, 424, 427, 429, 439, 443, 445, 454, 455, 456, 457, 460, 463, 465, 469, 475, 481, 482, 484, 485, 486, 504, 510, 511, 513, 514, 515, 516, 517, 518, 519, 523, 524, 525, 528, 529, 530, 531, 533, 534, 535, 540, 543, 544, 545, 550, 551, 556, 572, 579, 581, 586, 587, 590, 591, 592, 593, 596, 607, 608, 615, 622, 637, 647, 652, 657, 658, 659, 660, 662, 663, 666, 670, 679, 680, 686, 687, 691, 701, 702, 703, 708, 716, 717, 719, 720, 729, 733, 734, 749, 750, 751, 752, 759, 760, 774, 802, 803, 809, 815, 817, 824, 829, 841, 842, 853, 858, 863, 866, 867, 868, 869, 872, 878, 879, 882, 883, 885, 886, 887, 888, 892, 893, 894, 923, 924, 928, 929, 931, 932, 933, 934, 935, 936, 938, 943, 945, 947, 953, 955, 956, 957, 958, 966, 968, 970, 971, 972, 973, 976, 981, 983, 984, 985, 986, 989, 991, 992, 993, 994, 999, 1000, 1001, 1003, 1006, 1007, 1008, 1009, 1012, 1014, 1015, 1017, 1022, 1023, 1029, 1030, 1031, 1032, 1034, 1039, 1050, 1053, 1057, 1062, 1071, 1072, 1075, 1078, 1080, 1086, 1087, 1091, 1092, 1095 activation state, 934 active oxygen, 624 active site, xii, xiii, 46, 50, 75, 76, 78, 79, 89, 114, 117, 121, 124, 125, 126, 129, 130, 131, 132, 141, 142, 167, 169, 172, 173, 175, 211,
1106 212, 223, 261, 309, 331, 333, 447, 460, 520, 568, 720, 939, 970, 984, 1006, 1016, 1018, 1028, 1078 active transport, 1024, 1035 activity level, 654, 996 acute ischemic stroke, xxxvi, 1017, 1018, 1019, 1021, 1026, 1029, 1030, 1037, 1039 acute leukemia, 1041, 1051 acute lymphoblastic leukemia, 510 acute renal failure, 966, 983 acute stress, 458 adaptability, 514 adaptation, 247, 369, 503, 504, 508, 841 adenine, 791 adenocarcinoma, 290, 304 adenoma, xxxii, 876, 892 adenosine, 2, 3, 5, 37, 42, 160, 208, 465, 664, 716, 893, 922, 1070 adenosine triphosphate, 2, 3, 5, 37, 42, 160, 208, 716, 922 adenovirus, 54, 170, 180, 181, 197, 257, 331 adhesion, xxxv, 387, 389, 433, 662, 668, 842, 938, 945, 946, 957, 990, 995, 1000, 1003, 1005, 1009, 1012, 1014, 1057 ADP, 118, 127, 136, 160, 162, 208, 209, 213, 214, 220, 284, 396, 450, 452, 453, 460, 461, 465, 480, 522, 611, 777, 923, 944, 958 adrenal gland, 927 adrenal glands, 927 adrenocorticotropic hormone, 1071 adulthood, 902, 1074 adults, 1042 adverse event, 998, 1017, 1038, 1043, 1045, 1047 Africa, 906 age, xxv, xxx, 8, 11, 40, 67, 317, 340, 404, 409, 430, 469, 487, 489, 502, 510, 543, 545, 546, 548, 551, 565, 568, 570, 600, 632, 641, 676, 691, 698, 707, 715, 716, 721, 762, 763, 764, 767, 776, 777, 790, 791, 806, 807, 808, 812, 849, 902, 906, 923, 1068, 1074, 1097 ageing, xxii, 127, 318, 409, 446, 478, 523, 549, 580, 581, 582, 635, 676, 790, 824, 874, 1021, 1063 agent, xxxvi, 110, 177, 483, 663, 815, 825, 869, 889, 940, 967, 968, 970, 973, 994, 1016, 1037, 1038, 1044, 1072, 1084 aggregates, xiv, xv, xviii, xix, xx, xxi, xxii, xxv, xxvii, xxviii, xxx, 6, 7, 8, 9, 12, 48, 84, 85, 98, 103, 104, 110, 127, 175, 180, 188, 197, 207,
Index 208, 216, 241, 253, 254, 256, 270, 273, 274, 281, 282, 287, 289, 299, 315, 316, 317, 320, 321, 336, 337, 339, 390, 394, 401, 402, 403, 404, 409, 430, 437, 444, 446, 459, 464, 466, 467, 472, 473, 474, 521, 522, 533, 537, 539, 540, 541, 543, 545, 547, 553, 554, 558, 559, 563, 564, 568, 569, 572, 580, 581, 593, 595, 599, 602, 611, 612, 614, 616, 671, 672, 675, 676, 677, 678, 679, 680, 681, 682, 684, 685, 687, 688, 689, 690, 691, 692, 694, 699, 701, 702, 704, 708, 710, 712, 713, 720, 721, 725, 726, 727, 734, 738, 741, 742, 743, 745, 746, 748, 749, 750, 751, 757, 762, 764, 774, 777, 794, 797, 798, 800, 802, 805, 806, 808, 809, 810, 811, 812, 813, 815, 819, 820, 822, 849, 851, 905, 925, 926, 927, 930, 933, 939, 952, 991, 1024, 1085 aggregation, xv, xix, xx, xxi, xxiv, xxv, xxvi, 6, 7, 8, 9, 12, 55, 135, 148, 178, 195, 197, 203, 204, 208, 209, 212, 214, 216, 218, 221, 223, 224, 231, 232, 238, 241, 242, 246, 252, 253, 254, 256, 257, 258, 260, 266, 267, 268, 269, 304, 307, 315, 316, 317, 318, 319, 320, 321, 326, 327, 328, 333, 334, 335, 336, 337, 339, 340, 351, 403, 404, 408, 409, 430, 441, 442, 443, 445, 446, 447, 449, 451, 452, 458, 459, 464, 467, 469, 474, 475, 477, 478, 487, 491, 492, 493, 521, 522, 537, 539, 542, 549, 552, 554, 558, 561, 564, 566, 567, 569, 572, 577, 591, 593, 603, 604, 611, 612, 614, 615, 616, 617, 618, 623, 624, 625, 671, 672, 673, 674, 675, 676, 678, 681, 682, 684, 687, 688, 689, 691, 694, 695, 696, 697, 698, 702, 704, 706, 708, 715, 717, 719, 720, 721, 723, 724, 725, 726, 727, 734, 735, 736, 737, 738, 741, 742, 743, 745, 746, 747, 748, 749, 750, 751, 756, 758, 765, 766, 769, 780, 801, 804, 807, 808, 809, 812, 817, 821, 824, 925, 926, 948, 1006, 1024, 1077, 1083, 1086, 1090 aggregation process, 675, 694, 743, 751 aging, xxi, xxx, xxxiv, 127, 135, 336, 466, 468, 477, 484, 487, 490, 523, 524, 537, 539, 540, 541, 543, 544, 545, 546, 547, 548, 549, 553, 554, 561, 565, 569, 575, 593, 594, 597, 685, 688, 691, 700, 703, 705, 706, 711, 716, 727, 731, 733, 753, 779, 790, 791, 812, 947, 949, 951, 989, 991, 1005, 1031, 1052, 1077 aging process, 477, 543, 791 agonist, 193, 371, 388, 437, 659, 663, 664 AIDS, xxxv, 198, 972, 1011, 1016, 1025, 1035
Index airway epithelial cells, 892 airways, 55 akinesia, 600 alanine, 79, 85, 106, 123, 170, 198, 308, 340, 473, 1082 albumin, 16, 18, 483 alcohol, 898, 975, 986 alcohol withdrawal, 898 alcoholic liver disease, 571, 727 aldehydes, 124, 128, 132, 729, 868, 965, 966, 967, 981, 1016, 1074, 1085 aldosterone, 497, 504, 506, 507, 1076 algorithm, 312, 332, 1085 allele, xviii, 84, 102, 351, 394, 399, 564, 628 alpha1-antitrypsin, 303 ALS, 2, 6, 272, 289, 336, 412, 429, 432, 442, 444, 446, 462, 467, 473, 492, 493, 554, 565, 568, 597, 628, 635, 640, 649, 682, 685, 687, 689, 702, 707, 716, 717, 720, 726, 728, 736, 737, 834, 842, 990, 991, 1057 alternative, xvi, 6, 20, 21, 56, 103, 110, 140, 173, 188, 200, 212, 278, 282, 308, 311, 366, 416, 427, 431, 498, 507, 541, 674, 693, 738, 774, 817, 820, 837, 877, 913, 919, 1061, 1066, 1095 alternative hypothesis, xvi, 308 alternatives, 648 alters, 62, 331, 340, 359, 398, 419, 502, 503, 504, 506, 560, 561, 574, 577, 625, 638, 674, 709, 809, 812, 830, 894, 975 aluminium, 1057 Alzheimer’s disease, ix, xix, xxi, xxv, xxvi, xxx, 2, 55, 72, 98, 104, 118, 127, 216, 228, 242, 247, 256, 289, 429, 444, 467, 503, 545, 579, 581, 586, 587, 589, 591, 594, 597, 601, 672, 715, 716, 717, 735, 742, 750, 765, 776, 794, 804, 809, 811, 812, 834, 835, 842, 849, 935, 991, 1012, 1056, 1069, 1077, 1083, 1084, 1090 amendments, 13 amines, 73 amino acid, ix, x, xii, xxxii, 1, 4, 10, 11, 16, 21, 23, 36, 38, 41, 44, 48, 50, 53, 60, 64, 72, 73, 74, 75, 78, 86, 99, 117, 118, 125, 126, 132, 133, 140, 148, 152, 160, 173, 174, 177, 179, 181, 186, 232, 243, 244, 254, 259, 286, 292, 308, 309, 311, 312, 313, 314, 315, 316, 320, 321, 323, 325, 326, 329, 331, 332, 333, 348, 349, 359, 374, 383, 401, 452, 454, 459, 498, 499, 603, 628, 637, 638, 663, 664, 667, 673,
1107 677, 681, 682, 683, 690, 693, 703, 724, 731, 738, 739, 746, 747, 764, 772, 773, 814, 815, 816, 830, 831, 836, 838, 839, 864, 880, 897, 899, 929, 956, 978, 1007, 1058, 1059, 1060, 1061, 1065, 1069, 1075, 1077, 1078, 1079, 1085, 1087, 1089, 1090, 1093, 1094, 1095, 1096, 1099, 1100 amino acid side chains, 673, 724 amino acids, x, xii, xxxii, 16, 21, 23, 36, 41, 48, 50, 60, 73, 74, 75, 78, 99, 117, 118, 125, 126, 133, 148, 160, 173, 174, 232, 243, 244, 259, 311, 312, 313, 314, 315, 325, 326, 348, 349, 359, 383, 452, 454, 628, 637, 638, 663, 682, 683, 690, 703, 731, 738, 739, 747, 772, 773, 816, 836, 839, 864, 880, 897, 899, 1058, 1059, 1060, 1061, 1065, 1077, 1085, 1089, 1090, 1093, 1094, 1095, 1096, 1099, 1100 ammonium, 22, 686 amnesia, xvii, 374, 381 amphibians, 1083 amplitude, 378, 386, 1045, 1074 amputation, 663 amygdala, 376, 471, 509, 653, 663, 1056 amyloid beta, 105, 114, 335, 491, 699, 700, 701, 949 amyloid deposits, 287, 678, 681, 750, 765, 781, 842 amyloid plaques, 98, 558, 746, 819, 828, 842, 1077 amyloidosis, 247, 287 amyotrophic lateral sclerosis, xix, xxv, xxx, 2, 6, 242, 256, 268, 269, 272, 289, 304, 318, 336, 441, 442, 444, 446, 473, 492, 493, 518, 554, 584, 587, 595, 601, 635, 645, 649, 682, 702, 707, 708, 709, 715, 716, 717, 734, 834, 842, 849, 991, 1014, 1057 analgesic, xxiv, 652, 662, 663 androgen, 242, 256, 304, 483, 801, 803, 811, 885, 959, 978, 1051, 1090, 1097 androgen receptors, 811 anemia, 69, 92, 1025 aneuploidy, xxxi, 875, 883, 1088 angiogenesis, xxxii, 197, 234, 876, 881, 882, 927, 949, 968, 978, 983, 992, 1007, 1013, 1087 aniline, 1078 animal diseases, 247, 815 animal models, xxxi, xxxiii, xxxiv, xxxv, 318, 320, 321, 430, 522, 525, 559, 584, 587, 609, 611, 612, 635, 653, 682, 685, 688, 690, 738,
1108 743, 802, 807, 844, 850, 875, 877, 898, 912, 922, 944, 965, 966, 968, 969, 990, 993, 997, 998, 1011, 1019, 1022, 1023 animals, xxiv, xxviii, 34, 104, 175, 400, 430, 432, 501, 502, 521, 562, 606, 609, 610, 612, 651, 655, 656, 754, 763, 766, 797, 800, 801, 813, 814, 815, 913, 919, 925, 930, 996, 998, 1002, 1004, 1007, 1045, 1062, 1084, 1091 anion, 24, 251, 265, 566 annotation, 64 ANOVA, 582, 657 anoxia, 950 antagonism, 505, 543 anterior cingulate cortex, 653, 663 anterior pituitary, 885, 888, 1071 anti-apoptotic, 103, 113, 175, 283, 369, 396, 461, 473, 489, 540, 604, 817, 869, 871, 936, 947, 967, 1014, 1015, 1059, 1061 anti-apoptotic role, 817 antibiotic, 179 antibody, 292, 352, 377, 420, 471, 850, 852, 854, 855, 856, 858, 1072 anti-cancer, xxxiv, xxxvi, 961, 1037, 1038, 1043, 1064 anticancer drug, 594, 859, 980, 1031 anticoagulation, 923 anticonvulsant, 910, 912, 918 antidepressant, 502, 508, 509, 511 antigen, ix, xiii, xvii, xxix, xxx, 1, 3, 17, 72, 82, 91, 118, 126, 134, 151, 154, 169, 170, 171, 174, 178, 179, 180, 181, 191, 193, 195, 197, 198, 199, 239, 242, 249, 250, 256, 263, 268, 269, 300, 308, 311, 330, 331, 373, 382, 383, 389, 447, 464, 480, 489, 665, 759, 798, 833, 834, 835, 836, 837, 838, 839, 840, 843, 844, 845, 846, 850, 854, 855, 856, 857, 864, 869, 874, 924, 962, 966, 973, 985, 1001, 1009, 1034, 1062, 1073, 1074, 1080, 1085, 1092, 1100 antigen-presenting cell, 798 anti-inflammatory agents, xxxiv, 961, 970, 1001, 1009 anti-inflammatory drugs, 652, 653, 965, 1001, 1017 antioxidant, 127, 128, 386, 449, 523, 528, 529, 535, 565, 588, 592, 718, 731, 842, 932, 952, 974, 1015, 1033 antiphospholipid syndrome, 859 antisense, 511, 883, 902, 916 antisense RNA, 511
Index antitumor, 133, 483, 534, 536, 973, 982, 985, 1028, 1031, 1049 antitumor agent, 483, 536, 982, 1031, 1049 anxiety, xx, 495, 497, 502, 511 aorta, 966 apathy, 1083, 1085 APC, xvii, xxxii, 16, 34, 140, 160, 182, 192, 344, 350, 362, 412, 415, 422, 423, 426, 434, 437, 438, 876, 878, 882, 883, 887, 893, 1057, 1058, 1066 apoptosis, x, xiii, xv, xviii, xx, xxii, xxiii, xxix, xxx, xxxi, xxxii, xxxiv, 3, 6, 7, 31, 63, 69, 71, 80, 81, 103, 105, 136, 169, 171, 175, 179, 185, 191, 198, 216, 224, 230, 258, 267, 272, 275, 279, 282, 284, 286, 288, 289, 296, 297, 302, 303, 322, 335, 337, 339, 340, 344, 355, 369, 374, 376, 384, 386, 391, 393, 396, 399, 406, 429, 444, 445, 446, 447, 449, 456, 460, 468, 472, 473, 475, 476, 477, 489, 498, 501, 503, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 543, 547, 550, 552, 555, 572, 580, 582, 585, 586, 587, 591, 594, 595, 596, 607, 609, 613, 619, 628, 636, 637, 640, 647, 648, 662, 680, 687, 688, 690, 696, 698, 701, 702, 703, 707, 708, 711, 712, 725, 727, 734, 749, 759, 770, 775, 787, 808, 809, 814, 817, 820, 821, 824, 827, 829, 830, 831, 841, 842, 850, 863, 864, 866, 867, 868, 869, 872, 873, 876, 881, 883, 886, 887, 888, 889, 895, 905, 922, 924, 933, 936, 937, 947, 949, 951, 952, 953, 954, 955, 957, 961, 963, 967, 968, 972, 973, 978, 979, 981, 982, 985, 992, 1007, 1009, 1010, 1012, 1013, 1014, 1021, 1022, 1023, 1031, 1033, 1034, 1049, 1050, 1053, 1056, 1057, 1059, 1060, 1061, 1062, 1069, 1071, 1073, 1083, 1086, 1087, 1089, 1090, 1091, 1092, 1093, 1095, 1096 apoptosis pathways, 535 apoptosis proteins, 69, 344, 355, 386, 445, 922, 924, 949, 1012, 1071 apoptotic pathway, xx, 203, 319, 406, 513, 514, 515, 516, 517, 519, 524, 525, 526, 527, 528, 680, 689, 803, 924, 966, 967, 968, 983 appetite, 1025, 1083 apraxia, 1064 Arabidopsis thaliana, 61, 86 arachidonic acid, 718, 923, 956, 1072 archaebacteria, xi, 27, 117, 154, 222, 799, 1081
Index arginine, 48, 123, 177, 179, 197, 691, 693, 940, 962, 973, 986 argument, 24, 281, 311, 857, 924 arrest, xviii, xxxii, 29, 53, 80, 83, 245, 286, 394, 399, 400, 408, 445, 535, 592, 759, 786, 873, 876, 889, 890, 964, 1022, 1050, 1062 arteries, 923 artery, xxxv, 922, 923, 954, 958, 968, 984, 994, 995, 997, 1002, 1008, 1009, 1012, 1017, 1029, 1030, 1032 arthritis, xx, xxxiv, 234, 852, 1023 articular cartilage, 1003 aryl hydrocarbon receptor, 922, 927 ascorbic acid, 449 aspartate, 123, 349, 374, 441, 442, 445, 460, 514, 600, 664, 665, 666, 667, 755, 911, 955, 956 assessment, 948, 996, 1000, 1010, 1019, 1038, 1046 assets, 866 assignment, 504 assumptions, 314 asthma, xxxiv, 989, 992, 1009, 1023, 1032 astrocytes, 335, 375, 382, 385, 386, 405, 467, 473, 478, 517, 522, 679, 699, 700, 757, 815, 820, 842, 949, 955, 999, 1015 astrocytoma, 234, 240, 509, 865, 866, 873, 1068 asymmetry, 456 asymptomatic, 1042 ataxia, ix, xxxii, 2, 7, 9, 85, 92, 100, 104, 114, 308, 315, 338, 412, 435, 506, 507, 554, 560, 564, 628, 630, 632, 636, 643, 672, 709, 710, 772, 794, 809, 811, 898, 901, 904, 905, 906, 912, 917, 1058, 1065, 1068, 1071, 1074, 1082, 1090 atherogenesis, 1033 atherosclerosis, 288, 1023 atomic force, 676 atoms, 900, 939 atonic, 905 ATP, x, xi, xii, xiii, xv, xvi, 2, 3, 5, 8, 16, 22, 24, 25, 26, 27, 29, 36, 37, 38, 39, 42, 43, 45, 46, 57, 63, 65, 76, 79, 86, 94, 117, 119, 126, 128, 129, 137, 140, 142, 144, 145, 147, 148, 149, 150, 151, 152, 155, 156, 157, 160, 161, 162, 163, 164, 166, 167, 169, 171, 172, 178, 193, 194, 208, 209, 210, 212, 213, 214, 217, 218, 219, 220, 221, 222, 235, 236, 242, 248, 251, 253, 256, 261, 262, 265, 266, 272, 280, 282, 284, 301, 308, 309, 310, 323, 326, 329, 331, 340, 377, 448, 450, 451, 452, 453, 454, 455,
1109 460, 461, 464, 465, 469, 480, 481, 486, 520, 522, 524, 538, 541, 555, 585, 597, 610, 611, 613, 675, 699, 716, 718, 722, 736, 744, 745, 768, 769, 772, 773, 776, 777, 791, 870, 879, 880, 899, 922, 923, 924, 926, 928, 930, 933, 934, 936, 937, 951, 994, 1055, 1056, 1059, 1063, 1070, 1071, 1073, 1080, 1085, 1086, 1092, 1093 atrophy, xxvii, 8, 9, 242, 256, 316, 401, 445, 462, 469, 690, 793, 794, 959, 1057, 1090 attachment, xii, 44, 45, 46, 47, 49, 57, 60, 62, 94, 137, 143, 144, 145, 171, 412, 424, 447, 515, 744, 767, 769, 816, 904, 1000, 1024 attacker, 79 attacks, 79, 123 attention, xvii, xxxiv, 218, 276, 345, 393, 850, 962, 964, 979, 992, 1083 Australia, 11, 307, 793 autoantibodies, 858, 859, 860, 861 autocatalysis, 154 autoimmune disease, xxx, 12, 234, 239, 849, 850, 851, 852, 853, 856, 857, 858, 859, 972, 1057 autoimmune diseases, xxx, 12, 234, 239, 849, 850, 851, 852, 856, 858, 859, 972, 1057 autoimmune hepatitis, 851 autoimmunity, 330, 856 autolysis, 23, 123 autonomic neuropathy, 1046, 1047 autonomy, 414 autooxidation, 610, 615, 720 autophagic cell death, 619, 790, 1058 autophagy, xxvii, 10, 12, 19, 21, 31, 36, 40, 42, 59, 63, 64, 231, 317, 319, 335, 368, 401, 407, 408, 444, 449, 466, 472, 492, 558, 603, 618, 619, 681, 682, 684, 685, 687, 703, 704, 705, 706, 707, 762, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 790, 791, 795, 805, 925, 926, 929, 932, 945, 1034, 1058 autopsy, 734, 1077 autoreactive T cells, xxx, 850, 857 autosomal dominant, 84, 102, 242, 256, 267, 288, 399, 562, 563, 564, 605, 764, 877, 1054, 1067, 1069, 1075, 1081 autosomal recessive, 42, 55, 111, 223, 272, 279, 368, 428, 439, 440, 444, 473, 520, 554, 560, 572, 573, 580, 581, 600, 619, 630, 633, 692, 711, 772, 785, 788, 789, 906, 1068, 1075, 1081 availability, xii, 20, 159, 165, 395, 404, 521, 799, 934, 997, 1023
Index
1110 averaging, 139 avoidance, 381 axon terminals, 1048 axonal degeneration, xviii, 171, 191, 358, 394, 395, 397, 398, 562, 564, 642, 645, 647, 648, 772, 942, 996, 1054, 1068 axons, xvi, xxiii, 7, 8, 189, 273, 316, 344, 350, 351, 352, 355, 356, 357, 359, 360, 365, 368, 369, 375, 376, 380, 385, 395, 398, 399, 403, 407, 412, 510, 516, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 646, 647, 648, 649, 650, 653, 670, 768, 770, 774, 825, 828, 905, 925, 926, 933, 1048, 1058, 1063, 1065, 1069, 1076, 1077, 1078, 1088, 1089
B Bacillus subtilis, 476 bacteria, 24, 27, 476 bacterial cells, 36 bacterial infection, 383, 835 basal ganglia, 610, 624, 773, 1064 basal lamina, 399 basement membrane, 234 basic fibroblast growth factor, 834, 839 basic research, 3, 770, 946, 968, 979, 992 BBB, 834, 922, 943, 1012, 1022, 1025, 1026 BD, 115, 192, 196, 260, 367, 371, 391, 406, 480, 506, 535, 550, 551, 594, 648 behavior, 50, 233, 324, 325, 328, 391, 397, 414, 423, 430, 435, 502, 508, 609, 622, 681, 786, 898, 918, 1059, 1064, 1083, 1085 behavioral disorders, 85 beneficial effect, 12, 290, 525, 1017, 1023 benign, xxxi, 474, 875, 877, 879, 1093 benign tumors, 1093 benzodiazepines, 902 bicarbonate, 251 bile, 236 Bim, 529, 929, 950 bioavailability, 248, 973, 978 biochemistry, xxiv, 35, 256, 466, 478, 622, 628, 658, 734, 780 bioinformatics, 87, 575 biological activity, xxv, 592, 671 biological processes, xiii, 43, 169, 171, 190, 362, 446, 555, 556, 590, 910, 1012, 1056 biological systems, 405 bioluminescence, 693, 945
biopsy, 829, 906, 917, 1053 biosynthesis, 35, 38, 46, 53, 63, 185, 203, 210, 259, 414, 416, 417, 424, 432, 573, 589, 590, 591, 592, 647, 700, 784 biosynthetic pathways, 63, 417 biotechnology, 581, 695 biotin, 970 birds, 1077, 1083 birth, 259, 497, 633 blindness, 905 blocks, xvi, xvii, xxiii, 58, 163, 194, 340, 344, 373, 456, 511, 524, 628, 637, 641, 642, 710, 757, 775, 882, 936, 958, 965, 970, 973, 978, 984, 987, 1007, 1022, 1029, 1067, 1088 blood, xxv, xxxiii, xxxiv, 22, 288, 289, 375, 382, 484, 523, 525, 608, 716, 776, 834, 856, 859, 921, 922, 923, 932, 942, 943, 945, 972, 990, 993, 996, 997, 998, 1000, 1003, 1004, 1012, 1019, 1020, 1022, 1025, 1035, 1040, 1046, 1048, 1057, 1064, 1067, 1072, 1073, 1078, 1088 blood flow, xxxiii, 921, 923, 993, 1072, 1088 blood pressure, 998, 1019, 1046 blood stream, 1003 blood supply, 932, 1072 blood vessels, 375, 382, 1057 blood-brain barrier, 289, 375, 382, 523, 834, 856, 859, 922, 945, 1012, 1022 BMPs, 360 body fluid, 234, 718 body temperature, 998 body weight, 942, 996 bonding, 244, 677 bonds, xii, 43, 50, 72, 117, 125, 126, 132, 133, 243, 244, 274, 292, 309, 310, 322, 333, 457, 1065, 1093 bone marrow, 719, 962, 967, 1031, 1040, 1043, 1061, 1067 Bortezomib, viii, xxxv, xxxvi, 12, 302, 305, 479, 634, 709, 889, 895, 940, 942, 943, 951, 967, 982, 1011, 1016, 1021, 1026, 1031, 1032, 1037, 1038, 1039, 1040, 1045, 1047, 1049, 1052, 1053, 1061 boutons, 358, 397 bovine spongiform encephalopathy, 814, 815, 828, 1084 bowel, 1046 boys, 1082 bradykinesia, 606, 763, 966, 1064
Index brain, xvi, xvii, xx, xxv, xxvii, xxix, xxx, xxxii, xxxiii, xxxiv, xxxv, 6, 12, 55, 74, 98, 101, 103, 112, 127, 136, 173, 175, 178, 179, 186, 188, 189, 190, 204, 208, 234, 235, 279, 288, 289, 304, 311, 315, 334, 340, 343, 352, 358, 367, 368, 373, 375, 376, 377, 378, 379, 382, 383, 384, 385, 387, 390, 391, 394, 398, 408, 412, 428, 430, 433, 434, 440, 441, 457, 465, 467, 469, 470, 471, 484, 495, 496, 497, 498, 499, 500, 502, 504, 505, 507, 508, 509, 510, 516, 519, 523, 524, 525, 526, 531, 533, 545, 550, 554, 566, 587, 594, 597, 600, 605, 608, 611, 612, 624, 631, 632, 635, 642, 649, 650, 654, 677, 678, 680, 681, 685, 687, 692, 694, 697, 698, 701, 705, 710, 711, 715, 718, 721, 727, 738, 745, 754, 760, 761, 763, 766, 771, 772, 773, 776, 781, 789, 797, 802, 804, 806, 807, 812, 815, 820, 828, 829, 830, 833, 835, 836, 839, 842, 843, 849, 853, 869, 870, 874, 877, 897, 898, 899, 902, 903, 904, 905, 910, 916, 921, 923, 925, 927, 929, 931, 932, 939, 942, 943, 945, 946, 947, 948, 949, 950, 951, 954, 955, 956, 957, 958, 959, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1012, 1014, 1020, 1022, 1025, 1027, 1028, 1029, 1030, 1031, 1035, 1039, 1045, 1048, 1050, 1056, 1057, 1061, 1064, 1066, 1067, 1068, 1070, 1073, 1074, 1077, 1083, 1085, 1086, 1090, 1091 brain activity, xxxv, 902, 990 brain damage, xxvi, 136, 716, 957, 993, 1000 brain development, 738, 899 brain stem, 377, 1057 brain tumor, 1091 brainstem, 55, 100, 228, 230, 635, 642, 743, 1081 branching, 351, 359, 369, 382, 423, 905 breaches, 1083 breakdown, xiv, xxx, 3, 6, 23, 37, 38, 39, 65, 68, 80, 86, 94, 128, 167, 207, 209, 210, 233, 234, 317, 397, 659, 775, 784, 850, 872, 932, 936 breast cancer, 177, 499, 781, 893, 978, 979 budding, 43, 45, 47, 51, 57, 58, 59, 60, 62, 74, 80, 96, 97, 141, 149, 177, 183, 191, 204, 457, 556 buffer, 449 building blocks, xvii, 373 burning, 1061 buttons, 378
1111
C Ca2+, 273, 292, 348, 406, 416, 419, 658, 667, 680, 701, 773, 911, 923, 924, 931, 932, 938, 994, 1061, 1086, 1090 cables, 394 cachexia, 53 cadherin, 433 cadherins, 1056 cadmium, 61, 535 calcium, 153, 213, 273, 283, 286, 288, 378, 386, 427, 429, 468, 591, 592, 654, 665, 667, 679, 689, 701, 750, 820, 869, 904, 916, 923, 928, 930, 931, 933, 936, 938, 955, 956, 958, 1061, 1073, 1081 calculus, 845 Canada, 190, 729, 730, 945 cancer, xi, xxxiv, xxxv, xxxvi, 12, 40, 53, 54, 68, 69, 71, 81, 90, 102, 105, 112, 115, 128, 177, 180, 230, 236, 287, 290, 302, 305, 483, 516, 534, 536, 547, 709, 845, 846, 851, 853, 855, 858, 869, 872, 873, 881, 888, 892, 895, 936, 943, 949, 962, 963, 966, 967, 970, 973, 976, 979, 980, 987, 989, 992, 1011, 1013, 1016, 1021, 1022, 1023, 1026, 1027, 1028, 1029, 1033, 1037, 1044, 1049, 1051, 1052, 1053, 1062, 1081, 1086, 1087 cancer cells, 12, 69, 81, 115, 290, 302, 516, 547, 855, 858, 949, 967, 976, 992, 1016, 1021, 1022 cancer treatment, 1021, 1026 candidates, 132, 163, 340, 462, 872, 888, 913, 966, 970, 976, 980, 1028 CAP, 69, 914 capillary, 959 carbohydrate, 400, 906, 917 carbohydrates, 681 carbon, 16, 123, 1072, 1079 carbon atoms, 1072 carbonyl groups, 566, 567, 720, 724 carcinogenesis, xxxi, 105, 456, 476, 536, 875, 986 carcinoma, 290, 304, 305, 344, 355, 895, 971, 1065 cardiac arrest, 923 cardiovascular disease, 288, 980, 981 cardiovascular function, 503 carrier, 28, 30, 45, 171, 263, 631, 770, 967 casein, 125, 164, 240 caspase-dependent, 515
1112 caspases, xx, 185, 279, 286, 513, 514, 516, 517, 518, 519, 520, 525, 527, 646, 688, 695, 737, 749, 750, 824, 868, 873, 924, 934, 935, 937, 951, 1057, 1059, 1060, 1062 catabolism, 23, 36, 37, 751 catalase, 718 catalysis, 43, 78, 274, 447, 566, 576, 597, 731, 914 catalyst, 743 catalysts, 301 catalytic activity, xxvii, 29, 32, 83, 101, 106, 123, 145, 175, 190, 223, 328, 694, 761, 882, 930, 965, 1059, 1085 catalytic properties, xi, 72, 330 catecholamines, 720, 1066 cathepsin B, 18, 940, 965, 966 cation, 129, 235 cattle, 650, 815, 816, 823, 1084 Caucasians, 288, 463 causality, 1000 C-C, 156, 423 CD14, 719 CD45, 679 CD8+, 530, 835, 838 CD95, 864, 869, 1091 cDNA, 152, 197, 198, 505, 728, 752, 754, 781, 1080 CE, 108, 239, 265, 302, 335, 364, 365, 368, 369, 370, 406, 483, 492, 620, 699, 758, 919, 950, 1031 cell adhesion, xxxv, 433, 492, 587, 840, 938, 976, 1001, 1002, 1009, 1012, 1014, 1017, 1030, 1031, 1057, 1058 cell body, xxiii, 189, 350, 352, 375, 413, 414, 419, 420, 423, 425, 627, 628, 629, 633, 637, 639, 1048, 1058, 1059, 1065 cell culture, 6, 407, 468, 469, 474, 475, 498, 522, 562, 587, 594, 818, 827, 855, 858, 873, 935, 973, 1059 cell cycle, ix, x, xiii, xvi, xvii, xviii, xx, xxi, xxxi, xxxiv, xxxv, 1, 3, 15, 17, 29, 31, 39, 47, 53, 54, 59, 61, 62, 65, 110, 111, 160, 169, 171, 175, 182, 192, 195, 200, 202, 216, 224, 229, 230, 232, 238, 239, 250, 263, 298, 299, 343, 351, 362, 363, 373, 376, 377, 386, 387, 394, 400, 407, 411, 422, 430, 445, 446, 449, 455, 481, 513, 517, 518, 524, 525, 530, 535, 539, 579, 592, 644, 751, 760, 863, 864, 865, 867, 875, 876, 877, 878, 879, 882, 883, 886, 887, 888, 890, 891, 896, 951, 963, 989, 1011, 1013,
Index 1014, 1021, 1022, 1023, 1031, 1033, 1049, 1050, 1054, 1062, 1064, 1087, 1090, 1092, 1096 cell death, ix, x, xx, xxi, xxii, xxvii, xxviii, 1, 2, 7, 41, 85, 101, 103, 104, 111, 112, 185, 198, 237, 258, 279, 283, 286, 287, 288, 296, 297, 299, 300, 301, 312, 317, 336, 369, 386, 395, 396, 399, 405, 430, 440, 441, 442, 446, 455, 460, 468, 472, 473, 474, 476, 477, 481, 492, 497, 506, 514, 516, 520, 521, 522, 524, 525, 526, 527, 528, 529, 530, 533, 537, 538, 539, 542, 543, 546, 553, 558, 562, 563, 565, 566, 579, 580, 581, 586, 587, 589, 591, 593, 596, 597, 599, 603, 604, 609, 613, 614, 615, 616, 621, 622, 624, 633, 640, 642, 645, 677, 685, 688, 689, 690, 700, 701, 704, 705, 706, 719, 721, 723, 727, 743, 747, 748, 749, 758, 762, 764, 775, 778, 786, 787, 795, 798, 810, 811, 813, 815, 820, 830, 832, 842, 857, 868, 871, 872, 888, 905, 923, 924, 929, 930, 931, 932, 934, 936, 937, 944, 946, 947, 948, 949, 952, 955, 957, 959, 983, 991, 992, 993, 994, 1001, 1006, 1008, 1024, 1025, 1026, 1028, 1029, 1033, 1057, 1059, 1081 cell differentiation, 1090 cell division, x, xxxi, 42, 65, 160, 191, 233, 351, 368, 384, 391, 395, 399, 407, 466, 548, 592, 677, 863, 864, 1021, 1068, 1069 cell fate, ix, xix, xxxiii, 348, 364, 366, 371, 444, 922, 924, 983, 1030, 1069 cell growth, ix, 3, 80, 87, 236, 408, 484, 534, 759, 869, 873, 879, 886, 887, 1013, 1014, 1026, 1090 cell line, 105, 113, 189, 228, 230, 236, 238, 256, 267, 289, 383, 431, 458, 460, 465, 473, 474, 499, 500, 501, 516, 517, 520, 521, 528, 532, 586, 590, 592, 603, 634, 644, 680, 685, 688, 722, 750, 760, 770, 775, 795, 819, 822, 823, 824, 825, 830, 868, 869, 883, 885, 888, 892, 893, 911, 944, 956, 957, 959, 966, 967, 968, 970, 972, 973, 1016, 1021, 1024, 1033, 1035, 1054 cell lines, 105, 113, 228, 236, 238, 256, 267, 431, 458, 465, 473, 474, 499, 500, 501, 516, 517, 520, 521, 528, 532, 586, 592, 603, 634, 680, 685, 722, 750, 775, 819, 822, 823, 824, 825, 868, 869, 883, 888, 892, 911, 957, 966, 967, 968, 970, 972, 973, 1021, 1024, 1033, 1035 cell membranes, 318, 679, 967 cell metabolism, 749
Index cell organelles, 774 cell signaling, 447, 700 cell surface, ix, xvi, xxxi, 1, 54, 58, 110, 128, 192, 203, 250, 288, 344, 345, 346, 349, 355, 356, 360, 361, 365, 378, 383, 388, 412, 413, 414, 416, 417, 418, 419, 420, 421, 422, 423, 424, 431, 432, 433, 666, 679, 785, 817, 837, 863, 864, 884, 920, 925, 1003, 1056, 1063, 1074, 1075 cellular adhesion, xxxv, 990, 997, 1001, 1002, 1003, 1004 cellular homeostasis, xiv, xix, 171, 207, 444, 445, 726, 964, 990 cellular immunity, 174, 844 cellular processes, ix, x, xvi, xxxi, 1, 3, 12, 14, 15, 17, 54, 56, 71, 72, 94, 216, 242, 248, 250, 309, 312, 315, 343, 362, 363, 414, 453, 466, 467, 555, 557, 569, 584, 673, 692, 863, 864, 966, 1083, 1090 cellular regulation, xxi, 218, 579, 593, 785, 954 cellular signaling pathway, xxii, 580 cellulose, 24, 480 central nervous system, iv, ix, xvii, xxviii, xxix, xxxii, xxxiv, 2, 6, 85, 102, 104, 118, 171, 188, 189, 228, 229, 237, 272, 335, 355, 358, 370, 373, 374, 375, 380, 382, 386, 387, 389, 393, 394, 478, 496, 514, 517, 523, 529, 538, 595, 597, 643, 652, 653, 683, 705, 707, 716, 728, 738, 739, 791, 793, 813, 814, 815, 833, 834, 835, 843, 850, 874, 897, 905, 906, 922, 947, 949, 962, 980, 990, 1025, 1038, 1056, 1068, 1072, 1074, 1078, 1079, 1082, 1092 centrosome, xv, xxvii, 186, 188, 228, 231, 232, 238, 241, 242, 253, 255, 256, 257, 258, 266, 268, 400, 487, 532, 548, 762, 769, 786, 1024, 1076, 1087 cerebellar astrocytoma, 571, 727 cerebellar granule cells, 441, 516, 517, 528, 529, 534, 535, 936, 952, 1024, 1033 cerebellum, 375, 376, 377, 385, 433, 654, 853, 948, 1088 cerebral blood flow, 993 cerebral cortex, 8, 288, 517, 642, 677, 758, 946, 999, 1015, 1056, 1057, 1064, 1066 cerebral hemisphere, 376, 1077 cerebral hemorrhage, 943 cerebral ischemia, xv, xxxiii, xxxv, 12, 127, 272, 279, 296, 315, 334, 447, 460, 478, 525, 587, 596, 697, 700, 921, 925, 926, 930, 931, 934, 937, 938, 939, 941, 942, 943, 944, 946, 947,
1113 948, 950, 951, 952, 954, 956, 957, 958, 959, 968, 971, 984, 991, 993, 994, 995, 1001, 1004, 1007, 1008, 1009, 1011, 1022, 1028, 1029, 1030, 1032, 1050 cerebrospinal fluid, 545, 853, 854, 855, 861, 1035, 1079 cerebrovascular disease, 331, 851, 859, 928, 947, 1009, 1017, 1029, 1030 cerebrum, 375 certainty, 145 cervical ganglia, 395 changing environment, 247, 352 channel blocker, 260, 653, 993, 1010 channels, ix, xv, 1, 54, 66, 212, 220, 260, 271, 274, 276, 309, 370, 378, 387, 395, 405, 416, 425, 436, 439, 659, 680, 703, 733, 796, 911, 912, 913, 1073 chaos, 836 chaperones, xiv, xv, xviii, xix, xx, xxii, xxiv, xxviii, 7, 9, 21, 36, 48, 67, 103, 113, 121, 148, 161, 173, 175, 187, 194, 207, 208, 209, 210, 211, 214, 215, 218, 219, 221, 249, 252, 253, 254, 256, 260, 266, 267, 268, 271, 273, 274, 275, 276, 277, 278, 279, 283, 284, 285, 286, 288, 289, 291, 293, 301, 317, 318, 334, 335, 338, 394, 403, 404, 409, 443, 446, 447, 449, 451, 452, 453, 454, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 468, 469, 472, 474, 475, 476, 477, 478, 479, 480, 481, 482, 485, 486, 487, 488, 489, 490, 491, 492, 493, 521, 534, 537, 538, 539, 540, 541, 542, 543, 544, 546, 547, 549, 570, 576, 580, 581, 584, 585, 591, 594, 595, 620, 671, 675, 678, 681, 682, 691, 696, 697, 698, 709, 745, 746, 749, 757, 758, 759, 776, 793, 795, 797, 800, 801, 803, 804, 806, 811, 812, 925, 928, 934, 936, 944, 954, 959, 1024, 1056, 1059, 1062, 1063, 1069, 1070, 1071, 1072, 1076, 1084, 1089, 1092, 1095 chemical composition, 243 chemical interaction, 35 chemical structures, 663 chemokines, 662, 840, 842, 1001, 1003, 1009, 1017 chemotherapeutic agent, 653, 982, 1022, 1032, 1044, 1045 chemotherapeutic drugs, 663, 1045 chemotherapy, 12, 516, 709, 872, 873, 967, 1022, 1044, 1045 chicken, 91, 193, 477, 816, 1006, 1034, 1054
1114 childhood, 898, 902, 914, 1068, 1082 children, 899, 901, 915, 1042 chimera, 267, 487 chloroquine, 22 CHO cells, 187, 204 cholangitis, 852 cholera, 204, 281, 298 cholesterol, 274, 288, 303, 589, 590, 591, 592, 597, 1074 cholinergic neurons, 701, 703 chorea, 642, 851, 1065, 1071 choriomeningitis, 972 choroid, 1035 chromatid, 54, 644, 883, 893, 1088 chromatography, 26, 144, 214, 970 chromosome, xxxii, 38, 283, 401, 478, 497, 575, 694, 736, 737, 738, 753, 765, 789, 864, 876, 877, 881, 883, 890, 902, 903, 904, 1054, 1056, 1065, 1068, 1069, 1077, 1082, 1087, 1088, 1089, 1091, 1093 chronic lymphocytic leukemia, xxxv, 1011 chronic pain, xxiv, 652, 654, 655, 656, 658, 660, 662 chymotrypsin, xii, xxxi, 117, 118, 123, 132, 133, 135, 170, 172, 180, 238, 249, 258, 259, 267, 333, 338, 404, 408, 519, 532, 535, 592, 605, 706, 786, 863, 869, 870, 871, 922, 930, 940, 952, 962, 963, 984, 985, 986, 1012, 1016, 1018, 1033, 1038, 1055 cilia, 1076 circadian rhythm, xiii, 3, 54, 169, 171, 191 circadian rhythms, xiii, 54, 169, 171 circular dichroism, 673 circulation, 497, 966, 981, 1022 cirrhosis, 180 cisplatin, 519, 1044, 1045, 1047, 1053 citalopram, 502, 509 classes, 20, 21, 22, 37, 45, 59, 123, 200, 248, 273, 500, 560, 564, 568, 675, 681, 744, 900, 903, 939, 940, 953, 979, 992, 1016, 1074 classification, 9, 234, 483, 672, 872, 964 cleavage, xi, xvi, 4, 6, 10, 11, 71, 74, 82, 88, 123, 125, 131, 144, 149, 173, 175, 178, 249, 250, 261, 263, 273, 277, 301, 308, 309, 311, 312, 313, 314, 320, 321, 322, 323, 324, 325, 326, 327, 328, 331, 332, 350, 388, 396, 437, 468, 519, 520, 558, 590, 598, 674, 691, 692, 695, 749, 750, 759, 760, 789, 822, 839, 840, 841, 845, 882, 893, 920, 937, 938, 948, 1057, 1085, 1088, 1095
Index cleavages, 311, 312, 314, 326, 332, 514, 536, 846 clients, 465, 489 clinical diagnosis, 861, 872 clinical symptoms, 584, 763, 972 clinical syndrome, 694 clinical trials, xxxvi, 13, 178, 516, 634, 641, 889, 945, 946, 964, 965, 966, 969, 979, 980, 993, 998, 1012, 1019, 1021, 1022 clonality, 890 clone, 959 cloning, 108, 115, 152, 155, 197, 263, 264, 348, 349, 787, 890, 918 closure, 452, 453, 902 cluster of differentiation, 834 clustering, 436, 661 clusters, 232, 452, 650, 669 CNS, xvii, xx, xxiv, xxvi, xxxiv, xxxv, 2, 6, 8, 9, 12, 118, 127, 228, 230, 235, 272, 290, 303, 349, 369, 373, 374, 375, 376, 377, 378, 382, 383, 384, 386, 387, 393, 394, 396, 398, 399, 400, 405, 408, 416, 418, 437, 495, 496, 497, 501, 503, 504, 514, 526, 527, 528, 538, 545, 552, 628, 630, 631, 632, 634, 638, 642, 651, 652, 683, 694, 703, 716, 717, 718, 719, 726, 788, 795, 814, 815, 816, 826, 834, 835, 836, 839, 842, 843, 850, 858, 861, 905, 919, 922, 927, 929, 939, 945, 946, 947, 962, 972, 990, 991, 992, 999, 1003, 1004, 1005, 1006, 1030, 1038, 1047, 1048, 1059, 1082 cocaine, 502, 509 cocaine use, 509 codes, 131, 223, 681, 690 coding, 10, 34, 243, 286, 451, 564, 637, 737, 801, 884, 901, 906, 1062, 1084 codon, 102, 788, 831 coenzyme, 449 cognition, 646 cohesion, 1085 cohort, 19, 20, 413, 420, 425, 539, 760, 837, 1035 collaboration, 83, 585 collagen, 234, 240, 246 collateral, 923 colon, 179, 659, 971 colorectal adenocarcinoma, 290 colorectal cancer, 1052 combined effect, 469 combustion, 17 communication, xviii, 298, 374, 393, 395, 397, 414, 944, 1064
Index community, 3, 13 compensation, 583 competence, 345 competition, 56, 252, 253, 424, 430, 431, 432, 463, 547, 866 competitor, 431 complement, 140, 229, 425, 765 complementary DNA, 264, 504 complex interactions, xx, 456, 495 complex partial seizure, 905 complexity, xiii, xvi, xxvii, 5, 6, 31, 43, 55, 57, 65, 120, 159, 161, 165, 171, 189, 253, 308, 310, 330, 343, 355, 562, 762, 857, 861, 944 complications, 601, 617, 906 components, x, xi, xii, xiii, xiv, xv, xvi, xviii, xix, xxi, xxii, xxv, xxviii, xxxi, 3, 6, 7, 8, 9, 21, 22, 25, 28, 33, 34, 36, 41, 44, 47, 49, 53, 57, 58, 60, 62, 72, 93, 105, 109, 124, 125, 126, 131, 132, 133, 137, 140, 141, 143, 145, 147, 151, 155, 157, 162, 164, 170, 171, 174, 175, 178, 183, 184, 185, 187, 188, 189, 190, 200, 208, 209, 227, 228, 229, 231, 232, 241, 243, 252, 253, 254, 256, 272, 275, 278, 280, 282, 284, 286, 287, 290, 300, 316, 317, 318, 319, 344, 346, 347, 348, 351, 352, 358, 359, 362, 363, 377, 387, 397, 403, 411, 412, 413, 419, 420, 427, 444, 455, 459, 460, 461, 462, 467, 474, 476, 496, 499, 500, 519, 521, 543, 553, 555, 557, 558, 559, 560, 561, 565, 567, 568, 580, 581, 582, 584, 592, 599, 637, 638, 641, 655, 662, 671, 674, 677, 678, 681, 687, 689, 691, 692, 717, 721, 744, 752, 793, 795, 796, 797, 798, 799, 801, 804, 805, 806, 807, 811, 828, 844, 853, 861, 875, 879, 899, 913, 925, 945, 1024, 1034, 1055, 1059, 1065, 1072, 1076, 1084, 1087 composition, xiii, xxiv, 37, 120, 139, 157, 159, 229, 243, 254, 314, 348, 383, 388, 414, 422, 433, 436, 500, 558, 651, 667, 672, 739, 795, 807, 872, 910, 948, 1092 compound eye, 773 compounds, xxxiii, xxxiv, 12, 16, 455, 613, 659, 669, 700, 718, 722, 912, 922, 938, 939, 940, 944, 946, 961, 968, 972, 974, 975, 976, 992, 993, 1000, 1001, 1005, 1043, 1063, 1070, 1072 COMT inhibitor, 734 concentrates, 242 concentration, xv, xxix, xxxi, 29, 84, 119, 125, 171, 241, 323, 324, 397, 445, 468, 481, 488,
1115 507, 517, 545, 581, 603, 610, 631, 675, 678, 726, 746, 758, 772, 776, 814, 818, 822, 856, 859, 875, 878, 883, 884, 904, 929, 967, 968, 1013, 1038, 1039, 1086, 1093 concordance, 763, 899 condensation, 400, 515, 1057 conditioning, 428, 653 conduction, 401, 1038, 1045, 1048, 1073 configuration, 47, 172, 328, 456, 910 conflict, xxix, 230, 814, 907 conformational diseases, 7, 9, 112, 674, 1063 conformational stability, 259 confusion, 28, 140, 906, 1056 conjugation, xxxiv, 3, 10, 23, 28, 29, 30, 31, 37, 40, 42, 43, 45, 46, 47, 53, 56, 57, 58, 60, 61, 63, 64, 65, 73, 86, 87, 94, 114, 171, 183, 210, 215, 248, 264, 293, 296, 308, 337, 389, 439, 555, 556, 588, 597, 655, 708, 732, 779, 780, 782, 810, 894, 920, 951, 961, 1058, 1065, 1076 connective tissue, 851, 860, 1057 connectivity, 55, 350, 363, 364, 365, 371, 396, 406, 412, 428, 433, 912, 919 consciousness, 1038 consensus, 258, 584, 766, 797, 900, 915, 1088, 1092 conservation, 44, 131, 447, 773, 823, 1094, 1095 consolidation, 388 constipation, 1025, 1047 constitutive enzyme, 235 constraints, 107, 130, 131, 147, 183, 261, 317, 363, 459, 460, 685, 698, 727, 944 construction, 40, 65, 107, 236, 330, 363, 384, 477, 478, 527, 570, 664, 695, 756, 872, 960, 980, 1005, 1027 consumption, 937 contamination, 27 continuity, 630 control, ix, x, xiii, xv, xvi, xvii, xix, xx, xxxi, xxxiv, 1, 4, 7, 9, 14, 15, 17, 31, 36, 37, 42, 48, 54, 56, 65, 71, 72, 73, 79, 81, 86, 94, 101, 103, 110, 133, 165, 167, 170, 171, 172, 173, 175, 190, 191, 192, 208, 219, 223, 232, 237, 239, 241, 242, 246, 250, 251, 252, 253, 254, 256, 265, 266, 267, 271, 272, 273, 274, 281, 283, 291, 292, 293, 301, 302, 309, 334, 343, 349, 352, 353, 355, 365, 369, 378, 384, 388, 391, 393, 395, 397, 400, 405, 407, 408, 414, 416, 417, 424, 426, 434, 438, 444, 445, 446, 451, 453, 457, 459, 460, 469, 485, 486, 502, 505,
1116 515, 518, 535, 539, 540, 541, 542, 544, 545, 547, 548, 560, 570, 582, 584, 596, 607, 620, 638, 641, 662, 667, 668, 669, 703, 742, 744, 745, 750, 785, 788, 802, 803, 806, 823, 832, 834, 836, 844, 847, 853, 863, 869, 870, 871, 872, 877, 878, 883, 888, 889, 890, 899, 905, 918, 924, 925, 930, 950, 951, 957, 958, 963, 972, 979, 989, 990, 992, 996, 997, 1001, 1003, 1023, 1025, 1063, 1066, 1074, 1081, 1085, 1090 control group, 1023 controlled trials, 835 convergence, 551, 694 conversion, xix, 17, 247, 443, 452, 459, 544, 610, 676, 678, 770, 818, 820, 821, 827, 926 copper, 589, 612, 826 correlation, 29, 259, 329, 403, 623, 632, 754, 854, 856, 881, 886, 996, 1017, 1045 correlations, 313, 336, 702, 858, 916 cortex, 8, 230, 288, 375, 376, 385, 509, 577, 605, 635, 649, 653, 743, 777, 791, 926, 930, 938, 1068, 1071, 1073, 1083, 1088 cortical neurons, xxii, 189, 228, 460, 517, 522, 524, 525, 529, 532, 536, 580, 582, 591, 592, 597, 680, 686, 706, 726, 759, 828, 898, 951, 953, 955, 1034 corticobasal degeneration, 467, 753, 757, 1090 Corticobasal Degeneration, 736, 737 corticosteroids, 497 cortisol, 497, 507, 508, 510, 1069, 1071 coumarins, 483 couples, 88, 130, 153, 184, 193, 262, 541 coupling, 42, 79, 113, 162, 163, 165, 175, 184, 187, 216, 221, 273, 274, 335, 452, 454, 461, 489, 534, 548, 698, 911, 926, 952 covalent bond, 1018 coverage, 13 covering, 13, 209, 449, 840, 1073 COX-2 enzyme, 1017 CpG islands, 902 creatinine, 1023 credit, 309 Creutzfeldt-Jakob disease, 552, 685, 814, 815, 825, 828, 829, 1084 critical period, 371 cryptococcosis, 480 crystal structure, 60, 77, 78, 119, 139, 142, 146, 151, 173, 211, 285, 480, 620, 964, 967 crystalline, 473 crystallization, 5, 699
Index crystals, 970 CSF, 850, 853, 856 C-terminal hydrolase (UCH), xi, 71 cues, xvi, 266, 344, 351, 352, 354, 355, 356, 362, 416, 420, 1069 culture, xxix, 6, 10, 37, 186, 239, 258, 291, 321, 335, 352, 355, 401, 419, 431, 468, 470, 500, 508, 595, 605, 622, 639, 644, 648, 659, 661, 688, 699, 701, 814, 909, 945, 983, 1025, 1054, 1059 curing, xxxiii, 898, 909, 912 cycles, 46, 148, 253, 450, 452, 538, 585, 816, 1043 cyclic AMP, 412, 427, 439, 587, 652, 658, 666, 1086 cyclin-dependent kinase inhibitor, 53, 54, 128, 210, 530, 873, 890, 891, 892, 953, 1013, 1049 cycling, xx, 284, 362, 463, 513, 611, 669, 890, 958 cyclins, xxi, xxxi, 53, 54, 233, 239, 250, 362, 518, 524, 579, 866, 867, 875, 878, 879, 924, 963, 1013, 1024 cyclooxygenase, 576, 580, 587, 597, 652, 662, 679, 716, 717, 728, 729, 834, 840, 959, 1014, 1030 cyclooxygenase-2, 580, 587, 597, 652, 662, 717, 729, 959, 1014, 1030 cyclopentenone prostaglandins, 720, 729 cystatins, 698 cysteine residues, 77, 268, 457, 566, 568, 720, 724 cystic fibrosis, 42, 170, 180, 208, 221, 242, 251, 264, 265, 266, 272, 287, 294, 463, 488, 548, 802, 1064 cytochrome, 103, 113, 337, 469, 515, 517, 518, 524, 528, 529, 533, 535, 572, 584, 624, 680, 702, 759, 770, 787, 802, 809, 873, 931, 936, 937, 951, 952, 955, 1033 cytokine, xii, xxix, xxxv, 63, 118, 174, 249, 386, 648, 670, 717, 833, 834, 923, 967, 970, 971, 976, 987, 990, 1001, 1002, 1004, 1008, 1029, 1068, 1091 cytokine receptor, 1091 cytokines, xxvi, 386, 522, 662, 716, 718, 835, 839, 840, 841, 842, 938, 945, 946, 957, 997, 999, 1000, 1001, 1002, 1003, 1009, 1014, 1015, 1063, 1073, 1090, 1093 cytokinesis, 604 cytomegalovirus, 34, 269, 272, 280, 297, 303, 814, 823
Index cytopathology, 755 cytoplasm, xiv, xxviii, 19, 53, 81, 100, 105, 118, 227, 228, 230, 232, 233, 254, 273, 288, 298, 322, 375, 376, 384, 429, 450, 451, 457, 458, 459, 465, 519, 522, 523, 602, 612, 661, 674, 682, 684, 689, 691, 703, 737, 770, 778, 779, 781, 784, 794, 802, 803, 840, 842, 856, 867, 868, 881, 882, 887, 905, 912, 938, 1013, 1024, 1058, 1076, 1078, 1079 cytoskeleton, 98, 237, 397, 436, 449, 591, 670, 680, 924, 926, 932, 933, 1058, 1059, 1076 cytostatic drugs, 871 cytotoxic action, 234 cytotoxic agents, 718, 842 cytotoxic effects, xxxiv, 521, 858, 961, 972 cytotoxicity, 319, 559, 679, 689, 721, 725, 801, 806, 807, 858, 964, 970, 1039, 1078
D daily living, 1041, 1042 dardarin, 602, 605, 773 database, 259, 448, 478, 917 daughter cells, 233 DCI, 118, 123, 124, 125 death, ix, x, xx, xxi, xxii, xxiii, xxvi, xxvii, xxviii, xxx, xxxiii, 1, 2, 7, 17, 41, 84, 85, 101, 103, 104, 111, 112, 127, 185, 195, 198, 237, 258, 259, 279, 283, 286, 287, 288, 293, 296, 297, 299, 300, 301, 302, 304, 312, 317, 336, 339, 359, 369, 386, 395, 396, 399, 405, 430, 440, 441, 442, 446, 455, 460, 469, 473, 484, 516, 517, 526, 527, 528, 532, 535, 541, 562, 579, 581, 589, 600, 604, 611, 613, 614, 627, 629, 633, 634, 636, 638, 639, 642, 677, 680, 685, 686, 706, 711, 727, 729, 735, 744, 747, 748, 749, 761, 763, 766, 775, 808, 827, 842, 857, 906, 921, 923, 924, 926, 931, 932, 934, 935, 937, 944, 952, 953, 955, 957, 983, 992, 993, 1006, 1015, 1025, 1033, 1034, 1035, 1072, 1077, 1085, 1091, 1092 decisions, 113, 217, 221, 355, 366, 368, 465, 487, 506, 548 deconstruction, 664 defects, xxiii, 8, 9, 72, 85, 92, 114, 164, 177, 195, 316, 345, 350, 351, 356, 360, 382, 400, 426, 430, 435, 452, 469, 506, 546, 627, 629, 630, 632, 634, 635, 636, 637, 643, 687, 692, 701, 707, 764, 766, 770, 773, 854, 899, 900, 901, 903, 910, 912, 913, 1060, 1066
1117 defense, xxi, xxviii, 10, 54, 69, 127, 285, 375, 382, 445, 524, 537, 565, 717, 794, 926, 1024, 1078 defense mechanisms, 54, 69, 445 deficiency, xvi, xxvi, 96, 303, 308, 329, 428, 466, 500, 610, 612, 674, 692, 703, 736, 791, 858, 881, 905, 907 deficit, 385, 429, 469, 508, 544, 943, 955, 1022, 1030 definition, 6, 18 deformability, 1061 degenerate, xviii, xxii, 106, 312, 393, 395, 600, 629, 633, 636, 639, 653, 705 degenerative conditions, 935 degradation mechanism, xv, 64, 307 degradation pathway, ix, xiii, xviii, xix, xxxiii, xxxiv, 2, 64, 170, 182, 188, 204, 209, 210, 217, 218, 219, 411, 420, 443, 446, 453, 460, 543, 612, 686, 694, 706, 775, 921, 964, 980, 989, 1094 degradation process, xxv, 7, 45, 126, 149, 208, 211, 212, 314, 320, 322, 323, 324, 463, 671, 930 degradation rate, xvi, 11, 21, 36, 308, 321, 323, 324, 325, 326, 327, 328, 329, 383, 688, 694, 1013 dehydration, 719 delivery, xii, 4, 11, 12, 45, 49, 51, 53, 54, 56, 97, 109, 138, 148, 183, 190, 201, 204, 209, 215, 218, 257, 275, 297, 380, 415, 417, 418, 419, 420, 421, 422, 431, 440, 490, 522, 556, 609, 628, 659, 774, 779, 976, 1048, 1096 demand, 768 dementia, xv, 8, 235, 272, 288, 289, 299, 303, 304, 435, 444, 469, 490, 601, 618, 642, 672, 694, 734, 737, 753, 758, 780, 804, 811, 815, 905, 906, 1025, 1057, 1067, 1068, 1069, 1071, 1083, 1085 demyelinating disease, 653 demyelination, 394, 399, 404, 407, 563, 1048 denaturation, 29, 244, 259, 308, 447, 457, 776, 930 dendrites, 8, 189, 273, 315, 316, 350, 357, 374, 375, 376, 380, 382, 412, 433, 655, 659, 660, 677, 733, 905, 925, 1078, 1088 dendritic cell, 269, 296, 331, 843, 846 dendritic spines, xxiv, 273, 428, 433, 435, 652, 655 Denmark, 93, 307
1118 density, xxiv, 229, 230, 282, 325, 378, 379, 381, 382, 389, 412, 423, 437, 463, 633, 651, 652, 655, 666, 669, 919, 956, 1038, 1045, 1084 deoxyribonucleic acid, 42 Department of Energy, 309 dephosphorylation, 4, 45, 48, 72, 214, 413, 468, 747, 907, 908, 933, 936 depolarization, 679, 923 depolymerization, 730, 768, 771, 785 deposition, xxviii, 252, 554, 611, 672, 676, 700, 743, 748, 813, 815, 820, 828, 829, 835, 931, 952, 1091 deposits, xxv, 7, 8, 248, 287, 316, 319, 463, 672, 677, 678, 689, 707, 715, 716, 737, 741, 743, 776, 819, 825, 831, 925, 926, 1056 depressants, 502 depression, xxiv, xxxi, 374, 379, 386, 412, 413, 416, 433, 435, 436, 437, 497, 502, 508, 511, 652, 655, 659, 863, 1074, 1085 deprivation, 36, 398, 516, 646, 684, 817, 820, 930, 1007, 1024, 1069 deregulation, xxxi, 680, 865, 875 derivatives, 260, 940, 967, 972, 992 dermatomyositis, 852 desensitization, 369, 420 destruction, xiii, xviii, xix, xxxv, 3, 20, 29, 40, 42, 48, 49, 52, 63, 107, 169, 171, 181, 184, 191, 204, 234, 236, 250, 294, 310, 330, 333, 363, 370, 393, 411, 423, 424, 434, 443, 462, 465, 477, 506, 527, 570, 622, 630, 637, 641, 664, 695, 696, 756, 769, 829, 872, 883, 893, 907, 920, 927, 960, 962, 975, 980, 986, 1005, 1012, 1013, 1027, 1065, 1093 destruction processes, 637 destructive process, 931 detachment, 106, 747, 1075 detection, 64, 230, 432, 552, 567, 624, 781, 837, 853, 855, 882, 943, 1039, 1072, 1078 detergents, 146, 456, 1085 deubiquitinating enzymes, xi, 5, 11, 71, 745, 1072, 1095 developing brain, 1075 developmental process, 362 diabetes, 653, 852 diabetes mellitus, 852 diacylglycerol, 1086 diagnostic criteria, 915 diagnostic markers, 224, 856 dialysis, 623 diapedesis, 1000, 1003, 1004, 1017
Index diarrhea, 1040, 1041 diet, 23 differentiated cells, 9, 516 differentiation, ix, x, xvi, xxxi, xxxiv, 3, 6, 17, 31, 42, 54, 71, 192, 237, 273, 292, 343, 359, 365, 370, 395, 398, 399, 446, 484, 555, 634, 644, 647, 729, 739, 740, 858, 863, 864, 865, 868, 959, 961, 978, 983, 1048, 1061, 1063, 1064, 1077, 1091 diffraction, 123, 673, 678, 698 diffusion, 1029 digestion, xxxi, 18, 21, 23, 173, 490, 751, 863, 864, 924 dihydroxyphenylalanine, 716 dilation, 931 diluent, 1020 dimer, xv, 84, 133, 250, 272, 282, 384, 449, 454, 455, 456, 772, 841, 868, 937 dimerization, xxvii, 122, 564, 755, 762, 772, 840, 1060 diploid, 1024 direct action, 476, 939 disability, 992 discharges, 815, 904, 906 discomfort, 998, 1020 discrimination, 489 discs, 669 disease activity, 234, 851, 856 disease gene, 111, 439, 440, 441, 573, 593, 619, 705, 711, 783, 785 disease model, xxix, xxxv, 195, 300, 339, 462, 572, 646, 708, 809, 814 disease progression, 11, 305, 503, 510, 558, 601, 617, 648, 692, 828, 991, 992, 1004, 1022, 1047 disinhibition, 1083 dislocation, xv, 202, 252, 253, 271, 276, 287, 294, 295, 421, 1088 disorder, xv, xxvii, 55, 81, 127, 185, 222, 248, 272, 287, 288, 469, 564, 571, 600, 629, 633, 636, 737, 761, 807, 809, 842, 852, 857, 877, 898, 900, 906, 1053, 1057, 1064, 1065, 1066, 1068, 1069, 1071, 1073, 1075, 1078, 1081, 1082, 1085, 1090, 1093, 1097 displacement, 174 disposition, 901 dissociation, 53, 62, 103, 113, 145, 161, 162, 163, 164, 178, 368, 424, 427, 449, 456, 457, 460, 464, 479, 566, 611, 796, 930, 932, 934, 937
Index distress, 429, 430 distribution, xiv, xvii, 5, 10, 129, 173, 186, 189, 190, 227, 228, 229, 230, 233, 235, 237, 239, 295, 312, 315, 321, 330, 373, 376, 379, 386, 387, 390, 437, 465, 504, 589, 643, 667, 677, 690, 691, 694, 726, 729, 799, 801, 802, 803, 818, 819, 828, 830, 840, 874, 1006, 1035, 1039, 1040 divergence, 140 diversification, 452 diversity, xx, 86, 96, 97, 135, 222, 229, 275, 294, 495, 500, 505, 645, 686, 768, 828, 832 division, x, xv, xxxi, 15, 31, 42, 54, 65, 90, 145, 160, 191, 233, 241, 247, 298, 368, 384, 391, 395, 399, 407, 448, 548, 637, 878, 1057, 1092 division of labor, 145 dizygotic, 763, 899 dizygotic twins, 763 dizziness, 1046 DNA, xi, xiii, xxxi, xxxii, xxxiii, 3, 42, 43, 45, 48, 54, 56, 71, 72, 81, 82, 91, 94, 96, 100, 103, 108, 109, 110, 127, 134, 136, 160, 170, 176, 177, 182, 195, 196, 198, 200, 223, 238, 243, 257, 264, 285, 299, 384, 386, 445, 446, 447, 455, 456, 457, 458, 476, 483, 484, 496, 498, 499, 505, 515, 516, 524, 528, 535, 543, 544, 545, 550, 551, 556, 580, 586, 612, 614, 624, 680, 681, 729, 770, 771, 840, 850, 860, 863, 864, 866, 876, 878, 881, 883, 889, 890, 902, 904, 921, 929, 937, 950, 997, 999, 1001, 1008, 1028, 1054, 1057, 1059, 1060, 1062, 1067, 1075, 1079, 1080, 1087, 1090, 1092, 1097 DNA damage, 43, 45, 81, 82, 100, 110, 160, 446, 476, 524, 612, 680, 883, 1057, 1062, 1090 DNA lesions, 1097 DNA polymerase, 82, 1062 DNA repair, xi, xiii, xxxi, xxxii, 3, 54, 56, 71, 72, 82, 91, 94, 96, 108, 109, 134, 170, 176, 177, 196, 200, 238, 257, 524, 556, 863, 876, 881, 889, 904, 937, 1087 DNA sequencing, 223 DNA strand breaks, 127 DNA damaging agents, 499 docetaxel, 1034, 1044, 1045, 1053 dogs, xxxii, 897, 901, 906 domain structure, xi, 71, 77, 451 dopamine, 258, 270, 335, 428, 472, 565, 566, 574, 575, 600, 601, 605, 606, 607, 609, 610, 612, 613, 615, 619, 622, 623, 624, 642, 686, 698, 705, 712, 716, 720, 725, 730, 734, 763,
1119 764, 765, 766, 767, 769, 771, 781, 785, 786, 789, 790, 887, 923, 944, 1012, 1026, 1079, 1090 dopaminergic, xx, xxii, xxvii, 84, 101, 216, 279, 289, 304, 440, 441, 460, 469, 470, 472, 513, 517, 520, 522, 523, 525, 561, 566, 568, 573, 599, 600, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 620, 621, 622, 623, 625, 686, 717, 720, 730, 761, 762, 763, 764, 765, 766, 767, 769, 770, 773, 777, 779, 785, 789, 835, 876, 884, 949, 968, 983, 1035, 1079, 1081 dopaminergic neurons, xx, xxii, xxvii, 84, 101, 216, 279, 304, 440, 441, 469, 470, 472, 513, 517, 520, 522, 523, 525, 561, 566, 573, 599, 600, 601, 602, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 622, 623, 686, 720, 730, 761, 763, 764, 765, 766, 770, 773, 785, 789, 835, 983, 1079, 1081 dorsal horn, xxiv, 651, 652, 653, 654, 655, 656, 657, 658, 659, 663, 664, 665, 666, 667, 669, 1062, 1084 dosage, 84, 396, 507, 558, 830 dosing, 998, 1004, 1019, 1020, 1021, 1022, 1040 double bonds, 1074 Down syndrome, 765, 780 downregulating, 203 down-regulation, 82, 83, 91, 365, 414, 473, 506, 576, 587, 589, 590, 724, 829, 884, 885, 894, 967, 1022, 1074 draft, xvii, 374 drainage, 834 Drosophila, xvi, xvii, xxiii, xxxiii, 75, 83, 84, 85, 86, 91, 92, 103, 130, 147, 149, 151, 153, 154, 157, 175, 191, 192, 195, 198, 203, 236, 237, 268, 282, 298, 300, 334, 337, 343, 345, 347, 348, 349, 350, 351, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 380, 388, 393, 396, 397, 406, 422, 424, 425, 429, 438, 440, 441, 472, 475, 478, 483, 490, 492, 493, 514, 519, 534, 543, 549, 559, 565, 575, 620, 628, 636, 639, 640, 646, 681, 695, 696, 701, 702, 708, 743, 756, 764, 770, 771, 773, 776, 779, 780, 787, 789, 791, 801, 810, 892, 898, 901, 909, 910, 912, 915, 918, 919, 1056, 1058, 1060, 1065, 1066, 1067, 1068, 1070, 1074, 1075, 1076, 1080, 1083, 1091, 1096, 1097 drug action, 511 drug design, 1013, 1017
Index
1120 drug discovery, 516, 791, 976, 1013, 1063 drug resistance, 889, 982, 1031, 1049, 1053 drug safety, 1019 drug targets, 874, 1027 drug toxicity, 1019 drugs, x, xx, xxx, 12, 13, 16, 17, 22, 34, 42, 235, 283, 502, 508, 513, 516, 526, 601, 609, 641, 653, 656, 725, 834, 871, 883, 889, 910, 923, 939, 952, 972, 996, 1007, 1016, 1017, 1021, 1023, 1025, 1026, 1027, 1045, 1047, 1082, 1090 duplication, xviii, 68, 394, 401, 402, 558, 571, 764, 780, 1054, 1081, 1082 duration, 289, 496, 515, 581, 818, 1022 dyes, 1056, 1078 dysarthria, 906, 1064 dysphagia, 92 dystonia, 1064
E E.coli, 540 EAE, 834, 842 early labor, 1017 early warning, 476 eating, 703, 1083 ECM, 188, 189 edema, xxvi, xxxv, 716, 970, 1003, 1012 EEG, 898, 902, 904, 906, 915, 917, 1012, 1019 EEG patterns, 902 egg, 91, 193, 477, 1006, 1034, 1054 elaboration, 366 elderly, 600, 811, 923, 1021 electric current, 1072 electrodes, 902, 1066 electroencephalogram, 815, 898, 902, 1012, 1019 electron, 121, 139, 176, 229, 235, 274, 281, 291, 323, 358, 387, 420, 437, 468, 611, 617, 633, 673, 676, 698, 720, 769, 790, 923, 1006, 1088 electron microscopy, 121, 139, 176, 229, 323, 358, 468, 673, 698 electronic circuits, 377 electrons, 274, 611 electrophoresis, 27, 74, 567, 802, 855, 861, 871 electrostatic interactions, 244, 246, 740 ELISA, 850, 852, 853, 855 elk, 815 elongation, xiii, 4, 46, 47, 63, 94, 102, 112, 170, 171, 182, 183, 201, 279, 557, 644, 873, 932, 1066, 1069
emboli, 941 embolism, 954 embolization, 941 embryo, 61, 91, 349, 350, 352, 364, 506 embryogenesis, 3, 456, 484, 738, 1057 embryonic development, xvi, 343, 364, 484 embryonic stem cells, 780, 881 emergence, 291, 1053 emerging issues, 13 emission, 1007 emotions, 1056 emphysema, 287, 288 employment, 452 encephalitis, 635, 649, 842 encephalomyelitis, xxxiv, 12, 850, 851, 852, 972, 985, 989, 1023, 1081 encephalopathy, 8, 303, 610, 672, 815, 830, 1067 encoding, xxiii, 6, 9, 44, 51, 85, 86, 92, 101, 114, 130, 318, 319, 338, 357, 387, 435, 438, 507, 540, 544, 558, 562, 563, 574, 581, 582, 583, 584, 586, 591, 592, 603, 628, 641, 643, 644, 682, 689, 692, 709, 720, 752, 754, 772, 788, 805, 810, 836, 868, 899, 917, 919, 1014, 1015, 1058, 1081 encouragement, 363 endocrine, 511, 876, 877, 885, 888, 889, 1043, 1061, 1075 endocrine glands, 1075 endocrine system, 877 endocytosis, xi, xiv, xvi, 3, 17, 18, 19, 31, 43, 56, 57, 58, 59, 64, 71, 83, 84, 91, 170, 187, 193, 296, 343, 345, 346, 348, 349, 350, 355, 356, 357, 359, 360, 361, 362, 364, 367, 368, 370, 371, 378, 380, 388, 398, 413, 414, 417, 419, 421, 422, 423, 425, 431, 434, 436, 437, 438, 556, 659, 661, 664, 668, 669, 816, 817, 818, 819, 826, 884, 894, 913, 1090, 1094 endonuclease, 100, 110, 286, 1095 endothelial cells, 198, 288, 301, 303, 447, 499, 518, 530, 788, 943, 945, 959, 973, 974, 986, 995, 1001, 1003, 1004, 1019, 1020, 1022 endothelium, xxxv, 526, 946, 1003, 1007, 1012, 1017 energy, x, xiv, xxxiii, 3, 16, 20, 21, 22, 23, 25, 28, 29, 30, 32, 41, 42, 43, 45, 51, 145, 149, 207, 208, 209, 212, 217, 244, 259, 326, 329, 345, 405, 447, 566, 585, 590, 611, 673, 683, 791, 899, 921, 923, 924, 928, 932, 937, 1092, 1093 energy supply, 23
Index engagement, 10, 294, 320, 539, 690 enlargement, 431, 884 entrapment, 404 environment, xvii, 48, 173, 244, 273, 274, 319, 352, 373, 375, 398, 446, 451, 460, 476, 480, 486, 521, 673, 674, 675, 680, 686, 688, 696, 765, 766, 774, 776, 778, 805, 835, 842, 857, 858, 878, 1069, 1074 environmental change, xviii, 443, 445 environmental conditions, 247, 445, 447 environmental factors, 8, 398, 445, 472, 565, 614, 615, 675, 763, 914, 1057 environmental influences, 502 enzymatic activity, 180, 185, 278, 565, 657, 806, 935, 1059, 1078 enzyme-linked immunosorbent assay, 855 enzymes, x, xii, xiii, xix, xxx, xxxii, 3, 4, 5, 11, 12, 14, 18, 21, 24, 28, 30, 31, 34, 42, 43, 44, 45, 46, 47, 48, 49, 54, 56, 58, 59, 60, 64, 66, 71, 72, 75, 76, 78, 79, 86, 87, 89, 90, 94, 98, 104, 106, 113, 114, 122, 138, 141, 142, 150, 153, 161, 170, 171, 183, 184, 190, 192, 199, 202, 203, 223, 229, 231, 243, 248, 264, 273, 277, 285, 291, 295, 297, 350, 357, 359, 363, 374, 378, 379, 381, 383, 386, 390, 398, 401, 406, 413, 415, 420, 429, 431, 444, 447, 461, 462, 469, 478, 486, 496, 499, 500, 506, 516, 518, 523, 527, 541, 555, 557, 560, 561, 562, 564, 566, 568, 569, 570, 583, 590, 592, 632, 634, 635, 654, 660, 662, 683, 684, 693, 717, 718, 720, 723, 726, 727, 732, 744, 745, 749, 806, 849, 880, 897, 913, 922, 923, 925, 928, 937, 938, 939, 963, 975, 991, 1023, 1061, 1065, 1066, 1071, 1072, 1073, 1074, 1079, 1080, 1081, 1083, 1086, 1087, 1090, 1094, 1095 eosinophilia, 1003 epidemiology, 914 epidermal growth factor, 91, 864, 888 epilepsy, xxxii, 12, 447, 897, 898, 899, 900, 901, 902, 904, 905, 906, 907, 909, 912, 913, 914, 915, 917, 918, 920, 1014, 1065, 1072, 1091 epileptic seizures, 288, 1015 epileptogenesis, 919 epinephrine, 1066 epithelia, 497, 687 epithelial cells, 251, 288, 375, 463, 485, 533, 722, 1067 epithelial ovarian cancer, 861 epithelium, 517, 529, 597, 732
1121 Epstein-Barr virus, 170, 180 equilibrium, 2, 229, 234, 718 equipment, 35 Erk, 407, 647, 716, 719, 720 erythrocytes, 127, 731, 1061 erythropoietin, 1067 Escherichia coli, 17, 36, 37, 87, 132, 476, 479, 538, 547, 620, 984, 1006, 1028, 1063 esophageal squamous cell carcinoma, 290 EST, 580 ester, 30, 43, 45, 46, 171, 438, 722, 973, 986, 1066 estimating, 311 estradiol, 885, 978, 979 estrogen, xxxii, 499, 509, 876, 884, 887, 894, 978, 979 ethanol, 259 ethanolamine, 909 etiology, xxxiii, 54, 469, 502, 718, 720, 730, 764, 780, 898, 903, 904, 914, 915, 1081 etiquette, 1083 EU, 35, 221 euchromatin, 232 eukaryote, 1092 eukaryotic cell, ix, xi, xx, xxxi, xxxiii, 23, 72, 93, 94, 117, 118, 138, 171, 172, 217, 228, 257, 275, 296, 309, 345, 445, 448, 453, 455, 463, 515, 537, 682, 704, 774, 850, 856, 863, 864, 921, 935, 1013, 1023, 1061, 1067, 1076 Europe, 777, 906 European Community, 777 European Union, 35 evoked potential, 378 evolution, xxxv, 22, 24, 31, 38, 64, 74, 113, 143, 451, 481, 482, 534, 638, 696, 925, 992, 1011 examinations, 966 excision, 96, 108, 196, 282, 896, 1097 excitability, 416, 497, 504, 679, 857, 911, 913, 918, 944 excitation, 385, 416, 422, 936 excitatory postsynaptic potentials, 1074 excitatory synapses, 358, 433, 668 excitotoxicity, 111, 431, 440, 442, 473, 596, 614, 616, 700, 783, 922, 923, 956, 1007 excitotoxins, 386, 514, 842, 1015 exclusion, 195, 244, 545 excuse, 292 execution, xx, xxiii, 513, 514, 517, 519, 628, 640, 868, 937, 1057, 1062 exercise, x, 41, 476
Index
1122 exocytosis, 378, 414, 421, 424, 425, 426, 435, 438, 661 exons, 564, 738, 771, 772, 773, 816 experimental autoimmune encephalomyelitis, 834, 842, 968, 1010, 1032 experimental condition, xxxvi, 101, 377, 396, 823, 1012 expertise, 13 exposure, xx, xxi, 27, 53, 105, 119, 126, 127, 144, 213, 221, 244, 252, 253, 257, 259, 288, 318, 396, 401, 427, 445, 447, 458, 460, 470, 496, 497, 502, 503, 515, 535, 543, 553, 555, 559, 561, 565, 568, 572, 581, 592, 609, 611, 622, 623, 625, 645, 679, 717, 718, 768, 778, 791, 835, 892, 898, 926, 937, 944, 955, 968, 981, 992, 1003, 1004, 1006, 1025, 1026, 1035, 1043, 1046, 1049 expressed sequence tag, 580, 582 extracellular matrix, 412, 829, 938, 1077 extraction, xv, 230, 233, 252, 253, 271, 295 extraction process, 253 extrapolation, 290 eyes, 1082, 1085
F FAD, 242, 256 failure, xiv, xxi, xxii, xxiii, xxvi, xxxiii, 7, 9, 79, 207, 208, 215, 218, 256, 357, 366, 446, 467, 492, 510, 522, 550, 553, 599, 601, 603, 605, 608, 609, 612, 613, 616, 627, 629, 631, 641, 689, 692, 736, 746, 776, 791, 808, 835, 877, 918, 921, 923, 926, 963, 993, 1030, 1046, 1056, 1078 family, x, xi, xii, xix, xxvi, 7, 44, 45, 47, 49, 51, 59, 60, 61, 64, 66, 71, 74, 75, 76, 77, 78, 79, 81, 83, 84, 85, 86, 88, 90, 93, 106, 108, 110, 112, 115, 117, 140, 152, 182, 192, 201, 220, 224, 250, 251, 262, 279, 282, 284, 294, 296, 298, 300, 301, 310, 348, 359, 360, 365, 367, 369, 389, 433, 436, 437, 438, 440, 444, 446, 448, 450, 451, 453, 457, 461, 463, 464, 468, 478, 479, 480, 484, 485, 498, 514, 517, 518, 519, 522, 526, 530, 531, 538, 540, 541, 542, 543, 550, 552, 563, 584, 586, 590, 604, 621, 632, 633, 643, 644, 653, 655, 664, 720, 732, 735, 738, 740, 753, 759, 765, 770, 771, 774, 781, 787, 789, 801, 803, 822, 840, 878, 879, 884, 890, 911, 916, 918, 919, 924, 929, 934, 937, 954, 957, 1013, 1034, 1055, 1056, 1057,
1058, 1059, 1060, 1062, 1067, 1068, 1069, 1070, 1071, 1074, 1076, 1078, 1079, 1080, 1081, 1083, 1086, 1088, 1089, 1090, 1091, 1092, 1093, 1096 family history, 84 family members, xi, 59, 64, 71, 74, 75, 76, 77, 78, 86, 90, 106, 437, 463, 468, 485, 517, 522, 878, 916, 924, 1070, 1071, 1090 FAS, 519, 1057 fasciculation, 351 fasting, 53 fat, xvii, 91, 344, 359, 361, 371, 393, 397, 632, 1074 fatal familial insomnia, 815, 830 fatigue, 1040, 1041 fatty acids, 124, 125, 718 FDA, 12, 644, 940, 967, 1016, 1021, 1038, 1039 FDA approval, 644, 1039 fear, 428 febrile seizure, 902 feedback, xxii, 457, 485, 503, 580, 582, 583, 878, 883, 884, 885, 907, 909, 927, 932, 936, 1071, 1075, 1091 feet, 829, 1045 females, 1082 fencing, 20 fermentation, 971 fermentation broth, 971 ferritin, 723 fertility, 484 fertilization, 234, 239 fever, xxxii, 897, 898 fibers, 233, 398, 402, 403, 472, 608, 655, 664, 1046, 1073 fibrillation, 625 fibrinolysis, 946 fibroblast growth factor, 882, 888 fibroblasts, 175, 179, 290, 305, 400, 437, 508, 543, 549, 584, 593, 683, 843, 1024, 1049 fibrosis, 42, 170, 180, 208, 221, 242, 246, 251, 264, 265, 266, 272, 287, 288, 294, 463, 488, 935, 1062, 1064 fidelity, xi, xxv, 7, 71, 73, 273, 500, 671 filament, 136, 237, 254, 267, 269, 571, 658, 727, 752, 753, 755, 757, 926, 994 film, 27 filtration, 26 first generation, 965 fission, 80, 89, 96, 97, 141, 157, 177, 184 fixation, 229, 230, 236, 237
Index flank, 331 flavopiridol, 524, 873, 953 flexibility, 75, 355, 417, 447, 673 flexor, 664 flight, 549, 770, 910 fluctuations, 445, 617, 673, 675, 878 fluid, 19, 850, 1003, 1041, 1072, 1079 fluorescence, 74, 223, 255, 320, 327, 468, 636, 673, 688, 693, 712, 822, 835 fluoxetine, 502, 508, 509 focusing, ix, 15, 96, 210, 601, 630, 857, 861, 1026, 1072, 1079 folded conformations, 454 folding intermediates, xix, 253, 259, 444, 449, 462 food, 1038 Ford, 110, 166, 826, 1050 forebrain, xxii, 376, 502, 508, 595, 600, 606, 608, 662, 947, 956, 1009 fragmentation, xviii, 304, 358, 393, 515, 528, 633, 640, 929, 950, 997, 1008 frameshift mutation, 409, 903 France, 985 free calcium level, 938 free energy, 244, 259, 673 free radicals, 611, 612, 614, 629, 717, 924, 936, 1074, 1080 freezing, 291, 601 friends, 575 frontal cortex, 502, 509 frontal lobe, 902 frontotemporal dementia, xv, 8, 272, 289, 299, 304, 692, 711, 753, 755, 1069 fructose, 21 fruit flies, xxxii, 897, 899, 901 fuel, 16 functional analysis, 112, 152 functional aspects, 107, 258 fungal metabolite, 986 fungi, 247 fusion, 10, 43, 44, 46, 57, 58, 59, 60, 73, 74, 151, 160, 182, 216, 224, 230, 232, 257, 270, 282, 299, 340, 358, 398, 412, 413, 421, 424, 637, 652, 661, 683, 687, 693, 710, 757, 766, 789, 823, 936, 1065, 1076, 1094, 1096
G gait, 763, 1085 gametogenesis, 902
1123 ganglion, 345, 365, 398, 666, 670, 790, 1068 garbage, 555, 571 gases, 998 gastrointestinal tract, 43, 853 GDP, 286, 932, 1092 gel, 26, 27, 74, 567, 861, 871 gene, xvii, xviii, xxi, xxvi, xxxi, xxxii, xxxiv, xxxv, 2, 25, 44, 48, 54, 55, 67, 68, 81, 82, 83, 84, 90, 91, 92, 101, 102, 104, 106, 109, 111, 113, 115, 131, 133, 135, 139, 140, 147, 152, 155, 170, 175, 176, 177, 181, 185, 223, 224, 239, 248, 257, 260, 264, 265, 279, 282, 288, 296, 297, 298, 303, 304, 308, 318, 322, 336, 338, 344, 345, 346, 347, 348, 349, 350, 351, 356, 359, 363, 364, 365, 366, 367, 368, 371, 373, 376, 378, 386, 388, 389, 394, 396, 397, 399, 401, 406, 407, 408, 412, 423, 427, 428, 429, 435, 439, 440, 441, 446, 451, 457, 458, 472, 473, 477, 479, 481, 483, 484, 485, 497, 498, 499, 501, 503, 504, 505, 507, 508, 509, 511, 520, 527, 532, 533, 538, 543, 544, 545, 546, 548, 550, 551, 552, 555, 557, 558, 560, 561, 562, 563, 572, 573, 574, 575, 579, 581, 582, 589, 591, 595, 596, 597, 598, 602, 603, 614, 616, 618, 619, 620, 621, 628, 633, 637, 638, 640, 641, 643, 644, 645, 646, 647, 648, 661, 662, 664, 666, 681, 684, 687, 689, 690, 691, 692, 695, 703, 706, 709, 711, 716, 720, 729, 730, 735, 737, 738, 753, 763, 764, 766, 768, 770, 771, 772, 773, 778, 779, 781, 782, 783, 784, 785, 786, 787, 788, 789, 791, 798, 805, 808, 810, 814, 816, 821, 830, 831, 842, 843, 845, 864, 866, 874, 876, 877, 879, 884, 885, 888, 890, 891, 893, 898, 899, 900, 901, 902, 903, 904, 906, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 922, 923, 924, 927, 929, 934, 935, 937, 938, 949, 955, 958, 959, 973, 974, 989, 991, 992, 995, 996, 997, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1011, 1013, 1025, 1026, 1035, 1048, 1056, 1058, 1060, 1062, 1064, 1065, 1066, 1067, 1068, 1071, 1072, 1075, 1076, 1077, 1081, 1082, 1083, 1085, 1088, 1089, 1091, 1092, 1093, 1095, 1096, 1097 gene amplification, 879 gene expression, xvii, xxxiv, xxxv, 2, 68, 224, 298, 346, 367, 373, 376, 386, 388, 389, 408, 423, 427, 477, 479, 484, 485, 498, 504, 505, 507, 508, 527, 546, 548, 552, 582, 591, 595, 596, 638, 661, 662, 729, 770, 771, 772, 843,
1124 866, 874, 885, 955, 958, 959, 973, 974, 989, 991, 992, 995, 996, 997, 999, 1000, 1001, 1002, 1004, 1005, 1011, 1025, 1026, 1035, 1058, 1075 gene promoter, 457, 545, 1064 gene silencing, 82, 83, 91, 557 gene therapy, 647, 938 gene transfer, 408, 472, 473, 533 generalization, 276 generalized tonic-clonic seizure, 905 generation, xi, xxix, 13, 71, 74, 94, 98, 105, 114, 126, 128, 129, 134, 136, 151, 194, 240, 248, 250, 251, 261, 287, 316, 318, 331, 332, 370, 382, 383, 405, 458, 464, 469, 523, 527, 554, 587, 614, 624, 666, 677, 678, 679, 680, 709, 756, 769, 772, 798, 817, 823, 831, 833, 836, 837, 839, 844, 845, 846, 869, 870, 874, 878, 879, 889, 924, 932, 935, 951, 966, 981, 1015, 1033, 1065 genes, xx, xxii, xxiii, xxvii, xxxi, xxxii, 4, 9, 34, 44, 51, 54, 68, 74, 81, 82, 83, 86, 92, 98, 102, 105, 106, 119, 130, 135, 140, 152, 153, 177, 203, 243, 250, 260, 285, 286, 287, 303, 319, 338, 344, 345, 348, 349, 359, 360, 362, 366, 367, 369, 385, 400, 427, 428, 430, 433, 438, 452, 456, 457, 458, 467, 476, 485, 492, 495, 498, 499, 502, 504, 505, 509, 510, 518, 523, 524, 525, 540, 543, 544, 546, 547, 550, 552, 558, 560, 571, 572, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 595, 596, 599, 602, 614, 628, 683, 687, 689, 709, 732, 754, 761, 763, 764, 767, 769, 774, 775, 776, 778, 782, 786, 790, 800, 810, 818, 828, 836, 837, 840, 842, 843, 847, 866, 868, 872, 874, 875, 883, 884, 897, 899, 900, 901, 902, 903, 910, 911, 912, 913, 914, 917, 918, 920, 924, 926, 930, 933, 938, 945, 964, 978, 979, 990, 992, 999, 1001, 1014, 1015, 1022, 1060, 1065, 1070, 1071, 1075, 1078, 1080, 1081, 1086, 1087, 1092, 1095, 1101 genetic abnormalities, 84 genetic alteration, xx, 398, 537, 865, 866 genetic code, ix, 15, 243, 1067 genetic defect, 9, 555, 560, 562, 565, 901, 905 genetic disease, xxxii, 402, 463, 877, 898 genetic disorders, xxxi, 287, 630, 875, 899 genetic factors, xxvii, xxxii, 614, 761, 763, 767, 897 genetic information, 42
Index genetic mutations, xxi, 553, 554, 565, 567, 764, 766, 780, 903 genetics, 92, 269, 280, 334, 357, 362, 575, 697, 899, 913, 914 genome, 4, 31, 44, 64, 74, 75, 76, 91, 141, 183, 184, 185, 279, 357, 524, 557, 582, 844, 877, 925 genotype, 398, 1082 germ line, 102 Germany, 16, 67, 93, 373, 495, 752 germline mutations, 1092 gift, 35, 255 gland, 29, 368 glass, 360, 361, 1072, 1075 glia, xvii, 318, 374, 383, 386, 394, 395, 447, 468, 666, 676, 679, 807, 835, 866, 939, 991, 1001 glial cells, xiv, xvii, xviii, xxx, 6, 227, 232, 351, 355, 373, 374, 375, 377, 378, 382, 386, 389, 393, 394, 395, 399, 405, 523, 544, 550, 610, 816, 849, 870, 968, 1001, 1014, 1091 glioblastoma, 864, 865, 868, 873, 881 glioblastoma multiforme, 873 glioma, xxxi, 525, 536, 863, 865, 868, 869, 873, 951, 972, 985 globus, 623 glucagon, 682 glucocorticoid receptor, xx, 478, 482, 495, 496, 497, 499, 504, 505, 506, 507, 508, 509, 510, 511, 542, 876, 887, 894, 1034 glucocorticoids, 496, 497, 501, 503, 504, 506, 894, 1071 glucose, 16, 20, 21, 62, 272, 274, 277, 284, 301, 444, 448, 449, 479, 491, 538, 704, 775, 907, 923, 927, 949, 1071, 1086 GLUT4, 62 glutamate, xxiv, 7, 48, 123, 125, 171, 187, 291, 345, 348, 370, 374, 378, 379, 381, 387, 388, 389, 412, 416, 418, 423, 431, 432, 434, 435, 436, 437, 441, 442, 473, 504, 516, 517, 528, 649, 652, 653, 658, 661, 662, 663, 667, 668, 669, 679, 680, 700, 701, 911, 912, 913, 919, 920, 922, 923, 935, 936, 956, 1056, 1075, 1078 glutamate receptor antagonists, 667, 936, 937 glutamic acid, 308, 312, 716 glutathione, 74, 127, 274, 444, 449, 492, 580, 588, 597, 679, 717, 720, 952 glutathione peroxidase, 952 glycans, 273, 274, 276, 277, 279, 296 glycerol, 189
Index glycine, 28, 44, 60, 68, 73, 79, 82, 170, 184, 198, 259, 309, 374, 379, 388, 412, 414, 437, 473, 555, 663, 666, 775, 913, 920, 1082, 1096 glycogen, 20, 538, 551, 898, 901, 905, 906, 907, 908, 917, 1069 glycolysis, 927 glycoproteins, 109, 182, 201, 277, 278, 292, 294, 507, 1097 glycosylation, 178, 273, 274, 283, 414, 428, 742, 816, 822 goals, 945, 1013 gold, 18, 176, 470 grades, 1041 grants, 35, 65, 86, 107 granules, 551, 607, 608, 705, 1078 granulosa cells, 239 gravity, xxvi, 735 gray matter, 375, 1073, 1090 grey matter, 815 groups, xiii, xxxiv, 16, 21, 22, 34, 43, 44, 47, 54, 95, 96, 139, 141, 159, 171, 244, 287, 317, 318, 394, 460, 520, 557, 567, 677, 679, 692, 720, 724, 740, 768, 835, 851, 853, 854, 858, 880, 903, 919, 933, 962, 965, 1019, 1020, 1061, 1064, 1085, 1087, 1090 growth, ix, xiv, xvi, xvii, xxxi, xxxiii, 3, 31, 81, 83, 85, 87, 90, 91, 102, 147, 177, 191, 192, 204, 207, 229, 230, 236, 273, 286, 291, 344, 345, 347, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 365, 366, 368, 369, 370, 371, 374, 391, 393, 394, 395, 396, 397, 398, 399, 400, 406, 407, 408, 414, 417, 422, 434, 438, 446, 456, 483, 527, 535, 552, 589, 630, 636, 645, 655, 666, 754, 755, 820, 834, 840, 858, 864, 869, 873, 875, 876, 877, 878, 879, 883, 884, 887, 888, 889, 891, 894, 895, 911, 922, 948, 967, 970, 978, 982, 985, 998, 1031, 1033, 1048, 1049, 1050, 1056, 1058, 1064, 1069, 1070, 1077, 1083, 1086, 1092 growth factor, xxxi, 83, 91, 102, 345, 360, 365, 374, 391, 394, 395, 407, 417, 535, 645, 666, 820, 834, 864, 875, 876, 877, 878, 883, 884, 887, 888, 891, 894, 922, 998, 1069, 1070, 1077 growth factors, 878, 888, 998 growth hormone, xxxii, 204, 876, 887, 888, 889, 894, 895, 948 growth rate, 879 guanine, 370
1125 guardian, 524 guidance, xvi, 3, 128, 191, 343, 347, 350, 351, 352, 354, 355, 356, 357, 362, 368, 369, 370, 371, 396, 397, 1065, 1067, 1069, 1088 guidelines, 1053 gynecomastia, 1097
H hairpins, 1069, 1096 half-life, 2, 20, 21, 164, 248, 340, 416, 417, 419, 427, 628, 686, 768, 805, 812, 816, 869, 1039, 1048 hallucinations, 905 hands, 1045, 1064 haplotypes, 618 harm, 521 HBV, 199 HE, 298, 479 head trauma, xxxii, 897, 898, 993 headache, 998, 1025, 1047, 1048 healing, 1003 health, 265, 266, 389, 432, 477 hearing impairment, 1047 hearing loss, 1047, 1053 heart attack, xxxvi, 1012 heart disease, 288 heart rate, 998 heat, xix, xx, xxii, 2, 9, 25, 27, 29, 37, 53, 61, 63, 74, 113, 118, 119, 148, 160, 170, 178, 196, 214, 221, 223, 254, 268, 272, 283, 301, 308, 316, 317, 322, 334, 444, 445, 446, 448, 449, 456, 457, 458, 459, 467, 473, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 490, 491, 492, 493, 506, 513, 517, 521, 522, 525, 533, 537, 538, 539, 540, 543, 544, 545, 547, 548, 549, 550, 551, 552, 580, 581, 585, 586, 593, 594, 595, 596, 602, 649, 663, 674, 684, 691, 692, 697, 727, 732, 746, 758, 759, 798, 803, 811, 814, 815, 828, 922, 924, 926, 928, 933, 935, 948, 953, 954, 992, 994, 1007, 1012, 1024, 1034, 1062, 1069, 1071, 1072, 1089, 1090, 1093 heat shock protein, xx, xxii, 2, 9, 113, 118, 170, 178, 223, 272, 308, 316, 317, 334, 444, 445, 446, 448, 449, 458, 476, 477, 478, 479, 480, 481, 483, 487, 488, 490, 491, 492, 493, 513, 517, 521, 522, 525, 533, 537, 538, 543, 547, 548, 549, 550, 552, 580, 581, 585, 586, 595, 596, 602, 649, 674, 697, 727, 746, 758, 798,
1126 811, 928, 933, 953, 954, 992, 994, 1007, 1012, 1024, 1034, 1062, 1089 heating, 74, 663 heavy metals, 283, 445, 612, 616 Hedgehog signaling, 166 helical conformation, 765, 766 heme, 449, 600, 607, 927 heme oxygenase, 600, 607 hemisphere, 942, 996 hemoglobin, 22, 29, 36, 39 hemoglobinopathies, 22 hemorrhage, 943, 1045 hemorrhagic stroke, 1019 hepatitis, 170, 180, 181, 199, 839, 846 Hepatitis C virus, 197 hepatocellular carcinoma, 290, 305 hepatocytes, 37, 230, 543, 550 hepatoma, 37, 501 heptane, 1018 herbicide, 611, 624 herpes, 77, 180 herpes virus, 180 heterochromatin, 232 heterogeneity, 823, 866, 899 high-performance liquid chromatography, 214 hippocampus, 127, 366, 381, 385, 391, 433, 471, 500, 502, 504, 505, 507, 508, 509, 510, 519, 654, 655, 663, 773, 777, 791, 827, 904, 911, 926, 929, 930, 932, 934, 947, 950, 952, 1028, 1074 histidine, 74, 75, 77, 78, 79, 123, 223, 566, 816, 900, 1096 histocompatibility antigens, 835 histology, 622, 1030 histone, 28, 29, 38, 43, 45, 58, 74, 82, 91, 105, 115, 125, 126, 127, 134, 283, 300, 546, 552, 556, 873 histopathology, 478 HIV, xxxv, 134, 170, 180, 181, 198, 199, 268, 389, 635, 649, 842, 936, 940, 962, 972, 984, 985, 992, 1006, 1011, 1012, 1016, 1025, 1026, 1034, 1035 HIV infection, 1025 HIV-1, 170, 180, 181, 198, 199, 268, 940, 972, 985, 992, 1034 HLA, 331, 836 HO-1, 449, 600, 607 homeostasis, xiv, xv, xix, xxiv, xxxvi, 91, 171, 193, 207, 274, 286, 288, 307, 390, 444, 445, 458, 581, 589, 591, 597, 598, 602, 612, 613,
Index 614, 625, 671, 678, 680, 689, 706, 709, 726, 923, 924, 927, 931, 952, 979, 993, 1012, 1038, 1049 homocysteine, 288, 304 hormone, xx, xxxii, 24, 38, 103, 204, 215, 449, 454, 455, 482, 495, 496, 497, 498, 499, 500, 501, 503, 505, 506, 507, 510, 517, 732, 876, 877, 883, 885, 886, 894, 916, 1067, 1071, 1072, 1079 host, xiii, xv, xxxiii, 22, 170, 173, 181, 190, 198, 241, 378, 683, 795, 818, 820, 830, 840, 922, 924, 925, 930, 936, 993, 1001, 1073, 1078 housing, xiii, 169 HPA axis, 502, 503, 510 HPV, 257 human behavior, 502 human brain, xxx, 74, 112, 340, 377, 383, 390, 440, 468, 477, 549, 619, 620, 718, 754, 783, 828, 849, 870, 874, 904, 948, 959 human condition, 904, 923 human genome, 4, 31, 44, 75, 76, 184, 279, 498, 499, 557, 900, 1066 human immunodeficiency virus, 170, 180, 198, 389, 732, 962, 972, 985, 1012 human leukocyte antigen, 836 human papillomavirus, 39, 439, 902, 915 human subjects, 549 Huntington’s disease, ix, xxv, xxvii, xxx, xxxiv, 2, 55, 72, 195, 247, 289, 308, 315, 429, 430, 521, 543, 554, 601, 672, 715, 793, 794, 809, 812, 849, 989, 991, 1071 hyaline, 764, 886 hybrid, 89, 138, 162, 164, 172, 175, 177, 179, 180, 187, 189, 194, 229, 285, 311, 350, 356, 360, 429, 531, 782, 832 hybridization, 882 hydrogen, 16, 244, 274, 335, 457, 473, 524, 576, 625, 677, 679, 697, 718, 788, 841, 939, 971, 1060, 1088 hydrogen bonds, 244, 939, 971, 1060 hydrogen peroxide, 335, 457, 473, 524, 576, 625, 679, 697, 718, 788, 841, 1088 hydrolysis, xii, 22, 23, 43, 87, 117, 126, 129, 133, 135, 145, 146, 148, 149, 150, 155, 161, 162, 163, 164, 166, 167, 172, 176, 178, 211, 212, 213, 214, 217, 248, 249, 253, 280, 282, 284, 310, 332, 333, 448, 451, 452, 453, 454, 455, 460, 480, 481, 585, 636, 759, 764, 766, 768, 923, 935, 1070, 1080, 1087 hydroperoxides, 612
Index hydrophobic interactions, 450, 1086 hydrophobicity, xii, 118, 125, 244, 259, 678, 721, 728, 731, 733 hydroxyl, 123, 124, 624, 668, 965, 968, 1012, 1018, 1026, 1064, 1088 hyperalgesia, xxiv, 648, 651, 653, 654, 655, 656, 657, 659, 661, 662, 663, 664, 670 hyperphosphorylated tau protein, 749 hyperplasia, 879, 888, 1061, 1075 hypersensitivity, 177, 648, 659, 666, 670, 1023 hypertension, 966 hyperthermia, 449, 478, 550, 954 hypertrophy, 966, 1086 hypoglossal nerve, 648 hypokalemia, 1041 hypokinesia, 1081 hypotension, 1040, 1041, 1046 hypothalamus, 189, 884, 885, 1071 hypothesis, xvi, xix, xxii, 17, 20, 21, 23, 27, 29, 31, 55, 165, 173, 175, 180, 186, 214, 217, 267, 281, 308, 318, 323, 327, 334, 348, 351, 360, 361, 384, 404, 430, 444, 456, 467, 502, 508, 559, 571, 599, 612, 614, 616, 639, 681, 691, 694, 697, 701, 723, 724, 726, 728, 731, 743, 745, 750, 780, 796, 803, 822, 844, 854, 907, 915, 924, 944, 980, 1081, 1084 hypoxia, xii, 102, 112, 138, 150, 179, 234, 279, 289, 334, 445, 449, 499, 514, 517, 525, 645, 698, 729, 841, 919, 922, 924, 925, 926, 927, 929, 947, 948, 949, 962, 977, 979, 1072 hypoxia-inducible factor, 112, 179, 729, 927, 962, 977, 979 hypoxic cells, 958
I iatrogenic, 815 ICAM, xxxv, 938, 941, 949, 959, 973, 990, 995, 1002, 1010, 1012, 1014, 1017, 1019, 1030, 1056 identification, xxv, xxvii, 12, 22, 28, 29, 31, 34, 39, 86, 142, 156, 164, 165, 166, 190, 196, 200, 203, 214, 263, 264, 332, 346, 348, 358, 360, 362, 397, 475, 482, 488, 489, 531, 548, 555, 558, 560, 563, 565, 576, 581, 603, 660, 672, 694, 720, 732, 752, 761, 776, 825, 877, 884, 890, 899, 912, 929, 948, 976, 979, 1051 identity, 3, 9, 38, 60, 61, 63, 355, 419, 420, 479, 907, 911, 912, 925
1127 idiopathic, 7, 258, 472, 576, 663, 767, 788, 899, 913 idiosyncratic, 1047 IFN, 121, 131, 170, 175, 180, 242, 249, 250, 308, 311, 374, 383, 839, 870, 1024 IFNγ, 118, 119, 120, 122, 125, 126, 128, 250 IL-6, xxxv, 374, 386, 485, 533, 662, 719, 941, 967, 990, 995, 1001, 1014, 1017, 1022 IL-8, 662 ILAR, 946 illumination, 56 images, 77, 190, 690 imaging, 356, 631, 693, 812, 945, 1007, 1030 immersion, 1091 immune activation, 851 immune reaction, 835 immune response, x, xxix, xxx, xxxiv, 3, 27, 42, 54, 59, 179, 180, 181, 195, 311, 312, 330, 384, 450, 480, 833, 835, 839, 840, 843, 850, 851, 852, 853, 854, 856, 857, 858, 869, 870, 975, 989, 1026, 1057 immune system, xxx, 52, 292, 701, 835, 847, 850, 854, 857, 858 immunity, xxi, 174, 250, 312, 375, 579, 835, 837, 845, 859, 1009 immunocompetent cells, 288, 382 immunocytochemistry, 253, 256, 505, 828 immunodeficiency, 170, 180, 198, 199, 389 immunoglobulin, 302, 444, 449, 836, 1003 immunoglobulin superfamily, 1003 immunohistochemistry, 375, 471, 855 immunomodulation, 835 immunomodulatory, xii, 118 immunophilins, 539 immunoprecipitation, 278, 401 immunoproteasomes, xxix, 119, 120, 122, 126, 129, 175, 178, 179, 180, 181, 229, 231, 236, 250, 257, 269, 311, 313, 330, 331, 383, 390, 833, 839, 840, 845, 846, 874, 1040, 1085 immunoreactivity, xxxiii, xxxv, 228, 230, 235, 375, 376, 558, 650, 670, 757, 759, 811, 828, 870, 886, 895, 922, 925, 926, 930, 932, 941, 948, 959, 990, 1001, 1004 immunostimulatory, 249 immunosuppressive agent, 972 immunosurveillance, 128 immunotherapy, 713, 746, 757, 860 impairments, 605 implementation, 964 imprinting, 902, 903, 904, 916, 1068
1128 in situ, 656, 1048, 1058 in vitro, xiii, xix, xx, xxii, xxiii, xxv, xxx, 21, 37, 40, 56, 61, 74, 76, 79, 83, 84, 98, 101, 104, 127, 135, 142, 144, 150, 159, 161, 164, 165, 172, 173, 175, 176, 178, 179, 180, 181, 197, 213, 214, 217, 221, 223, 224, 228, 233, 249, 253, 264, 280, 300, 311, 312, 313, 315, 318, 320, 326, 335, 337, 349, 351, 352, 356, 358, 362, 395, 396, 399, 402, 444, 446, 456, 463, 464, 468, 469, 473, 474, 475, 484, 488, 490, 495, 500, 501, 503, 520, 522, 523, 547, 549, 558, 559, 563, 564, 566, 567, 568, 571, 577, 585, 595, 596, 598, 599, 604, 605, 607, 609, 611, 613, 614, 625, 627, 630, 633, 634, 636, 637, 639, 640, 671, 673, 676, 679, 681, 686, 687, 699, 708, 719, 723, 740, 741, 746, 748, 755, 757, 770, 782, 785, 795, 799, 805, 806, 809, 831, 842, 844, 849, 858, 868, 889, 906, 907, 925, 927, 929, 933, 936, 954, 964, 967, 968, 972, 980, 985, 992, 1001, 1013, 1016, 1022, 1035, 1038, 1056, 1080 in vivo, xix, xx, xxii, xxiii, xxv, 9, 21, 36, 49, 50, 52, 55, 56, 67, 72, 83, 129, 132, 142, 152, 161, 164, 165, 172, 173, 175, 178, 179, 185, 212, 213, 224, 249, 253, 264, 282, 310, 318, 320, 321, 326, 328, 335, 338, 348, 349, 352, 355, 356, 358, 362, 368, 370, 391, 400, 402, 409, 426, 430, 444, 454, 459, 463, 464, 473, 479, 480, 486, 495, 500, 501, 503, 508, 521, 522, 523, 547, 558, 559, 564, 569, 585, 595, 596, 598, 599, 605, 606, 608, 609, 613, 614, 627, 630, 633, 634, 636, 637, 638, 639, 640, 655, 671, 673, 679, 681, 687, 688, 692, 693, 698, 709, 712, 719, 722, 741, 743, 756, 765, 766, 775, 788, 800, 806, 807, 809, 824, 826, 842, 843, 844, 856, 868, 873, 888, 906, 907, 929, 932, 933, 936, 945, 946, 954, 964, 972, 976, 984, 985, 993, 1025, 1028 incidence, xxxvi, 305, 446, 565, 815, 899, 923, 941, 1019, 1026, 1038, 1041, 1043, 1044, 1045, 1046, 1048, 1049 inclusion, xiv, xv, xxi, xxii, xxvii, xxviii, xxxiii, 6, 8, 55, 69, 175, 178, 185, 195, 197, 204, 207, 238, 253, 254, 259, 267, 268, 269, 272, 288, 289, 303, 304, 308, 315, 317, 319, 336, 337, 338, 339, 340, 395, 442, 467, 469, 472, 474, 475, 520, 521, 522, 524, 531, 532, 533, 543, 553, 558, 559, 571, 572, 576, 593, 600, 601, 610, 612, 614, 621, 622, 623, 643, 646, 672, 677, 678, 681, 686, 688, 689, 692, 697, 702,
Index 706, 708, 709, 710, 712, 717, 721, 727, 762, 765, 769, 776, 786, 787, 794, 795, 797, 798, 800, 801, 804, 805, 808, 809, 811, 812, 819, 898, 912, 917, 933, 949, 953, 960, 968, 983, 1033, 1034, 1056, 1067, 1069, 1072, 1084 inclusion bodies, xiv, xxi, xxxiii, 6, 8, 55, 175, 185, 204, 207, 238, 268, 269, 303, 308, 315, 317, 336, 338, 340, 442, 467, 469, 474, 475, 520, 521, 522, 543, 553, 558, 559, 571, 576, 593, 610, 612, 622, 643, 672, 677, 689, 697, 702, 709, 717, 721, 727, 776, 786, 787, 812, 819, 898, 912, 917, 933, 960, 1033, 1056, 1067, 1072, 1084 incubation period, xxviii, 813, 815 incubation time, 516, 830, 1024 independence, 640, 808 indexing, 448 India, 906, 917 indication, 238, 258, 338, 350, 395, 398, 408, 532, 691, 706, 913, 1033 indicators, 467 indirect effect, 24, 745 indolent, 1050 inducer, 103, 530, 568, 723, 967 inducible protein, 114, 210, 263, 433, 664 induction, ix, xx, xxi, xxxi, 1, 13, 82, 103, 104, 110, 181, 193, 236, 258, 275, 278, 282, 284, 285, 286, 287, 289, 301, 311, 381, 383, 385, 390, 396, 399, 433, 434, 448, 456, 457, 458, 467, 468, 472, 473, 476, 478, 483, 491, 492, 498, 499, 503, 510, 513, 514, 517, 523, 524, 525, 533, 536, 546, 550, 579, 580, 581, 582, 591, 594, 595, 596, 663, 666, 676, 678, 679, 685, 691, 708, 717, 759, 776, 783, 790, 803, 809, 835, 850, 873, 885, 934, 938, 949, 954, 955, 956, 957, 967, 973, 981, 985, 992, 1010, 1031, 1034, 1050 industry, 13, 1007 inefficiency, 823 infancy, 64, 902, 1082 infarction, xxxiv, xxxv, 596, 923, 948, 952, 955, 958, 984, 990, 995, 996, 1004, 1007, 1008, 1012, 1030, 1032, 1050 infection, xxvi, xxix, 63, 106, 180, 257, 382, 701, 716, 815, 816, 817, 818, 819, 820, 821, 830, 1016, 1023, 1072 infertility, 879, 1097 inflammation, x, xxv, xxix, xxxi, xxxv, 42, 179, 250, 289, 311, 384, 477, 523, 568, 609, 625, 657, 658, 660, 664, 666, 700, 716, 717, 718,
Index 722, 723, 724, 725, 726, 728, 729, 833, 840, 842, 846, 847, 850, 856, 857, 858, 864, 922, 923, 924, 933, 935, 943, 946, 978, 979, 987, 992, 1000, 1001, 1007, 1009, 1011, 1019, 1027, 1029, 1072 inflammatory cells, 1003, 1004, 1005 inflammatory disease, xxxi, xxxiv, xxxv, xxxvi, 526, 847, 850, 852, 858, 962, 963, 968, 978, 979, 990, 1000, 1011, 1037, 1039, 1043, 1079 inflammatory mediators, 581, 679, 718, 719, 923, 938, 1017, 1018, 1019 inflammatory response, xxii, xxvi, xxx, xxxi, xxxv, 12, 31, 54, 334, 386, 501, 580, 587, 588, 591, 592, 594, 698, 716, 719, 834, 840, 843, 863, 868, 938, 941, 943, 945, 947, 984, 990, 993, 994, 999, 1003, 1005, 1014, 1022, 1029, 1057, 1073 inflammatory responses, xxii, xxx, xxxi, 12, 54, 386, 501, 580, 588, 591, 592, 719, 834, 840, 843, 863, 868, 943, 1022, 1073 infusion model, 610 inheritance, xxxii, 260, 473, 763, 897, 899, 902, 1067, 1068, 1075, 1082 inherited disorder, xxxii, 897, 1071 inhibitory effect, 178, 524, 689, 690, 741, 884, 926, 1001, 1039 initiation, xv, xxx, xxxii, 80, 103, 138, 143, 204, 234, 252, 272, 285, 301, 360, 361, 370, 517, 530, 644, 665, 842, 850, 876, 904, 925, 932, 933, 993, 1057 injections, 610, 996, 1002, 1004 injuries, 514, 585, 935, 946, 994, 1014 innate immunity, 478 inositol, xxix, 272, 275, 284, 680, 813, 814, 816, 886, 911, 1083 input, 275, 312, 412, 593, 654 insects, 345, 357, 1068, 1076 insertion, xxiv, 78, 98, 104, 273, 310, 416, 417, 418, 652, 658, 659, 661, 739, 1088 insight, xi, xxii, 93, 139, 201, 237, 328, 419, 423, 426, 464, 599, 700, 782, 899, 904, 913, 1083 insomnia, 829 instability, 160, 161, 401, 412, 415, 866, 1046, 1065, 1075, 1092 insulation, 1073 insulin, 125, 156, 240, 246, 467, 682, 876, 883, 894, 1069 insulin signaling, 467 integration, 448 integrin, 335, 439, 699
1129 integrity, xviii, 368, 394, 399, 414, 445, 447, 524, 850, 943, 945, 1022 intellect, 1074 intensity, 660, 830, 938, 1042, 1044 interaction, xiv, xxviii, 11, 14, 20, 35, 46, 51, 52, 58, 61, 67, 69, 72, 77, 78, 80, 96, 97, 98, 100, 101, 103, 105, 106, 107, 109, 110, 113, 120, 128, 142, 143, 146, 147, 148, 150, 153, 160, 161, 163, 166, 170, 178, 180, 181, 199, 200, 201, 202, 204, 209, 216, 218, 228, 243, 250, 255, 280, 283, 284, 300, 314, 318, 321, 322, 327, 328, 329, 348, 349, 360, 361, 367, 379, 413, 424, 429, 437, 438, 440, 445, 453, 454, 457, 458, 461, 464, 465, 467, 468, 473, 478, 505, 506, 507, 538, 542, 545, 613, 660, 661, 666, 667, 669, 691, 694, 716, 719, 725, 733, 739, 740, 745, 755, 766, 768, 770, 774, 794, 798, 805, 817, 818, 827, 843, 857, 877, 878, 880, 882, 885, 900, 907, 910, 911, 916, 927, 936, 937, 973, 986, 992, 993, 1003, 1017, 1026, 1029, 1060, 1064, 1065, 1080, 1089, 1091, 1092 interactions, xix, xx, xxiv, xxvii, 7, 49, 50, 59, 64, 75, 109, 122, 131, 139, 140, 142, 143, 149, 153, 167, 183, 187, 190, 201, 203, 218, 229, 244, 246, 259, 260, 274, 281, 283, 287, 297, 318, 322, 349, 351, 359, 367, 400, 429, 438, 443, 450, 455, 456, 457, 459, 462, 463, 464, 465, 470, 475, 486, 493, 505, 538, 539, 541, 542, 546, 556, 557, 575, 624, 625, 652, 661, 665, 668, 673, 678, 700, 762, 767, 770, 796, 818, 825, 856, 873, 908, 912, 913, 925, 927, 928, 936, 945, 955, 1056, 1058, 1092 intercellular adhesion molecule, 389 interdependence, 395 interface, 48, 452, 827, 847 interference, xxxi, 130, 147, 153, 157, 175, 224, 248, 267, 289, 300, 320, 445, 467, 505, 716, 725, 807, 850, 881, 910, 918, 1041, 1088 interferon, xii, xxix, 59, 63, 100, 106, 111, 115, 118, 119, 122, 129, 131, 134, 135, 152, 170, 174, 194, 195, 229, 231, 236, 242, 249, 263, 308, 311, 330, 331, 339, 374, 383, 390, 531, 833, 846, 853, 858, 869, 874, 1072 interferon (IFN), 174 interferon gamma, 129, 131, 242, 390 interferons, 106 interferon-γ, xxix, 118, 119, 194, 229, 231, 249, 383, 833 Interleukin-1, 1029
1130 interleukin-8, 953 interleukins, 1017 intermediate targets, 355 intermolecular interactions, 218 internal clock, 2 internalised, 356 internalization, 5, 58, 83, 94, 98, 187, 219, 364, 379, 380, 388, 415, 418, 420, 434, 437, 668, 888, 889, 895, 913, 920, 925, 1076 interneurons, 829, 1081 interphase, 233 interpretation, 886 interval, 1072 intervention, xxxiv, 13, 185, 387, 409, 432, 514, 516, 865, 911, 923, 925, 961, 963, 979 intestine, 179 intoxication, 898 intracellular cysteine proteases, 940 intracerebral hemorrhage, 1019, 1031 intracranial tumors, 877 introns, 106, 837 inventions, 64 invertebrates, 179, 346, 349, 357, 358 involution, 58 ion channels, ix, 1, 54, 378, 387, 395, 416, 425, 653, 680, 700, 864, 899, 912, 913, 920 ion exchangers, 680 ion transport, 680, 701, 913 ionizing radiation, 445, 476, 522, 815, 978 ions, 23, 125, 132, 136, 333, 416, 524, 589, 600, 610, 654, 678, 679, 841, 923, 931, 951, 1058, 1073, 1090 ipsilateral, xxiv, 609, 651, 656, 657, 658, 662, 942, 999 IR, 296, 336, 471, 478, 701, 736, 739, 827 iron, 449, 492, 554, 557, 592, 622, 624, 718, 922, 927, 930, 950 irradiation, 177 irreversible aggregation, 214 ischemia, xii, xv, xxxiii, xxxiv, xxxv, 12, 13, 127, 136, 138, 272, 275, 279, 289, 296, 315, 334, 445, 447, 449, 460, 514, 522, 527, 567, 568, 584, 587, 595, 677, 697, 698, 700, 922, 923, 924, 925, 926, 927, 928, 929, 930, 932, 933, 934, 935, 936, 937, 938, 939, 941, 942, 943, 944, 945, 946, 947, 948, 949, 952, 954, 958, 959, 968, 984, 990, 992, 993, 994, 996, 999, 1000, 1001, 1004, 1005, 1008, 1009, 1012, 1018, 1019, 1022, 1023, 1032, 1050
Index ischemic stroke, xxxiii, xxxvi, 517, 922, 934, 943, 945, 1017, 1022, 1029, 1030 isolation, 27, 39, 77, 88, 130, 235, 298, 422, 783, 902 isoleucine, 311 isomerization, 274 isotope, 16, 35 isozymes, 1086 Israel, 15, 35, 41, 65, 68, 199, 263, 436, 537 Italy, 1, 14, 117, 271, 307, 443, 671, 761, 863, 921, 1011, 1053
J Japan, 207, 343, 599, 617, 1065 joints, 1003 Jordan, 624, 891, 892, 894
K K+, 439, 648, 680, 956 kainate receptor, 668 Kaposi sarcoma, 180 ketones, 724 kidney, 179, 186, 287, 476, 716, 870, 927, 966 kidneys, 383 killing, 636, 1074 kinase activity, 285, 428, 532, 700, 773, 840, 869, 879, 880, 953, 983, 1014, 1034, 1090 kinases, xxvi, xxxi, 33, 48, 83, 250, 415, 424, 426, 439, 454, 455, 483, 485, 514, 524, 594, 668, 736, 739, 740, 741, 742, 743, 748, 750, 751, 841, 866, 867, 875, 878, 884, 887, 922, 937, 952, 953, 978, 999, 1061, 1064, 1071, 1079, 1083, 1085, 1090, 1091 kinetic model, 312, 328, 329, 332 kinetics, xvi, 17, 22, 36, 246, 308, 314, 323, 324, 325, 327, 328, 333, 441, 526, 535, 673, 675, 740, 826, 1033 kinetochore, 160 Korea, 71, 86 kuru, 828
L labeling, 228, 229, 230, 233, 422, 470, 504, 630, 970 labor, 145 language, 1068, 1083
Index latency, 18, 180, 609, 652, 657, 1082 lateral sclerosis, xix, xxv, xxx, 2, 6, 242, 247, 256, 268, 269, 272, 289, 304, 318, 336, 412, 441, 442, 444, 446, 628, 635, 649, 1057 laughter, 901 layering, 677 LDL, 204, 729 leakage, 923 learning, xvi, 25, 193, 313, 343, 357, 376, 381, 386, 412, 426, 427, 428, 433, 434, 435, 436, 500, 507, 664, 843, 904, 912, 914, 1062, 1074, 1076 lens, 132, 448, 482, 484, 490, 516, 528, 722, 732, 1024, 1034 lesions, xxxi, 55, 235, 315, 440, 567, 645, 680, 811, 830, 875, 877, 903, 1029, 1056, 1069 leucine, 17, 22, 97, 105, 106, 112, 182, 301, 311, 544, 550, 600, 605, 773, 837, 1081 leukemia, xxxv, 228, 232, 239, 242, 256, 517, 814, 851, 873, 971 leukemia cells, 873, 971 leukemic cells, 136, 235, 498, 503, 973 leukocytes, xxxv, 179, 587, 923, 938, 995, 999, 1000, 1001, 1003, 1004, 1009, 1012, 1020, 1063, 1067 Lewy bodies, xxii, 8, 42, 55, 84, 94, 98, 111, 228, 235, 315, 316, 319, 383, 435, 445, 520, 554, 558, 561, 562, 568, 571, 573, 599, 600, 601, 602, 603, 609, 612, 614, 616, 617, 632, 649, 672, 716, 717, 720, 762, 764, 780, 786, 804, 811, 955, 1073, 1081 liberation, 103, 1074 life cycle, 22, 208 life expectancy, 1026 life span, 467, 549, 707, 902 lifespan, 230, 543, 549, 807, 1082 lifetime, xxv, 715, 898 ligand, xvi, 18, 57, 81, 83, 129, 152, 330, 343, 346, 348, 360, 384, 408, 412, 416, 422, 454, 455, 482, 496, 497, 498, 525, 659, 719, 729, 733, 864, 869, 873, 874, 885, 886, 888, 894, 948, 977, 979, 993, 1057, 1075, 1079, 1092 ligands, 187, 263, 313, 332, 350, 464, 719, 837, 846, 887, 978, 1088, 1092 likelihood, 64, 328, 463, 603, 634, 693 limb weakness, 633 limbic system, 1056, 1070, 1081 limitation, 993 linkage, xix, 24, 28, 29, 38, 43, 44, 45, 47, 55, 57, 60, 63, 72, 75, 79, 80, 84, 171, 396, 398,
1131 444, 466, 555, 564, 677, 775, 890, 899, 927, 1066, 1095 links, xiii, xix, xxiv, 28, 108, 146, 170, 196, 200, 203, 274, 299, 300, 334, 390, 436, 443, 456, 461, 486, 540, 631, 633, 652, 697, 733, 911, 916, 1065 lipase, 783 lipid metabolism, 729 lipid peroxidation, 127, 136, 566, 567, 568, 588, 597, 612, 624, 680, 717, 722 lipid rafts, 382, 440, 468, 589, 1074 lipids, 523, 597, 601, 612, 614, 615, 683, 1074, 1083, 1088 lipoproteins, 463 lipoxygenase, 1014 liquid chromatography, 214 liver, 18, 36, 105, 119, 120, 129, 133, 153, 180, 186, 235, 236, 237, 238, 262, 293, 330, 463, 483, 590, 703, 704, 718, 844, 905, 943, 986, 1025 liver cancer, 180 liver disease, 180 localization, xiv, xvii, 14, 43, 53, 57, 59, 62, 64, 68, 70, 72, 75, 88, 103, 157, 175, 187, 204, 223, 228, 229, 230, 232, 233, 234, 235, 236, 237, 238, 239, 240, 254, 257, 267, 277, 293, 297, 299, 317, 335, 337, 339, 374, 375, 376, 377, 379, 385, 386, 387, 389, 397, 399, 433, 437, 448, 449, 467, 471, 504, 544, 550, 551, 565, 649, 678, 698, 702, 708, 717, 754, 768, 769, 771, 775, 788, 789, 796, 808, 809, 811, 812, 817, 819, 821, 822, 825, 828, 830, 831, 834, 840, 859, 860, 887, 911, 937, 1006, 1034, 1070, 1078, 1101 location, 52, 79, 113, 120, 121, 235, 251, 254, 257, 278, 295, 415, 420, 455, 467, 556, 767, 787, 817, 819, 822, 824, 826, 934, 1080, 1087 locus, 63, 160, 359, 366, 428, 440, 558, 571, 574, 601, 609, 618, 702, 764, 766, 772, 773, 779, 780, 789, 877, 890, 906, 909, 918, 1073, 1079, 1082 long distance, 274, 352 longevity, 2, 467, 490, 543, 549, 691 long-term memory, xvii, 54, 374, 381, 385, 389, 420, 427, 439 long-term potentiation (LTP), xxiv, 413, 436, 651, 653, 904 low-density lipoprotein, 463 LPS, 383, 834, 841, 962, 972, 973, 976
Index
1132 LTD, 374, 386, 412, 413, 415, 418, 422, 433, 668 luciferase, 693 lumen, 19, 21, 43, 58, 94, 183, 187, 231, 234, 273, 275, 277, 279, 280, 281, 284, 287, 294, 522, 540, 684, 774, 838, 938, 1067, 1092 lung cancer, 230, 236, 709, 853, 951, 979, 1044, 1053 lung disease, 510 lupus, xxx, 234, 850, 852, 859 lupus erythematosus, xxx, 234, 850, 852 lymph, 304, 1067 lymph node, 304 lymphatic system, xxv, 716 lymphoblast, 134 lymphocytes, xxx, 128, 170, 180, 331, 383, 840, 874, 1050, 1074 lymphoid, 311, 841 lymphoid organs, 311, 841 lymphoma, 450, 479, 527, 814, 922, 1033, 1039, 1050, 1060 lysine, 28, 38, 43, 44, 46, 48, 56, 57, 58, 61, 62, 64, 72, 94, 102, 109, 112, 123, 160, 166, 171, 202, 213, 322, 327, 403, 414, 416, 434, 541, 555, 556, 574, 654, 691, 716, 722, 775, 880, 899, 963, 975, 976 lysis, 234, 835 lysosomal enzymes, 3, 231, 945 lysosome, x, 15, 16, 17, 18, 19, 20, 21, 22, 24, 31, 33, 40, 58, 59, 65, 187, 270, 346, 349, 413, 415, 419, 434, 452, 466, 473, 477, 540, 548, 554, 562, 682, 687, 762, 774, 789, 816, 818, 819, 913 lysozyme, xiii, 27, 29, 39, 76, 125, 159, 164, 247, 259, 723
M machinery, xix, xxxi, xxxii, 9, 20, 22, 24, 31, 50, 51, 53, 57, 58, 59, 62, 64, 108, 144, 162, 164, 165, 175, 183, 190, 208, 216, 238, 242, 246, 252, 253, 255, 258, 273, 275, 278, 281, 283, 285, 291, 294, 317, 318, 319, 338, 352, 378, 380, 382, 402, 430, 431, 436, 438, 444, 458, 460, 462, 463, 466, 467, 469, 472, 479, 487, 500, 506, 514, 532, 547, 561, 566, 567, 568, 614, 674, 675, 682, 687, 690, 692, 703, 709, 721, 723, 744, 771, 776, 811, 819, 822, 823, 839, 843, 868, 875, 877, 879, 889, 897, 899, 902, 933, 937, 945
Mackintosh, 551, 648 macromolecules, 445, 446, 447, 459, 673, 674, 924 macrophage inflammatory protein, 662 macrophages, 22, 179, 407, 719, 729, 843, 1000, 1004 magnesium, 125, 132 magnetic resonance, 975 major depression, 502 major histocompatibility complex, xxix, 2, 42, 118, 131, 133, 134, 151, 198, 223, 242, 250, 269, 272, 285, 297, 308, 311, 374, 489, 833, 834, 843, 844, 845, 874, 962 malaise, 1040, 1041 malaria, 153 malate dehydrogenase, 214 males, 1097 malignancy, 857, 859, 870, 871, 1022, 1027 malignant cells, xx, 513, 853, 869, 888, 1039, 1040 malignant melanoma, 644, 851, 859, 1052 mammalian brain, 379, 398, 434, 771 mammalian cells, 58, 69, 85, 118, 129, 144, 177, 180, 186, 222, 230, 232, 236, 238, 249, 275, 278, 280, 285, 293, 297, 339, 408, 425, 451, 455, 489, 507, 540, 581, 582, 684, 710, 728, 785, 822, 823, 860, 878, 893, 1073, 1076 mammalian tissues, 6, 17, 1074 management, xxxvi, 617, 860, 902, 915, 1037 manganese, 136, 333, 374, 386, 763 manganese superoxide dismutase, 136, 374, 386 manipulation, 9, 350, 501, 835 manners, 51, 213 mantle, 1039, 1050 mapping, 359, 482, 722, 728, 755, 855, 1067, 1090 Marfan syndrome, 246 market, 17, 34 marriage, 1064 masking, 843 mass spectrometry, 196, 323, 567, 722 matrix, xiv, 192, 227, 233, 238, 308, 313, 412, 449, 450, 492, 660, 796, 809, 842, 945 matrix metalloproteinase, 842, 945 maturation, 18, 55, 60, 118, 120, 122, 130, 131, 154, 179, 180, 248, 252, 260, 265, 269, 273, 285, 368, 382, 418, 461, 464, 503, 511, 548, 668, 753, 784, 839, 844, 873, 1078, 1095 MBP, 394, 400, 403, 404 MCP, 16, 29, 237, 1100
Index measles, 842 measurement, 74, 691, 1082, 1091 measures, 657, 996 media, 234 median, 311, 1044, 1046 mediation, 666, 994 medical care, 993 Mediterranean, 906 Mediterranean countries, 906 medulla, 350, 630, 652, 653 medulla oblongata, 630 meiosis, 910, 1076, 1078 MEK, xxiii, 407, 627, 628, 647 melanoma, 305, 851, 873 membrane permeability, 319, 681, 978 membranes, 18, 20, 144, 204, 224, 230, 231, 278, 282, 297, 299, 318, 416, 417, 446, 448, 451, 589, 674, 680, 718, 781, 871, 925, 939, 1058, 1060, 1061, 1074 memory, xvi, xvii, xx, 3, 6, 54, 65, 193, 260, 343, 374, 376, 379, 381, 382, 385, 386, 389, 412, 420, 426, 427, 428, 433, 435, 439, 495, 497, 663, 664, 744, 756, 777, 843, 1047, 1048, 1056, 1062, 1070, 1074, 1076, 1083 memory formation, 386, 389 memory loss, 1048 mental activity, 791 mental illness, 509 mental retardation, xxxii, 382, 500, 898, 901, 912, 916, 1068 mental state, 1047 mesencephalon, 115, 228, 230, 377, 507, 959 messages, 274 messenger ribonucleic acid, 509, 547, 894 messengers, 1003 metabolic acidosis, 53, 949, 1010 metabolic disorder, 248, 870 metabolic pathways, 16, 17, 31, 944, 952 metabolism, iv, xxxiii, 9, 16, 35, 135, 146, 150, 286, 291, 292, 317, 337, 445, 449, 462, 503, 523, 531, 539, 563, 572, 612, 615, 641, 682, 706, 708, 728, 733, 737, 750, 751, 752, 764, 766, 771, 786, 791, 899, 906, 907, 908, 922, 923, 927, 944, 949, 950, 1014, 1023, 1051, 1078, 1094 metabolites, 726, 924, 938, 1039, 1051 metabolizing, 592 metalloproteinase, 143, 959 metals, 283, 445, 718
1133 metamorphosis, xvii, 344, 357, 358, 366, 406, 646 metaphase, 233, 535, 786, 878, 882 metastasis, 304, 978, 1013 methane, 170, 177 methionine, 48, 160, 567, 724, 728, 733, 952 methylation, 58, 546, 879, 891, 902, 1067 Mg2+, 654 MHC, xii, xiii, xxix, xxx, 2, 34, 42, 54, 63, 118, 126, 129, 135, 136, 151, 152, 169, 170, 174, 178, 179, 180, 181, 191, 194, 195, 231, 242, 250, 257, 261, 263, 272, 280, 288, 303, 308, 310, 311, 312, 313, 330, 331, 332, 374, 382, 383, 389, 464, 527, 756, 798, 833, 834, 835, 836, 837, 838, 839, 843, 844, 845, 846, 849, 850, 857, 858, 869, 870, 874, 951, 962, 966, 972, 973, 981, 985, 1033, 1074, 1085, 1092 MHC class II molecules, 835, 1074 mice, xxix, xxxii, 10, 61, 69, 84, 85, 92, 104, 106, 114, 174, 175, 180, 195, 284, 286, 291, 304, 335, 337, 338, 339, 348, 359, 366, 382, 385, 391, 398, 401, 402, 403, 404, 405, 407, 425, 428, 430, 432, 434, 435, 436, 440, 441, 442, 472, 473, 474, 480, 484, 492, 501, 502, 506, 507, 511, 520, 531, 534, 552, 562, 563, 564, 571, 573, 574, 577, 608, 610, 611, 612, 622, 623, 624, 630, 631, 632, 633, 635, 636, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 657, 664, 665, 683, 684, 687, 692, 693, 696, 700, 702, 704, 707, 708, 709, 712, 757, 758, 765, 766, 769, 776, 781, 786, 788, 790, 791, 796, 797, 806, 808, 810, 811, 812, 813, 816, 818, 819, 820, 821, 822, 823, 824, 827, 828, 829, 830, 832, 844, 879, 884, 897, 899, 901, 904, 905, 911, 912, 913, 914, 917, 920, 945, 953, 954, 957, 976, 1003, 1035, 1048, 1095, 1096 microarray, 406, 427, 502, 529, 580, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 594, 997 microenvironment, 1016 microglia, xxvi, 229, 236, 375, 382, 383, 385, 386, 389, 390, 478, 607, 608, 615, 666, 679, 716, 717, 719, 720, 815, 842, 870, 873, 945, 999, 1000, 1015 microinjection, 784, 976 microscopy, 32, 121, 139, 144, 149, 176, 179, 186, 229, 230, 254, 255, 323, 358, 397, 470, 472, 642, 655, 676, 678, 819, 822, 925 microsomes, 237, 282
1134 microtubules, xviii, xxvi, 229, 236, 254, 257, 393, 401, 455, 468, 491, 687, 735, 737, 738, 739, 740, 747, 756, 758, 767, 768, 769, 770, 785, 786, 787, 883, 994, 1075, 1076, 1092 midbrain, 106, 472, 500, 517, 529, 533, 768, 1075 Middle East, 906 migration, 103, 892, 1013, 1014 milligrams, 1040 mineralocorticoid, 496, 497, 504, 506, 508, 509, 1076 Ministry of Education, 617 minority, 1062 mitochondria, 20, 36, 103, 273, 336, 450, 517, 518, 533, 565, 572, 604, 611, 612, 613, 619, 625, 635, 645, 674, 680, 682, 696, 702, 709, 718, 730, 770, 772, 773, 774, 777, 787, 802, 825, 831, 924, 938, 1070 mitochondrial abnormalities, 521 mitochondrial damage, 604 mitochondrial DNA, 769, 786 mitogen, 457, 551, 652, 658, 666, 700, 864, 876, 878, 884, 1066, 1073 mitosis, xxxii, 201, 230, 232, 233, 239, 299, 437, 638, 751, 756, 760, 876, 878, 883, 924, 1021, 1068, 1076, 1092, 1096 mitotic checkpoint, 883 mixing, 673 mobility, 230 moclobemide, 502, 508 model system, xvii, 85, 252, 280, 350, 357, 370, 393, 397, 502, 889, 946, 968 modeling, 142, 322, 395, 480, 562, 993 models, xvi, xxviii, xxix, xxxi, xxxii, xxxiii, xxxv, 6, 10, 290, 307, 314, 318, 319, 320, 321, 322, 328, 356, 386, 395, 397, 409, 429, 430, 432, 462, 474, 487, 493, 502, 522, 559, 561, 576, 581, 601, 609, 610, 631, 653, 667, 669, 670, 673, 674, 677, 681, 687, 688, 689, 691, 743, 774, 794, 798, 799, 802, 806, 807, 814, 818, 856, 858, 876, 877, 888, 898, 901, 908, 909, 912, 921, 923, 925, 934, 939, 941, 942, 943, 946, 954, 958, 967, 990, 993, 994, 995, 999, 1000, 1002, 1012, 1015, 1017, 1018, 1019, 1025, 1026, 1032, 1056, 1085 modules, 140, 182, 186, 364, 900, 1095 molecular biology, 243, 368, 478, 734, 825 molecular changes, 877 molecular forces, 317, 678
Index molecular mass, 26, 27, 29, 139, 448, 469, 472, 538, 722, 834, 870, 1075, 1089, 1093 molecular mechanisms, xix, 213, 217, 251, 252, 317, 395, 425, 429, 444, 464, 526, 557, 562, 563, 581, 667, 682, 790, 844, 1015, 1029 molecular oxygen, 611 molecular pathology, 877 molecular structure, 118 molecular weight, 39, 105, 118, 129, 140, 151, 188, 221, 253, 261, 268, 269, 430, 441, 467, 520, 854, 855, 1092 molecules, x, xiii, xvi, xix, xxiv, xxix, xxx, xxxii, xxxiv, xxxv, 10, 11, 12, 13, 17, 27, 34, 42, 43, 45, 46, 52, 54, 56, 57, 59, 74, 79, 94, 129, 134, 151, 159, 161, 172, 173, 174, 178, 181, 186, 194, 195, 231, 243, 248, 250, 251, 253, 273, 276, 308, 314, 321, 322, 323, 324, 325, 326, 329, 360, 378, 382, 383, 387, 396, 397, 403, 412, 419, 431, 433, 443, 450, 456, 466, 473, 519, 527, 539, 542, 544, 555, 585, 590, 612, 631, 652, 660, 674, 676, 677, 679, 693, 739, 756, 771, 833, 834, 835, 836, 837, 838, 839, 840, 842, 844, 849, 850, 858, 868, 870, 878, 879, 887, 897, 899, 905, 911, 913, 937, 938, 945, 946, 958, 962, 964, 965, 966, 972, 973, 974, 975, 976, 981, 984, 987, 994, 997, 1000, 1001, 1003, 1009, 1012, 1014, 1015, 1017, 1030, 1033, 1056, 1060, 1063, 1064, 1065, 1074, 1078, 1085, 1087, 1088, 1089, 1092 mollusks, 439 monoamine oxidase inhibitors, 786 monoclonal antibody, 755, 871, 1017 monocytes, 382, 719, 966, 981 monomer, 44, 72, 73, 100, 384, 456, 487, 544, 551, 680, 764, 1061 monomers, ix, xi, 1, 71, 73, 143, 184, 456, 472, 564, 672, 678, 690, 692, 741, 742, 768, 934 monozygotic twins, 763 mood, 502, 509, 1085 mood disorder, 509 Moon, 387 morbidity, 923 morning, 497 morphine, 667 morphogenesis, 54, 423, 438, 1080 morphology, 352, 359, 365, 367, 368, 371, 441, 455, 496, 497, 504, 681, 764, 769, 770, 773, 778, 999, 1058 mortality, 632, 879, 923, 995, 996, 1017 morula, 362
Index mother cell, 344, 345, 1068 mothers, 345, 360, 1082, 1089 motion, 298, 932 motives, 740 motor behavior, 568 motor function, 600, 696, 776, 797, 801, 811, 1048 motor neuron disease, 639, 687, 707 motor neurons, xvii, 228, 230, 350, 358, 359, 375, 376, 393, 473, 492, 550, 564, 707, 720, 1057 mouse model, 268, 269, 319, 321, 395, 409, 428, 432, 441, 442, 472, 473, 493, 500, 501, 508, 511, 533, 559, 595, 613, 623, 624, 635, 636, 640, 646, 648, 681, 687, 688, 692, 693, 704, 707, 712, 744, 746, 756, 795, 800, 801, 805, 806, 807, 819, 820, 904, 905, 916, 920, 960, 973, 1003, 1023 movement, xxvii, 211, 329, 396, 424, 571, 761, 763, 807, 1064, 1066, 1081, 1090 movement disorders, 763 mRNA, 106, 243, 259, 284, 285, 286, 301, 322, 380, 382, 387, 414, 417, 435, 458, 477, 478, 497, 508, 509, 538, 540, 549, 656, 670, 690, 770, 772, 806, 820, 839, 869, 877, 879, 881, 882, 884, 885, 888, 890, 894, 924, 932, 934, 938, 941, 948, 972, 997, 1002, 1004, 1008, 1009, 1030, 1057 mucoid, 234 mucous membrane, 888, 1061, 1064 mucous membranes, 1064 multicellular organisms, 273 multiple factors, 183, 958 multiple myeloma, xxxiv, xxxv, xxxvi, 12, 479, 516, 525, 634, 644, 889, 895, 940, 943, 962, 967, 982, 990, 1011, 1012, 1016, 1021, 1031, 1032, 1037, 1038, 1041, 1049, 1050, 1052, 1053, 1061 multiple sclerosis, xxx, 503, 510, 649, 834, 835, 842, 847, 849, 851, 860, 968, 992, 1003, 1015, 1023, 1079 multiplication, 764, 766, 780 multiplicity, 377 mumps, 846 muscle tissue, 395 muscle weakness, 401, 1068, 1069 muscles, 230, 237, 360, 361, 516, 770, 910, 1063, 1066, 1097 muscular dystrophy, 1069 mustard oil, xxiv, 651, 655, 656, 658, 1056
1135 mutagenesis, 78, 123, 358, 910 mutant, xxiii, xxxi, 29, 39, 63, 65, 79, 80, 84, 101, 106, 122, 123, 146, 160, 163, 164, 178, 185, 187, 195, 204, 213, 224, 245, 248, 252, 256, 260, 265, 266, 267, 268, 269, 274, 275, 282, 288, 291, 293, 295, 300, 302, 303, 304, 311, 312, 318, 319, 320, 321, 326, 327, 329, 337, 338, 339, 345, 349, 350, 351, 356, 359, 360, 363, 390, 396, 398, 400, 402, 404, 409, 416, 420, 423, 432, 435, 441, 442, 452, 454, 462, 468, 472, 473, 474, 492, 493, 518, 521, 524, 533, 555, 560, 563, 564, 565, 571, 572, 574, 584, 602, 603, 605, 614, 618, 619, 620, 627, 631, 635, 636, 642, 645, 649, 665, 674, 681, 684, 685, 687, 688, 689, 691, 695, 696, 704, 706, 708, 709, 710, 711, 722, 723, 736, 743, 745, 746, 748, 751, 755, 766, 770, 772, 774, 782, 788, 789, 790, 795, 796, 799, 800, 801, 803, 805, 808, 810, 811, 817, 821, 823, 824, 830, 831, 844, 863, 864, 881, 909, 912, 918, 920, 953, 957, 1059, 1093 mutant proteins, 274, 319, 320, 404, 454, 474, 602, 687, 688, 689 mutation, 7, 34, 54, 55, 82, 84, 85, 92, 102, 104, 114, 123, 177, 184, 223, 242, 245, 251, 256, 283, 288, 289, 304, 348, 349, 350, 351, 357, 362, 366, 382, 402, 404, 416, 420, 435, 442, 473, 503, 507, 542, 561, 563, 574, 581, 586, 603, 604, 618, 620, 621, 630, 631, 632, 633, 634, 636, 637, 643, 644, 646, 647, 660, 664, 673, 687, 691, 706, 707, 720, 730, 743, 753, 757, 770, 771, 772, 788, 789, 800, 821, 822, 831, 842, 865, 877, 883, 903, 904, 906, 909, 910, 917, 918, 1060, 1068, 1082, 1084, 1091, 1093, 1097 mutations, xxi, xxvi, xxvii, xxix, 6, 7, 10, 34, 54, 55, 61, 62, 82, 83, 85, 92, 98, 101, 102, 111, 112, 177, 246, 247, 248, 251, 255, 258, 260, 267, 279, 280, 287, 288, 297, 298, 303, 316, 318, 319, 337, 348, 349, 359, 362, 363, 366, 383, 389, 396, 399, 401, 402, 409, 416, 423, 426, 428, 430, 439, 466, 500, 507, 520, 543, 554, 555, 558, 560, 561, 562, 563, 567, 569, 573, 574, 581, 584, 586, 601, 602, 603, 604, 605, 614, 616, 619, 621, 661, 672, 673, 676, 677, 678, 681, 684, 687, 689, 690, 692, 699, 701, 702, 707, 711, 735, 737, 741, 753, 755, 761, 763, 766, 767, 768, 772, 773, 774, 776, 782, 789, 797, 800, 801, 808, 814, 821, 822, 831, 832, 865, 866, 877, 879, 883, 884, 888,
Index
1136 893, 894, 895, 899, 901, 903, 904, 906, 909, 910, 912, 914, 916, 918, 927, 935, 991, 1056, 1064, 1066, 1081, 1082, 1085 myelin, xviii, xxxvi, 235, 256, 375, 382, 394, 395, 399, 400, 401, 402, 403, 404, 408, 409, 647, 1038, 1048, 1049, 1065, 1073, 1082, 1089 myelin basic protein, 235, 394, 400, 408 myoblasts, 238, 593 myocardial infarction, xxxiv, xxxv, 552, 969, 973, 979, 989, 992, 1011, 1080 myoclonic seizures, 902, 905 myoclonus, xxxii, 815, 898, 901, 905, 912, 915, 917, 918, 1064 myogenesis, 90 myopathy, xv, 272, 289, 298, 304, 692, 711, 1069 myosin, 230, 643, 918 myositis, 851, 859
N Na+, 416, 680, 910 NaCl, 768 NAD, 637, 641, 647, 650 NADH, 611, 679, 791 naming, 448, 1083 narcotic, 1045 narcotics, 898 National Institutes of Health, 190, 569 native protein conformation, 676, 1081 nausea, 1025, 1041 NCS, 1038, 1045 necrosis, 81, 90, 308, 311, 514, 516, 522, 528, 535, 585, 610, 652, 662, 680, 716, 936, 952, 1007, 1014, 1029, 1033, 1045, 1057, 1093 nematode, 283, 351 neocortex, 376, 1073 neoplasia, 876, 877, 888, 1075 nerve, xvii, xxv, xxxvi, 273, 357, 359, 365, 369, 374, 375, 377, 378, 379, 391, 393, 394, 395, 398, 399, 400, 401, 402, 403, 404, 407, 425, 434, 435, 535, 563, 603, 608, 634, 638, 639, 640, 642, 646, 647, 648, 649, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 666, 669, 670, 671, 694, 700, 733, 766, 772, 843, 910, 911, 918, 1024, 1038, 1045, 1048, 1049, 1058, 1062, 1065, 1073, 1076, 1077 nerve cells, 374, 377, 843, 1076, 1077 nerve fibers, 398, 402, 1073
nerve growth factor, 365, 374, 391, 394, 395, 407, 535, 640, 647, 733, 911, 1024 nerves, xviii, 393, 394, 395, 398, 399, 400, 402, 403, 404, 407, 562, 633, 637, 640, 648, 653, 1045, 1076, 1077, 1078, 1082 nervous system, ix, xiv, xvi, xvii, xxviii, xxx, xxxii, xxxiv, 2, 3, 4, 6, 12, 13, 14, 85, 96, 102, 104, 118, 170, 171, 188, 189, 190, 228, 229, 237, 272, 273, 315, 335, 343, 344, 345, 350, 355, 357, 358, 359, 362, 363, 370, 371, 373, 374, 375, 380, 382, 384, 386, 387, 389, 393, 394, 395, 396, 397, 398, 405, 407, 408, 409, 439, 447, 511, 514, 523, 528, 586, 595, 628, 643, 653, 676, 705, 774, 777, 790, 834, 849, 850, 852, 858, 910, 911, 912, 913, 918, 944, 950, 1008, 1013, 1045, 1047, 1068, 1075, 1077, 1078 Netherlands, 18, 1037 network, xi, xix, 42, 58, 94, 160, 161, 204, 290, 295, 313, 356, 443, 455, 456, 486, 496, 514, 633, 767, 770, 868, 926, 932 neural development, xvi, 343, 345, 347, 351, 362, 526, 843, 1057, 1058, 1069 neural mechanisms, xviii, 394, 395 neural network, xxxii, 313, 332, 897, 898 neural networks, xxxii, 313, 332, 897, 898 neural tissue, xxviii, 793, 998 neuralgia, 653 neuroblastoma, 101, 264, 279, 297, 460, 530, 568, 603, 644, 680, 691, 700, 722, 723, 725, 726, 750, 754, 759, 760, 770, 822, 824, 967, 968, 982, 983, 1054 neuroblasts, 345, 364, 1073 neurodegeneration, xviii, xix, xxi, xxii, xxv, xxvi, xxvii, xxx, xxxiii, 7, 10, 17, 55, 69, 85, 92, 175, 185, 203, 219, 224, 255, 289, 290, 299, 300, 308, 318, 319, 334, 335, 336, 338, 368, 370, 411, 440, 441, 444, 466, 472, 475, 486, 487, 490, 519, 526, 530, 543, 546, 549, 553, 554, 555, 560, 561, 562, 563, 564, 565, 568, 569, 572, 573, 575, 576, 580, 581, 587, 593, 594, 599, 601, 602, 603, 605, 606, 609, 611, 612, 613, 614, 615, 616, 624, 625, 633, 646, 679, 680, 681, 682, 687, 692, 695, 697, 698, 699, 700, 701, 702, 703, 705, 707, 708, 709, 715, 716, 717, 719, 720, 721, 725, 726, 727, 735, 736, 737, 743, 746, 750, 751, 756, 758, 760, 763, 766, 776, 781, 784, 790, 791, 798, 808, 809, 810, 812, 820, 824, 830, 831, 832,
Index 842, 849, 851, 899, 906, 912, 917, 921, 936, 956, 959, 966, 1006, 1034, 1035 neurodegenerative dementia, 737 neurodegenerative diseases, xi, xv, xix, xxi, xxiv, xxv, xxviii, xxx, xxxiv, 6, 9, 12, 55, 69, 71, 72, 84, 85, 87, 91, 93, 96, 103, 107, 171, 193, 216, 219, 223, 248, 258, 289, 299, 302, 307, 312, 314, 315, 317, 318, 319, 320, 328, 333, 336, 351, 405, 409, 429, 444, 447, 466, 472, 475, 477, 490, 514, 523, 531, 532, 543, 545, 553, 555, 557, 558, 559, 560, 564, 565, 566, 567, 568, 569, 575, 576, 579, 580, 581, 582, 586, 589, 593, 594, 597, 601, 612, 643, 671, 672, 674, 676, 681, 684, 689, 690, 691, 693, 696, 697, 699, 701, 705, 715, 718, 731, 732, 757, 759, 760, 774, 779, 787, 801, 804, 806, 811, 812, 813, 818, 819, 825, 843, 849, 857, 927, 933, 935, 960, 979, 989, 991, 1006, 1034, 1054, 1056, 1081, 1084 neurodegenerative disorders, x, xiv, xv, xx, xxiii, xxv, xxviii, xxxiv, 6, 7, 9, 16, 42, 55, 85, 104, 135, 175, 180, 207, 208, 228, 267, 272, 300, 318, 319, 340, 391, 444, 460, 466, 474, 510, 520, 526, 560, 595, 614, 627, 634, 635, 636, 638, 640, 645, 681, 694, 700, 705, 710, 715, 716, 717, 718, 721, 724, 725, 726, 737, 745, 753, 757, 762, 804, 807, 813, 814, 842, 847, 851, 857, 945, 947, 956, 962, 963, 975, 991, 994, 1004, 1015, 1025, 1026, 1027, 1048, 1056, 1063 neurodegenerative processes, xxvi, 386, 735, 843 neuroendocrine, 1052, 1071 neuroendocrine cells, 1071 neurofibrillary tangles, xxvi, 55, 228, 235, 315, 467, 469, 471, 554, 558, 568, 672, 677, 680, 701, 717, 736, 754, 756, 757, 759, 760, 804, 811, 1060, 1077 neurofibromatosis, xviii, 394 neurofilament, 633, 652, 658 neurofilaments, 317, 602, 633, 642, 677 neurogenesis, xvi, 106, 115, 343, 345, 350, 362, 364, 368, 739, 740, 1068, 1073 neuroinflammation, xxiii, 407, 600, 608, 614, 615, 616, 717, 718, 726, 840, 1016, 1017, 1026 neurological condition, 717 neurological deficit, 943, 1022 neurological disease, xxx, 8, 12, 14, 85, 429, 432, 466, 475, 638, 797, 849, 851, 852, 912, 925, 1016, 1026, 1027
1137 neurological disorder, ix, xxx, xxxii, xxxiii, 2, 290, 524, 526, 728, 786, 834, 842, 897, 922, 1014, 1015, 1091 neuromuscular diseases, 733 neuron death, xxiii, xxx, 396, 600, 609, 610, 611, 613, 849, 958 neuron response, 656 neuronal apoptosis, 6, 406, 429, 514, 516, 517, 519, 521, 523, 524, 525, 526, 527, 531, 582, 586, 591, 592, 594, 701, 720, 730, 829, 832, 842, 873, 929, 947, 949 neuronal cells, xv, xvii, xx, xxi, 10, 107, 150, 171, 232, 234, 267, 271, 279, 289, 290, 322, 373, 374, 375, 377, 382, 386, 406, 460, 465, 474, 492, 513, 514, 516, 518, 519, 520, 522, 523, 524, 528, 529, 537, 543, 544, 545, 582, 594, 597, 688, 706, 723, 725, 729, 734, 739, 751, 776, 811, 820, 824, 829, 835, 933, 945, 1063 neuronal circuits, 496 neuronal death, xx, xxviii, xxix, xxxiii, 195, 302, 339, 395, 396, 405, 473, 513, 521, 526, 527, 529, 533, 571, 591, 601, 613, 645, 672, 676, 710, 717, 744, 754, 794, 809, 813, 857, 921, 926, 929, 931, 935, 936, 944, 945, 948, 951, 952, 968, 1014 neuronal excitability, 827, 899, 911, 913 neuronal plasticity, 385, 391, 413, 842, 847, 947, 1027 neuronal survival, 377, 396, 406, 524, 526, 642, 739, 765, 773, 944, 1048, 1054, 1072 neuropathic pain, xxiv, xxxvi, 634, 639, 648, 651, 653, 654, 655, 656, 657, 658, 662, 663, 664, 666, 667, 669, 670, 1037, 1042, 1045, 1049, 1056, 1071 neuropathies, xviii, 256, 394, 395, 400, 401, 402, 404, 405, 409, 525, 562, 639, 653, 844, 1045, 1048, 1054, 1063 neuropathy, xxiii, xxiv, xxxvi, 394, 396, 401, 404, 407, 408, 409, 543, 586, 628, 629, 633, 644, 648, 651, 663, 1022, 1037, 1038, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1052, 1053, 1054, 1065, 1068 neuropeptides, 1071 neuropharmacology, 432 neuroprotection, xx, 474, 475, 513, 523, 525, 526, 581, 587, 594, 641, 843, 857, 933, 935, 944, 950, 954, 955, 990, 992, 993, 994, 995, 996, 1000, 1008, 1019
1138 neuroprotective, xxii, xxiii, xxix, xxxiii, xxxiv, xxxv, 185, 386, 396, 406, 491, 517, 522, 524, 525, 529, 558, 565, 566, 580, 581, 582, 584, 585, 586, 587, 591, 592, 593, 594, 602, 604, 613, 625, 627, 637, 640, 647, 694, 700, 788, 814, 827, 842, 922, 923, 935, 938, 939, 941, 942, 943, 945, 947, 949, 953, 954, 990, 992, 994, 996, 997, 998, 1000, 1002, 1004, 1005, 1007, 1012, 1016, 1019, 1021, 1022, 1026, 1030 neuroprotective agents, 992, 1019 neuroprotective drugs, 1007 neuroscience, 2 neurotoxic effect, xx, 513, 610, 726 neurotoxicity, xxv, xxviii, xxix, xxxvi, 185, 261, 318, 440, 491, 493, 520, 523, 525, 561, 565, 576, 589, 611, 613, 620, 622, 625, 671, 680, 681, 694, 700, 734, 745, 748, 794, 796, 797, 798, 800, 801, 807, 814, 818, 821, 824, 830, 922, 936, 937, 944, 953, 956, 992, 1007, 1019, 1028, 1037, 1038, 1042, 1043, 1044, 1045, 1047, 1048, 1049 neurotransmission, 7, 171, 426, 432, 555, 574, 653, 680, 903 neurotransmitter, xviii, xxiv, 85, 362, 378, 379, 395, 397, 398, 411, 412, 413, 415, 419, 421, 423, 424, 425, 427, 438, 441, 651, 660, 924, 944, 1073, 1074, 1075, 1088, 1090, 1091 neurotransmitters, 9, 375, 378, 418, 425, 508, 653, 725, 923, 1079 neurotrophic factors, 842 neutrophils, 1000, 1004, 1007 nicotinamide, 394, 398, 637 nigrostriatal, xxii, 440, 520, 533, 573, 599, 601, 606, 608, 609, 611, 622, 634, 645, 720, 777, 778, 779, 781 nitric oxide, 127, 270, 288, 445, 454, 455, 460, 483, 524, 529, 535, 554, 566, 625, 646, 662, 679, 716, 719, 834, 922, 928, 936, 950, 1012, 1014, 1022, 1030, 1071 nitric oxide synthase, 270, 445, 454, 455, 483, 646, 662, 679, 716, 719, 936, 1012, 1022, 1030, 1071 nitrogen, 16, 23, 554, 566, 575, 679, 765 nitrosative stress, 566, 568, 596 NK cells, 234 NMDA receptors, 379, 418, 424, 615, 616, 653, 658, 659, 660, 665, 667, 679, 936, 956 N-methyl-D-aspartic acid, 412, 666, 1078
Index NMR, 76, 78, 101, 564, 673, 678, 698, 731, 826, 831, 975 Nobel Prize, 3 nociception, 666, 667, 669 nociceptive, 653, 655, 656, 657, 659, 664, 665, 666, 667, 669, 1045, 1078 nociceptive neurotransmission, 667 nodes, 290, 395 nondisjunction, 910 non-human primates, 622, 623 nonsense mutation, 903, 906 norepinephrine, 504, 1066 normal aging, xxx, 689, 849 normal development, 63, 514, 905, 1084 Norway, 849 NSAIDs, 652, 653, 938 nuclear receptors, xxxii, 391, 498, 505, 728, 729, 876 nucleation, 268, 340, 403, 678, 698, 699, 741, 742, 743, 755, 1092 nuclei, xiv, 8, 227, 230, 232, 233, 237, 238, 282, 315, 316, 375, 376, 377, 402, 456, 633, 677, 801, 804, 840, 884, 892, 925, 1024, 1025 nucleic acid, 523, 524, 815, 825, 1078, 1088 nucleolus, 106, 239, 377 nucleophiles, 73, 213 nucleophilicity, 123 nucleoplasm, xiv, 227, 273 nucleoprotein, 1078 nucleosomes, 28 nucleotide sequence, 498 nucleotides, 164, 213 nucleus, ix, xi, xiv, xx, xxiii, xxviii, xxxiii, 1, 3, 49, 53, 58, 81, 100, 105, 106, 117, 118, 127, 155, 171, 173, 186, 189, 203, 227, 228, 230, 232, 233, 237, 238, 250, 257, 285, 286, 299, 309, 320, 322, 339, 375, 376, 380, 385, 413, 414, 422, 423, 449, 457, 458, 465, 470, 481, 495, 523, 551, 559, 587, 590, 597, 601, 606, 609, 627, 636, 638, 639, 657, 674, 678, 691, 711, 770, 771, 787, 794, 795, 802, 806, 808, 821, 841, 856, 865, 868, 881, 882, 887, 921, 937, 948, 978, 999, 1014, 1024, 1062, 1076, 1078, 1079 nutrients, 20, 22, 682, 1058 nutrition, 142, 375, 382 nystagmus, 1082
Index
O OAS, 106 observations, xxiv, 56, 84, 97, 163, 164, 175, 210, 211, 213, 214, 249, 252, 254, 280, 282, 352, 355, 356, 363, 383, 396, 407, 466, 467, 525, 584, 606, 633, 635, 636, 648, 652, 658, 659, 744, 746, 773, 776, 800, 803, 806, 817, 818, 820, 822, 823, 825, 834, 837, 870, 871, 907, 926, 935, 937, 1051 obstruction, xxxiii, 55, 174, 921, 923, 1072 occlusion, xxxv, 145, 480, 577, 625, 922, 923, 942, 943, 951, 958, 968, 984, 994, 995, 996, 997, 1002, 1003, 1008, 1009, 1012, 1017, 1029, 1030, 1032 oil, xxiv, 655, 656 old age, 808 olfaction, 835 oligodendrocytes, 9, 375, 382, 385, 395, 400, 405, 478, 517, 595, 757, 874 oligomeric structures, 1089 oligomerization, 48, 436, 550, 551, 586, 596, 782, 1092 oligomers, xix, xxxi, 74, 180, 197, 247, 335, 339, 444, 446, 449, 468, 487, 493, 558, 559, 614, 615, 616, 672, 675, 676, 678, 682, 699, 702, 863, 864, 868, 1080, 1089, 1093 oligosaccharide, 277, 297 oncogenes, 454 oncoproteins, 40, 54, 250, 879, 924 oocyte, 753, 1080 oocytes, 265, 1076 oogenesis, 237, 787 opioids, 653, 663 optic chiasm, 877 optic nerve, 646, 1039 optical density, 1001 optimization, 940, 972 organ, xxx, 330, 366, 367, 389, 412, 497, 844, 850, 852, 858, 877, 972, 994, 1045, 1088 organelles, xiv, 8, 12, 18, 19, 54, 150, 186, 227, 231, 254, 291, 316, 400, 448, 451, 642, 680, 682, 683, 703, 774, 785, 819, 829, 925, 926, 1058, 1059, 1076 organic compounds, 16 organism, xvi, 52, 280, 290, 343, 466, 545, 636, 901, 964, 1024, 1026, 1090 organization, xiv, xv, 78, 196, 227, 238, 241, 267, 292, 370, 378, 387, 426, 431, 498, 532, 1034, 1079
1139 organizations, 135, 672 orientation, 120, 142, 314, 791, 1092 orthostatic hypotension, 1046 oscillation, 1082 ototoxicity, 1047 output, xvi, xx, 343, 496, 1088 ovarian cancer, 851, 853, 857 ovarian tumor, xi, 71, 74, 75 ovaries, 910 overload, xv, xxviii, 6, 241, 274, 285, 319, 604, 689, 794, 798, 931, 993, 1090 overproduction, 523, 784 overtime, 945 oxidation, 23, 53, 136, 178, 213, 215, 459, 524, 535, 554, 557, 558, 566, 567, 568, 576, 612, 720, 722, 723, 724, 728, 731, 732, 733, 742, 765, 766, 767, 769, 771, 788, 931, 950 oxidative damage, xxv, 234, 275, 283, 524, 529, 535, 565, 566, 568, 604, 612, 613, 624, 700, 715, 718, 720, 721, 722, 732, 786, 931 oxidative stress, xx, xxi, xxiii, xxv, 53, 68, 127, 274, 283, 288, 292, 318, 322, 449, 454, 459, 469, 475, 476, 479, 492, 513, 522, 523, 525, 535, 537, 539, 543, 544, 549, 553, 555, 558, 561, 565, 566, 567, 568, 569, 576, 584, 588, 589, 591, 592, 593, 594, 595, 597, 600, 604, 608, 609, 610, 611, 612, 613, 614, 615, 616, 621, 623, 642, 678, 679, 680, 691, 700, 701, 712, 715, 716, 717, 718, 720, 721, 722, 723, 724, 726, 727, 728, 730, 732, 764, 766, 772, 773, 791, 820, 922, 927, 931, 934, 944, 959, 978, 1006, 1007, 1013, 1052, 1057, 1060 oxygen, xxvii, 79, 112, 274, 318, 386, 445, 575, 576, 709, 718, 731, 764, 765, 771, 870, 922, 923, 927, 928, 930, 951, 952, 1007, 1014, 1069, 1072, 1079, 1088 oxygen consumption, 718
P p53, xxxii, 34, 35, 39, 54, 62, 66, 77, 80, 83, 90, 181, 198, 246, 251, 264, 428, 434, 439, 456, 477, 484, 499, 500, 506, 507, 518, 519, 521, 524, 525, 530, 532, 534, 535, 536, 729, 770, 787, 802, 865, 866, 867, 873, 876, 881, 883, 901, 902, 904, 914, 915, 916, 924, 937, 963, 967, 973, 976, 981, 986, 1013, 1014, 1017, 1022, 1024, 1029, 1031, 1050, 1062, 1075, 1092 packaging, 12, 253, 397
1140 paclitaxel, 648, 1045 Paget’s disease, xv, 272, 289, 692 pain, xxiv, xxxvi, 639, 646, 651, 652, 654, 657, 659, 661, 662, 663, 664, 665, 666, 667, 670, 1038, 1042, 1045, 1046, 1047, 1049, 1056, 1072, 1078 pain management, 1045 pairing, 22, 58 Pakistan, 906 palliative, 906 PAN, 142, 154, 208, 210, 212, 222, 340, 1081 pancreas, 186, 457, 927 pancreatic cancer, 302, 528, 1022, 1031 paradigm shift, 434, 491 paralysis, 85, 92, 104, 368, 632, 909, 918, 1042, 1059 parameter, 324, 932 parasite, 22 parasites, 837 parathyroid, 1075 paresis, 630, 772 paresthesias, 1043, 1045 parietal cortex, 502 parietal lobe, 1064 Parkinson’s disease, ix, xix, xxii, xxv, xxvi, xxvii, xxx, 2, 55, 72, 84, 94, 101, 104, 127, 193, 216, 228, 258, 272, 287, 383, 429, 444, 445, 446, 469, 520, 522, 546, 557, 560, 581, 582, 588, 593, 594, 597, 599, 600, 602, 615, 672, 715, 716, 717, 720, 736, 761, 762, 801, 818, 834, 835, 849, 851, 922, 935, 968, 991, 1012, 1056, 1073, 1081 parkinsonism, xxii, 42, 55, 101, 111, 223, 272, 279, 289, 368, 439, 440, 520, 523, 525, 572, 574, 576, 580, 581, 584, 599, 600, 603, 604, 612, 614, 618, 619, 621, 622, 643, 692, 711, 753, 772, 776, 777, 786, 788, 789, 966, 1057, 1068, 1081 particles, xx, 3, 14, 18, 19, 29, 36, 118, 161, 162, 164, 173, 178, 208, 209, 212, 230, 234, 237, 242, 250, 257, 537, 676, 723, 744, 825, 907 partition, 212, 469 parvalbumin, 820, 829 passive, 835 pathogenesis, x, xv, xxi, xxii, xxiv, xxviii, xxxi, 7, 9, 12, 13, 15, 17, 34, 42, 54, 55, 69, 72, 84, 127, 171, 175, 195, 216, 234, 261, 286, 289, 290, 307, 315, 317, 318, 320, 321, 328, 334, 336, 339, 382, 399, 400, 404, 405, 468, 469, 470, 475, 477, 503, 535, 553, 555, 557, 558,
Index 559, 562, 563, 564, 565, 566, 568, 569, 579, 581, 588, 593, 599, 602, 603, 614, 615, 617, 622, 625, 629, 635, 636, 645, 671, 674, 676, 678, 679, 681, 688, 691, 693, 694, 697, 700, 702, 705, 710, 721, 730, 764, 770, 771, 780, 781, 793, 794, 795, 797, 804, 807, 812, 820, 822, 842, 851, 856, 857, 858, 861, 875, 877, 905, 925, 945, 963, 979, 980, 1013, 1015, 1026, 1027, 1049, 1063, 1084 pathogens, 514, 815 pathology, iv, ix, xxiii, xxvi, xxviii, xxxiv, 8, 9, 13, 14, 69, 84, 107, 258, 268, 287, 312, 315, 316, 318, 320, 337, 408, 431, 432, 465, 467, 490, 515, 520, 560, 563, 597, 617, 618, 621, 627, 630, 631, 632, 633, 635, 636, 638, 639, 642, 645, 649, 674, 676, 680, 687, 691, 702, 703, 737, 746, 755, 759, 780, 789, 790, 797, 800, 801, 805, 810, 813, 815, 820, 829, 830, 865, 904, 935, 962, 1000, 1054, 1058, 1064 pathophysiological mechanisms, 611, 923 pathophysiology, ix, xiv, 2, 7, 151, 170, 190, 317, 478, 498, 587, 593, 645, 682, 700, 711, 779, 1013, 1017, 1023 pathways, ix, x, xi, xii, xiii, xviii, xix, xx, xxii, xxiii, xxv, xxix, xxxi, xxxii, 2, 7, 9, 16, 17, 31, 34, 42, 48, 49, 54, 63, 64, 73, 83, 110, 117, 137, 166, 170, 187, 188, 198, 203, 210, 215, 223, 244, 246, 259, 274, 276, 278, 285, 286, 290, 293, 294, 299, 300, 309, 317, 319, 334, 337, 350, 352, 354, 355, 357, 359, 360, 361, 365, 379, 384, 389, 390, 399, 405, 408, 411, 414, 417, 418, 426, 427, 429, 430, 443, 445, 447, 455, 456, 457, 458, 460, 462, 463, 466, 475, 476, 478, 480, 486, 488, 489, 496, 516, 537, 541, 542, 544, 546, 551, 563, 565, 580, 587, 591, 593, 599, 618, 625, 627, 630, 636, 653, 658, 662, 667, 671, 674, 679, 680, 681, 682, 684, 686, 692, 695, 697, 702, 708, 719, 726, 739, 750, 763, 778, 782, 785, 810, 814, 825, 847, 857, 858, 863, 864, 865, 866, 876, 885, 917, 928, 933, 937, 947, 992, 1009, 1027, 1064, 1066, 1074, 1086, 1096 patterning, 84, 367, 434 PBMC, 1038 PCR, 505, 656, 882, 943, 1008, 1012, 1022 pediatric patients, 1040, 1041, 1042, 1051 penetrability, 1001 penetrance, 899 penicillin, 1079
Index peptidase, 125, 132, 134, 135, 140, 142, 167, 181, 330, 688, 732, 844, 845, 869, 870, 940, 968, 1059, 1080 peptide chain, 451, 1016 peptides, xii, xiii, xxix, xxxi, xxxii, 4, 5, 7, 28, 29, 30, 32, 45, 50, 52, 54, 73, 74, 98, 118, 119, 125, 126, 127, 128, 129, 131, 134, 136, 146, 148, 151, 152, 159, 161, 162, 163, 164, 167, 171, 172, 173, 174, 178, 194, 195, 197, 213, 231, 248, 249, 250, 259, 261, 308, 310, 312, 314, 318, 330, 331, 333, 382, 487, 527, 556, 558, 661, 676, 679, 680, 690, 692, 701, 703, 724, 751, 755, 756, 798, 799, 833, 835, 836, 837, 838, 839, 840, 844, 845, 850, 863, 864, 869, 870, 874, 897, 899, 927, 938, 939, 965, 966, 973, 975, 981, 986, 1033, 1055, 1085, 1092 perception, xxiv, 651, 653, 654, 1056, 1083, 1089 perinatal, 503, 919 periodicity, 1082 peripheral blood, 857, 1038, 1039, 1053 peripheral blood mononuclear cell, 857, 1038, 1039, 1053 peripheral nervous system, 85, 104, 358, 394, 407, 408, 409, 562, 630, 632, 636, 910, 1038, 1078, 1089 peripheral neuropathy, xxiii, xxxvi, 409, 627, 634, 641, 647, 663, 670, 1025, 1037, 1038, 1040, 1041, 1042, 1043, 1045, 1046, 1053 permeability, 319, 931, 997 permit, 9, 311, 455, 539, 540, 541, 546, 685 peroxidation, 127, 136, 568, 1074 peroxide, 222, 335, 457, 700 peroxynitrite, 136, 612, 679, 700, 924 personal communication, 640 pesticide, 623, 777, 778 PET, 618 PGE, 700 pH, 18, 21, 22, 24, 123, 231, 259, 445, 456, 678, 774, 841, 864, 923, 930, 994, 1074 phagocytosis, 18, 19, 335, 648, 699, 1003 pharmacokinetics, 946, 967, 982 pharmacological treatment, 993 pharmacology, 734, 967 phenotype, xviii, xxiii, 96, 147, 177, 247, 282, 289, 348, 349, 351, 356, 358, 359, 360, 394, 399, 403, 404, 423, 425, 472, 496, 499, 514, 559, 627, 628, 632, 633, 635, 637, 638, 681, 692, 772, 807, 815, 820, 899, 901, 1082, 1085
1141 phenylalanine, 48 pheochromocytoma, 102, 795, 972 phosphates, 737, 1086, 1087 phosphatidylcholine, 946, 1086 phosphatidylethanolamine, 63 phosphatidylserine, 515 phosphoenolpyruvate, 36 phospholipids, 124, 718, 765, 766, 900, 918 phosphorylation, xxvi, 4, 21, 23, 45, 47, 53, 72, 81, 86, 92, 154, 195, 229, 230, 232, 283, 285, 287, 289, 299, 304, 335, 384, 413, 415, 426, 427, 428, 431, 448, 457, 458, 469, 483, 485, 522, 524, 541, 544, 545, 551, 556, 557, 638, 639, 658, 661, 662, 664, 665, 666, 670, 680, 695, 699, 701, 704, 733, 735, 736, 739, 740, 741, 742, 743, 746, 747, 748, 749, 751, 753, 754, 755, 756, 760, 775, 839, 841, 866, 867, 878, 879, 880, 882, 883, 887, 891, 892, 923, 932, 933, 937, 956, 957, 993, 999, 1015, 1062, 1072, 1079, 1083, 1086, 1087, 1090 photoreceptors, 84 phylogenesis, 1093 physical interaction, 101, 146, 147, 464, 473, 690 physical properties, 275 physicochemical properties, 678 physiological factors, 998 physiological regulation, 499 physiology, iv, ix, xiv, xvii, xxii, xxvi, xxxiv, 2, 6, 13, 14, 170, 171, 290, 373, 375, 376, 476, 478, 498, 515, 580, 597, 735, 740, 745, 989, 1000, 1038, 1064 pigs, xxxv, 994, 1012 pituitary gland, 29, 886 pituitary tumors, xxxi, 875, 876, 877, 879, 881, 882, 883, 884, 886, 887, 888, 889, 891, 892, 893 placebo, 835, 998, 1019, 1020 plants, 31, 179 plaque, 757, 1003, 1058 plasma, xv, xix, xxviii, 58, 59, 83, 98, 110, 187, 216, 233, 234, 271, 273, 295, 348, 367, 388, 400, 413, 417, 418, 421, 424, 431, 432, 437, 443, 483, 504, 510, 586, 659, 668, 701, 739, 771, 813, 816, 821, 822, 824, 851, 859, 920, 952, 1020, 1029, 1038, 1039, 1067 plasma levels, 234 plasma membrane, xv, xix, xxviii, 58, 59, 83, 98, 110, 187, 216, 233, 234, 271, 273, 295, 348, 367, 388, 400, 413, 417, 418, 421, 424, 431,
1142 432, 437, 443, 483, 586, 659, 668, 701, 739, 771, 813, 816, 821, 822, 824, 920, 1067 plasminogen, 968, 996, 1019 plasticity, xvi, xvii, xviii, xxiv, 55, 66, 88, 171, 337, 343, 358, 362, 374, 376, 377, 378, 379, 380, 385, 386, 387, 388, 389, 391, 393, 398, 411, 412, 413, 415, 418, 422, 423, 425, 426, 432, 433, 435, 439, 440, 496, 500, 514, 595, 651, 652, 653, 658, 659, 660, 662, 663, 665, 669, 708, 808, 835, 843, 844, 924, 936, 1028, 1061 platelet aggregation, 966 platelets, 1067 platinum, 1043, 1044 PLP, 1082 PM, 39, 108, 110, 114, 130, 131, 191, 197, 198, 199, 220, 236, 237, 238, 239, 262, 264, 266, 268, 291, 294, 295, 302, 330, 331, 332, 335, 336, 365, 388, 389, 390, 436, 437, 438, 439, 489, 510, 526, 530, 574, 620, 621, 624, 644, 663, 667, 695, 699, 700, 702, 711, 756, 788, 789, 812, 827, 844, 845, 846, 859, 860, 861, 873, 895, 914, 915, 916, 953, 985, 1028, 1032, 1034, 1049, 1052, 1053 point mutation, 84, 184, 267, 401, 404, 428, 764, 765, 766, 767, 776, 821 Poland, 373, 833, 961 polarity, 53, 291, 1058, 1072 pollutants, 718 polyacrylamide, 27, 74 polycystic kidney disease, 287 polymer, xxv, 7, 243, 671, 677, 1076 polymerase, 106, 118, 127, 136, 396, 546, 882, 922, 924, 1012, 1083 polymerase chain reaction, 882, 1012 polymerization, 44, 45, 298, 303, 614, 615, 678, 728, 754, 768, 933 polymers, 44, 56, 73, 677, 880, 1067 polymorphism, xxii, 564, 580, 771, 788 polymorphisms, 543, 597, 821, 884 polymyositis, 852 polypeptide, xii, 3, 16, 27, 37, 38, 50, 58, 61, 65, 72, 82, 86, 94, 112, 137, 144, 148, 149, 186, 204, 224, 243, 244, 245, 246, 247, 248, 277, 278, 279, 284, 295, 302, 445, 447, 449, 450, 451, 452, 454, 459, 460, 474, 488, 490, 538, 555, 573, 593, 620, 673, 745, 756, 768, 769, 783, 899, 932, 992, 1055, 1077, 1080 polypeptides, xix, 38, 47, 60, 94, 144, 146, 209, 211, 249, 252, 253, 255, 278, 285, 444, 448,
Index 449, 450, 451, 459, 462, 480, 540, 674, 745, 768, 838, 840, 925, 933, 1063 polyphenols, 592, 594, 598 polyQ, xxvii, 10, 85, 170, 175, 185, 283, 289, 322, 327, 337, 383, 462, 463, 467, 474, 475, 493, 702, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 1084 pons, 471 pools, 49, 181, 230, 234, 417, 483, 679, 690 poor, 230, 290, 331, 684, 694, 846, 870, 966, 968, 976, 978, 993, 997, 1001, 1017 population, 21, 24, 45, 139, 239, 762, 788, 914, 1021, 1026, 1057, 1074, 1084 ports, 209, 309 positive feedback, 519, 678, 721, 839, 882, 937 postmitotic cells, xx, 230, 513, 524 postural hypotension, 1046 postural instability, 763, 1081 posture, 606, 966 potassium, 517, 523, 913, 920, 923 power, 274, 280, 336, 357, 374, 470 Prader-Willi syndrome, 916 precipitation, 497, 731 precursor cells, 345, 514, 776 prediction, 163, 243, 312, 313, 326, 327, 328, 332, 387, 1085 prediction models, 328 preference, 133, 180, 214, 309, 312, 314, 538, 684, 781, 906, 1083 prefrontal cortex, 624 pregnancy, 884 premature infant, 503, 510 prematurity, 510 pressure, xxxv, 394, 402, 445, 476, 998, 1012, 1023, 1082 prevention, xxxvi, 4, 9, 128, 215, 248, 404, 467, 492, 510, 514, 524, 525, 526, 842, 936, 938, 943, 956, 979, 1008, 1025, 1037 primary biliary cirrhosis, 860 primary cells, 1024 primate, 653, 663, 669 priming, 378, 398, 426, 438 prion diseases, ix, xxix, 2, 320, 446, 545, 685, 688, 804, 813, 815, 816, 818, 819, 820, 821, 822, 824, 825, 826, 828, 829, 831, 851, 1084 prions, 335, 698, 824, 830 probability, 9, 314, 658, 993 probe, 89, 114, 405, 693, 712, 970 processing pathways, 817
Index production, xxvi, xxvii, xxix, xxxvi, 136, 175, 257, 284, 288, 319, 320, 330, 331, 332, 335, 349, 352, 378, 386, 459, 462, 475, 524, 528, 529, 535, 559, 565, 581, 588, 597, 610, 611, 613, 614, 648, 674, 685, 689, 697, 716, 717, 718, 719, 726, 729, 762, 764, 768, 769, 770, 802, 833, 842, 854, 857, 858, 906, 923, 936, 938, 952, 955, 956, 966, 968, 973, 976, 1013, 1015, 1025, 1033, 1035, 1038, 1049, 1057 progenitor cells, 345, 365, 528 progesterone, 454, 482, 500, 507, 510, 885 prognosis, 290, 304, 305, 858 program, xxvii, 77, 446, 596, 762, 869, 937, 1033 progressive neurodegenerative disorder, 1057 progressive supranuclear palsy, 322, 467, 581, 650, 690, 1090 Progressive Supranuclear Palsy, 736, 737 pro-inflammatory, 587, 717, 718, 719, 726, 857, 938, 967, 968, 992, 999, 1000, 1001, 1002, 1003, 1017, 1063, 1073, 1074, 1079 proinflammatory effect, 526 pro-inflammatory response, 718, 1000, 1017 prokaryotes, 24, 27, 63, 208, 212, 309 prokaryotic cell, 1092 prolactin, 510, 876, 885, 887, 889, 894, 895 proliferation, x, xviii, xix, xxxi, xxxiii, xxxiv, 71, 102, 393, 395, 399, 444, 446, 449, 455, 518, 525, 549, 717, 759, 850, 857, 866, 867, 868, 869, 871, 875, 879, 881, 883, 885, 887, 888, 890, 918, 921, 961, 972, 973, 978, 979, 1001, 1056, 1061, 1073, 1077, 1083, 1091, 1093 prolyl endopeptidase, 235 promoter, 344, 457, 458, 483, 502, 546, 587, 645, 744, 765, 766, 776, 781, 823, 824, 879, 937 promoter region, 457, 458, 588, 765, 879, 937 propagation, 386, 818, 827, 1076 prostaglandins, 587, 716, 718, 719, 720, 722, 724, 726, 728, 729, 730, 938, 1072 prostate, 81, 90, 483, 547, 873, 887, 978, 1051 prostate cancer, 81, 90, 483, 873, 978, 1051 prostate carcinoma, 547 protease inhibitors, 37, 79, 353, 992, 1026, 1035 protective mechanisms, xxvii, 677, 688, 762 protective role, 242, 382, 403, 472, 503, 576, 608, 685 protein aggregation, xx, 9, 12, 178, 197, 204, 238, 266, 267, 268, 269, 304, 319, 320, 333, 334, 335, 336, 337, 340, 351, 404, 408, 409, 430, 441, 442, 446, 448, 449, 452, 459, 464, 466, 475, 487, 513, 533, 539, 544, 545, 549,
1143 550, 558, 560, 561, 562, 563, 569, 571, 572, 581, 614, 615, 616, 646, 675, 684, 687, 688, 689, 694, 695, 696, 697, 699, 707, 711, 712, 716, 717, 721, 727, 730, 734, 757, 786, 800, 804, 811, 812, 925, 926, 927, 929, 931, 948, 950, 952, 960, 991, 1033, 1034, 1070 protein binding, 64, 348, 445, 458, 541, 796 protein blocks, 163 protein conformations, 247, 248, 317, 446, 462, 554, 682 protein denaturation, 244, 447, 540, 550, 935 protein design, 179 protein family, xi, xix, 47, 93, 106, 140, 152, 220, 224, 262, 298, 437, 444, 1070 protein folding, xv, xix, 7, 8, 215, 221, 224, 241, 242, 243, 244, 245, 246, 248, 251, 256, 259, 260, 265, 266, 273, 274, 275, 285, 291, 292, 308, 316, 404, 408, 444, 446, 449, 459, 460, 461, 465, 466, 467, 469, 477, 479, 480, 481, 486, 538, 539, 540, 547, 575, 587, 590, 673, 695, 696, 745, 768, 788, 927, 933, 1070 protein function, 243, 413, 424, 547, 569, 720, 754, 832 protein kinase C, 264, 389, 436, 551, 664, 665, 669, 716, 733, 759, 787, 952, 953 protein kinases, 178, 264, 283, 318, 378, 389, 439, 454, 463, 544, 545, 678, 700, 737, 740, 787, 803, 920, 933, 956, 1061, 1073, 1085, 1086, 1087, 1090 protein misfolding, xx, xxii, 2, 7, 9, 110, 232, 242, 261, 272, 275, 283, 289, 316, 317, 318, 335, 336, 444, 445, 467, 486, 487, 555, 558, 564, 599, 604, 672, 673, 674, 677, 678, 681, 682, 696, 698, 699, 727, 801, 804, 809 protein oxidation, 612, 624, 680, 731, 857 protein sequence, xxviii, 105, 243, 423, 452, 794 protein structure, 147, 243, 244, 246, 247, 248, 318, 456, 459, 777, 1065 protein synthesis, ix, 1, 23, 28, 105, 106, 114, 259, 273, 283, 285, 352, 353, 354, 355, 365, 369, 370, 374, 381, 387, 406, 413, 414, 427, 435, 581, 584, 586, 751, 837, 844, 924, 929, 932, 933, 938, 952, 954, 1000, 1078, 1086, 1088 proteinase, ix, xi, 2, 3, 16, 29, 117, 129, 130, 131, 132, 133, 134, 196, 222, 223, 235, 236, 237, 333, 374, 390, 482, 535, 786, 811, 819, 822, 845, 859, 888, 951, 954, 955, 981, 986, 1033, 1055, 1059, 1061
1144 protein-protein interactions, 7, 59, 64, 75, 140, 142, 183, 259, 349, 351, 359, 464, 733, 841, 900, 906, 1071 proteolipid protein, 288, 303, 1082 proteolysis, x, xiv, xvi, xvii, xix, xx, xxvi, xxvii, xxx, 3, 6, 11, 12, 15, 17, 20, 22, 23, 25, 28, 31, 34, 37, 42, 43, 47, 48, 49, 50, 53, 54, 57, 65, 67, 68, 79, 80, 108, 109, 110, 111, 112, 118, 126, 144, 145, 147, 148, 149, 151, 152, 160, 161, 163, 164, 166, 167, 171, 172, 181, 183, 186, 193, 194, 196, 198, 201, 202, 203, 207, 208, 210, 211, 212, 213, 218, 222, 223, 224, 227, 231, 232, 236, 238, 250, 252, 253, 254, 256, 258, 261, 267, 276, 297, 298, 299, 300, 301, 307, 310, 314, 320, 331, 333, 338, 343, 345, 346, 349, 352, 354, 355, 357, 359, 360, 361, 362, 363, 373, 375, 378, 379, 381, 399, 404, 407, 408, 409, 436, 437, 439, 442, 443, 457, 464, 489, 506, 515, 524, 532, 535, 536, 537, 557, 567, 569, 575, 593, 625, 644, 657, 664, 678, 683, 689, 690, 696, 703, 706, 712, 717, 721, 728, 736, 742, 745, 749, 751, 759, 761, 762, 764, 769, 796, 799, 801, 808, 836, 839, 849, 850, 860, 878, 879, 881, 882, 883, 885, 887, 891, 892, 893, 894, 896, 926, 932, 933, 937, 938, 950, 951, 962, 963, 973, 975, 976, 977, 978, 985, 986, 987, 990, 1006, 1013, 1031, 1033, 1034, 1049, 1050, 1054, 1057, 1071, 1074, 1084, 1087, 1089, 1094 proteolytic enzyme, 24, 462, 515, 717 proteome, 52, 196, 204, 273, 540 proteomics, 69, 87, 156, 166, 200, 419, 488 protocol, 377, 610 protons, 123 proto-oncogene, 264, 405, 864, 1014 prototype, 44, 161, 852 provocation, 898 pruning, xxiii, 357, 358, 363, 366, 396, 397, 406, 407, 628, 629, 636, 640, 641, 646, 647, 650, 1059 PSD, xxiv, 187, 192, 371, 378, 388, 389, 412, 415, 418, 419, 420, 422, 424, 426, 429, 431, 433, 437, 441, 651, 652, 655, 658, 660, 664, 665, 668, 669, 912, 919, 922, 936, 956 psoriasis, xxxiv, 989, 992, 1003, 1009, 1023, 1032 psychiatric illness, 509, 510 psychostimulants, 502 pulse, 378, 389, 403, 417, 419, 425 PUMA, 596
Index pumps, xiii, 169, 680 purification, 22, 24, 38, 86, 121, 129, 151, 154, 170, 186, 194, 221, 261, 262, 1101 pyramidal cells, 376 pyrimidine, 108 pyrophosphate, 2, 5, 597
Q quality control, ix, x, xiii, xiv, xv, xix, xx, 1, 7, 9, 14, 15, 17, 31, 41, 48, 54, 169, 171, 208, 219, 223, 237, 241, 242, 246, 250, 251, 252, 253, 254, 256, 265, 266, 267, 271, 272, 291, 292, 293, 302, 309, 334, 414, 434, 444, 445, 446, 459, 460, 468, 481, 488, 537, 546, 552, 576, 584, 586, 593, 596, 674, 697, 708, 712, 756, 785, 939, 954, 955, 1063, 1066, 1067 quality of life, 248, 601 question mark, 747, 817 quinolinic acid, 431, 442 quinone, 566, 720 quinones, 718, 728, 766
R race, 635, 776 radiation, 96, 160, 445, 477, 888, 967, 1090 Radiation, 476 radiation therapy, 888 radical formation, 930, 993 radio, 1020 radioactive isotopes, 16 rain, xxv, 1091 range, ix, xxi, 2, 59, 94, 126, 139, 164, 179, 185, 190, 273, 285, 310, 351, 446, 452, 458, 474, 479, 537, 543, 630, 634, 635, 660, 681, 691, 718, 762, 806, 816, 841, 857, 884, 906, 910, 967, 968, 971, 1000, 1015, 1019, 1047, 1056, 1078, 1082, 1092 RANTES, 662 rapamycin, 775, 864, 1069 reaction mechanism, 78 reactive gliosis, 490, 758 reactive oxygen, xxvii, 318, 386, 445, 493, 514, 523, 549, 554, 565, 580, 588, 596, 600, 608, 609, 620, 678, 716, 761, 762, 764, 766, 771, 834, 841, 922, 931, 955, 956, 994, 999, 1003, 1015, 1017, 1088 reactivity, 402, 818, 819, 825, 853, 995
Index reading, 14, 35, 42, 187, 286, 363, 593, 772, 816, 821, 837, 1081 reagents, xiii, 53, 159 real time, 1012 reality, 2, 743, 905 reception, 375 receptor agonist, 601, 663, 669 receptors, ix, xiii, xvi, xviii, xx, xxxii, 1, 45, 54, 58, 66, 83, 98, 109, 147, 149, 152, 166, 170, 171, 182, 183, 187, 190, 191, 200, 201, 275, 278, 281, 291, 335, 344, 345, 346, 350, 356, 358, 361, 365, 367, 369, 370, 378, 379, 381, 385, 388, 389, 391, 395, 411, 412, 413, 415, 416, 417, 418, 419, 420, 423, 424, 430, 431, 434, 435, 436, 437, 441, 454, 482, 495, 496, 497, 498, 504, 506, 508, 511, 653, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 679, 680, 698, 719, 725, 835, 864, 876, 884, 885, 886, 887, 888, 894, 898, 911, 912, 913, 919, 920, 923, 925, 936, 956, 975, 1009, 1017, 1029, 1065, 1067, 1071, 1075, 1078, 1079, 1083, 1086, 1091, 1093, 1094, 1096 recognition, xii, xv, 3, 6, 11, 12, 28, 31, 34, 44, 45, 47, 52, 53, 54, 60, 67, 75, 89, 90, 94, 97, 109, 127, 137, 144, 146, 155, 157, 160, 171, 180, 210, 212, 213, 214, 216, 218, 224, 249, 252, 253, 271, 279, 285, 295, 307, 310, 314, 351, 408, 413, 415, 420, 422, 424, 426, 431, 438, 453, 462, 463, 466, 483, 489, 505, 556, 557, 561, 570, 674, 675, 721, 722, 723, 744, 745, 747, 748, 775, 801, 845, 865, 879, 885, 900, 904, 914, 925, 976, 978, 1015, 1059, 1096 recombination, 170, 176, 177, 196, 910 reconcile, 20 reconstruction, 161, 165, 176 recovery, xxxv, 104, 458, 486, 652, 909, 930, 941, 942, 943, 991, 993, 995, 996, 998, 1000, 1004, 1005, 1012, 1017, 1018, 1019, 1021 recruiting, 47, 98, 105, 177, 184, 209, 217, 463, 521, 522, 540, 1025, 1088 recurrence, 305 recycling, xxiv, 4, 45, 53, 73, 80, 83, 85, 104, 172, 242, 379, 381, 417, 421, 424, 556, 557, 564, 632, 652, 659, 765, 766 red blood cells, 22, 535, 854 redistribution, 256, 338, 709, 803, 806, 807, 811, 828, 929 reduction, xxxiv, xxxv, 11, 80, 85, 104, 126, 127, 185, 274, 275, 286, 292, 321, 324, 329, 348,
1145 356, 429, 430, 460, 468, 484, 517, 566, 568, 657, 660, 676, 688, 744, 772, 776, 777, 799, 800, 801, 881, 910, 923, 931, 934, 941, 942, 943, 990, 994, 995, 996, 997, 1001, 1003, 1004, 1012, 1017, 1018, 1022, 1023 redundancy, 173, 185, 349, 484, 923, 1001 reflexes, 652, 665, 1042, 1068 refractory, xxxiv, 178, 456, 881, 943, 961, 967, 982, 992, 1016, 1028, 1039, 1041, 1043, 1044, 1050, 1051, 1052, 1061 regenerate, 395, 398, 636, 637 regeneration, 105, 358, 370, 395, 398, 407, 637, 646, 648, 998, 1069 regression, xxxi, 863, 865 regrowth, 224, 298 regulation, ix, x, xi, xiv, xvi, xviii, xx, xxi, xxiv, xxx, xxxi, xxxiv, xxxv, 1, 3, 4, 14, 15, 17, 28, 31, 38, 43, 53, 54, 56, 57, 61, 62, 64, 66, 68, 69, 71, 72, 82, 83, 90, 91, 94, 103, 106, 108, 112, 113, 114, 128, 129, 144, 166, 181, 190, 191, 192, 200, 207, 212, 214, 216, 218, 238, 244, 245, 248, 250, 258, 260, 268, 274, 285, 318, 326, 333, 339, 343, 345, 347, 349, 356, 357, 359, 360, 362, 364, 365, 366, 367, 369, 374, 376, 377, 378, 379, 384, 386, 395, 406, 407, 411, 414, 415, 417, 418, 420, 422, 423, 425, 426, 427, 428, 432, 433, 434, 437, 446, 447, 449, 451, 453, 454, 455, 457, 458, 460, 464, 466, 472, 473, 474, 476, 477, 479, 481, 484, 485, 486, 489, 491, 492, 496, 497, 499, 500, 501, 503, 505, 506, 508, 509, 523, 531, 534, 539, 541, 544, 545, 546, 547, 549, 550, 552, 555, 557, 562, 563, 565, 570, 576, 579, 581, 582, 584, 586, 588, 589, 590, 591, 592, 593, 594, 597, 601, 623, 624, 644, 648, 651, 655, 657, 659, 661, 662, 678, 679, 696, 703, 705, 712, 719, 725, 727, 728, 732, 733, 734, 738, 739, 740, 759, 770, 787, 802, 834, 836, 839, 840, 841, 842, 843, 846, 847, 858, 863, 864, 865, 867, 868, 869, 872, 873, 874, 875, 878, 879, 881, 882, 883, 884, 885, 887, 888, 890, 891, 894, 902, 904, 906, 910, 912, 913, 928, 930, 933, 949, 951, 954, 975, 986, 989, 1000, 1009, 1011, 1013, 1015, 1021, 1022, 1026, 1027, 1073, 1086, 1087, 1090, 1096 regulations, xxi regulators, xi, xii, xv, xxiii, 31, 33, 54, 64, 72, 86, 87, 117, 128, 153, 155, 165, 167, 192, 194, 234, 241, 263, 301, 333, 406, 485, 518, 557,
1146 560, 596, 598, 628, 640, 712, 841, 879, 891, 900, 914, 927, 1013, 1014, 1059, 1091 rehabilitation, 1021 rejection, 835 relapses, 1003, 1023 relationship, xix, xx, xxv, xxxii, xxxiii, 7, 39, 56, 63, 171, 217, 237, 245, 395, 399, 444, 478, 486, 495, 496, 501, 504, 513, 528, 535, 571, 601, 602, 603, 604, 609, 611, 613, 615, 616, 648, 649, 674, 716, 717, 722, 746, 805, 865, 897, 899, 901, 907, 912, 913, 921, 932, 933, 937, 952, 1026, 1033, 1039 relationships, xxiii, xxvi, 600, 736, 738, 986, 1100 relatives, 61, 103, 251, 547, 872, 1064 relevance, xxxiv, 101, 127, 147, 178, 464, 498, 662, 703, 731, 790, 824, 853, 920, 962, 964 reliability, 314, 1053 remission, 525 remodelling, 198, 357, 360, 1000 renal cell carcinoma, 1052 repair, xi, xiii, xxii, xxxi, xxxii, xxxiii, 3, 9, 43, 45, 54, 56, 71, 72, 82, 91, 94, 96, 108, 109, 134, 170, 176, 177, 196, 200, 208, 212, 217, 218, 238, 252, 253, 257, 268, 282, 447, 459, 462, 523, 540, 580, 581, 630, 637, 717, 727, 745, 790, 864, 896, 922, 924, 944, 1057, 1062, 1080, 1096, 1097 replacement, xxix, 119, 125, 311, 383, 763, 833, 869 replication, 3, 82, 180, 182, 195, 257, 269, 282, 299, 455, 825, 842, 904, 1062 repression, 62, 83, 91, 181, 285, 336, 458, 545, 551, 701, 801 repressor, 39, 115, 193, 484, 544, 545, 712, 864, 876, 883, 887, 893, 1071 reproduction, 477 residues, xii, 5, 27, 28, 29, 43, 44, 48, 56, 57, 60, 62, 64, 72, 74, 75, 77, 78, 79, 94, 102, 117, 122, 123, 125, 126, 132, 133, 155, 173, 174, 175, 177, 181, 213, 244, 268, 277, 283, 309, 310, 311, 313, 314, 318, 322, 326, 327, 333, 348, 349, 416, 452, 457, 465, 514, 520, 541, 551, 556, 564, 566, 568, 584, 654, 662, 673, 681, 690, 691, 722, 724, 733, 740, 764, 769, 799, 837, 839, 880, 900, 927, 928, 936, 967, 976, 1061, 1062, 1063, 1069, 1073, 1082, 1084, 1086, 1087, 1089, 1091, 1094, 1095, 1096
Index resistance, 68, 125, 187, 245, 290, 292, 312, 323, 431, 449, 450, 479, 498, 501, 507, 510, 517, 534, 543, 549, 584, 610, 681, 732, 796, 799, 822, 828, 831, 885, 924, 934, 958, 967, 1056 resolution, xxxi, 22, 38, 88, 108, 119, 129, 130, 155, 173, 176, 193, 222, 244, 261, 262, 263, 329, 420, 541, 542, 546, 695, 718, 863, 925, 1009, 1046, 1101 respiration, 611, 612, 718, 936 respiratory, 68, 366, 510, 764, 771 respiratory distress syndrome, 510 respiratory failure, 366 responsiveness, xvii, xx, 344, 355, 357, 362, 369, 418, 495, 497, 498, 500, 501, 503, 506, 511, 655, 657, 658, 884, 885, 1074 retardation, xxxii, 382, 531, 901 retention, 252, 273, 288, 310, 404, 958, 1041, 1048 reticulum, ix, xiii, xiv, xv, xxi, xxix, 1, 2, 20, 42, 54, 56, 58, 97, 104, 112, 114, 152, 170, 175, 187, 191, 201, 204, 208, 216, 224, 227, 228, 229, 230, 236, 237, 264, 265, 266, 269, 271, 272, 273, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 336, 375, 380, 382, 412, 421, 434, 444, 449, 453, 491, 552, 554, 562, 573, 579, 580, 586, 587, 593, 594, 596, 597, 603, 604, 620, 628, 633, 647, 672, 684, 703, 711, 712, 759, 762, 769, 783, 785, 786, 794, 803, 811, 814, 816, 817, 818, 834, 836, 837, 838, 840, 844, 898, 912, 922, 925, 928, 938, 952, 954, 1012, 1024, 1058, 1067, 1071, 1084, 1092, 1097 retina, 102, 368, 387, 929 retinitis, 242, 256, 267 retinitis pigmentosa, 242, 256, 267 retinoblastoma, 524, 864, 878, 890, 976 retinoic acid, 239, 885 returns, 284 rheumatoid arthritis, xx, xxxiv, 234, 513, 526, 852, 856, 861, 989 rhodopsin, 180, 246, 256, 267 rhythm, 3, 191 rhythms, xiii, 54, 169, 171 ribonucleic acid, 42 ribose, 118, 127, 136, 396, 1078 ribosome, 243, 286, 459, 674, 768, 895, 1062 ribosomes, 7, 182, 273, 280, 421, 925 rigidity, 318, 600, 676, 763, 966, 1064, 1081 rings, xi, xiii, 29, 32, 50, 117, 119, 120, 121, 125, 126, 129, 130, 135, 144, 145, 163, 169, 172,
Index 174, 176, 178, 211, 212, 221, 249, 309, 333, 377, 450, 963, 1015, 1016, 1055, 1080 risk, 84, 195, 288, 339, 564, 571, 603, 611, 632, 705, 710, 726, 763, 772, 788, 842, 852, 860, 899, 1023 risk factors, 611 RNA, ix, 15, 42, 106, 130, 147, 153, 157, 175, 180, 224, 243, 267, 272, 284, 286, 289, 300, 445, 446, 467, 474, 535, 546, 549, 624, 638, 695, 716, 725, 841, 881, 888, 899, 910, 918, 933, 950, 954, 1088, 1089, 1096 RNA processing, 638, 1089 RNAi, 81, 82, 83, 122, 147, 175, 255, 259, 278, 281, 282, 445, 467, 468, 543, 716, 724, 725, 1088 rodents, xxii, 319, 395, 422, 424, 565, 600, 689, 801, 830, 835, 884, 994 Romania, 875 Rouleau, 915, 917 routing, 31, 187 Royal Society, 407 Russia, 575
S safety, xxxiv, 946, 990, 996, 998, 1019, 1021, 1026, 1040 salivary glands, 478 salpingitis, 480 salt, 25, 144, 244, 259, 933, 966 sample, 243, 403, 1040, 1075 sampling, 274, 462 saturation, 676, 823 schizophrenia, 502, 509, 510 school, 510 Schwann cells, xviii, xxxvi, 393, 394, 399, 400, 401, 402, 403, 405, 407, 408, 640, 790, 1038, 1048, 1049, 1063, 1065 scientific community, 3, 13 scientific understanding, 1013 scleroderma, 644, 852 sclerosis, xix, xxv, xxx, 2, 6, 242, 247, 256, 268, 269, 272, 289, 304, 318, 336, 412, 503, 635, 850, 852, 859, 1079 search, 3, 4, 152, 224, 567, 617, 776, 972, 976, 993 searches, 458, 673 searching, 359 secondary tissue, 523 secrete, 355
1147 secretion, 224, 251, 273, 292, 294, 298, 303, 399, 414, 425, 449, 463, 468, 491, 586, 885, 1067, 1071 sediment, 189 sedimentation, 416 seeding, 678 segregation, 345, 601, 910 seizure, xxxii, 12, 381, 478, 897, 898, 900, 901, 902, 905, 906, 909, 910, 911, 912, 913, 915, 918, 919, 1059 seizures, xxvi, xxxii, 288, 386, 716, 835, 897, 898, 899, 901, 902, 903, 904, 905, 906, 909, 911, 918, 1015, 1028, 1067, 1068, 1072 selecting, 463 selective serotonin reuptake inhibitor, 502 selectivity, xxvii, 21, 31, 37, 47, 109, 125, 166, 201, 265, 310, 333, 334, 489, 683, 684, 695, 755, 762, 940, 985, 1038 self-assembly, 493, 549, 756, 785 self-destruction, 370, 646 senescence, 222, 446, 549, 576, 782, 881, 1087 senile dementia, 752 senile plaques, 467, 468, 642, 672, 697, 717 sensation, 653, 664, 998, 1020, 1025, 1046, 1061, 1064, 1078 sense organs, 348 sensing, 288, 292, 301, 303, 457, 476, 927, 1069 sensitivity, xxiv, 9, 21, 96, 177, 423, 431, 442, 459, 507, 521, 522, 533, 572, 619, 634, 651, 653, 654, 657, 667, 688, 696, 773, 879, 906, 909, 928, 930, 931, 944, 982, 1004, 1016, 1022, 1032, 1060, 1071 sensitization, 415, 439, 662, 663, 665, 666, 1022 sensors, 273, 285, 425 separation, 21, 54, 56, 165, 214, 273, 641, 644, 883, 893, 1088 sepsis, 53 sequencing, 64, 75, 223, 722, 752, 754, 781, 884 series, xxxiii, 17, 46, 61, 67, 94, 109, 202, 243, 246, 248, 254, 274, 297, 382, 409, 520, 603, 639, 644, 654, 711, 730, 766, 768, 839, 868, 878, 888, 921, 940, 971, 972, 976, 1000 serine, 48, 123, 124, 133, 240, 361, 428, 483, 529, 551, 652, 658, 662, 716, 736, 755, 773, 778, 788, 803, 884, 968, 1028, 1056, 1061, 1069, 1079, 1080, 1085, 1086, 1091 serotonin, 381, 415, 510 sertraline, 502
1148 serum, xxx, 16, 18, 400, 412, 428, 516, 549, 592, 683, 725, 817, 849, 852, 853, 854, 855, 857, 1002, 1024, 1029, 1039, 1079 serum albumin, 16, 18 severe stress, 938 severity, 289, 409, 724, 737, 755, 851, 858, 1001, 1009, 1029, 1032, 1041, 1045, 1074, 1082 sex, 902 shape, xi, 77, 117, 119, 139, 312, 331, 374, 514, 1055, 1061, 1070 shares, 351, 358, 564, 640 sharing, 315, 382, 384, 467, 676, 695, 840 sheep, 815, 816, 821, 826, 1084 shock, xix, xx, xxii, xxxii, 2, 9, 53, 61, 113, 118, 160, 170, 178, 196, 221, 223, 254, 266, 268, 272, 283, 301, 308, 316, 317, 322, 334, 444, 445, 446, 448, 449, 456, 458, 467, 473, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 490, 491, 492, 493, 501, 506, 507, 521, 522, 533, 538, 539, 540, 543, 544, 545, 547, 548, 549, 550, 551, 552, 593, 594, 684, 691, 692, 727, 745, 747, 748, 758, 759, 803, 814, 828, 897, 898, 909, 922, 924, 926, 935, 948, 954, 955, 1024, 1034, 1064, 1069, 1070, 1071, 1090 sibling, 349 siblings, 84, 563, 720, 899 sickle cell, 22 side effects, 13, 634, 653, 967, 998, 1019, 1020, 1022, 1023, 1025, 1043, 1048 sign, 905, 1026, 1079 signal peptide, 816, 822, 823 signal transduction, x, xi, 2, 3, 17, 31, 42, 48, 56, 71, 72, 81, 161, 198, 378, 384, 428, 445, 449, 454, 455, 478, 518, 522, 544, 665, 679, 739, 803, 884, 894, 933, 937, 957, 1071, 1086, 1087, 1096 signaling pathway, xxii, xxxii, 166, 293, 300, 364, 367, 389, 390, 399, 447, 455, 467, 496, 499, 525, 534, 597, 639, 680, 720, 729, 779, 832, 841, 869, 876, 883, 1086 signalling, xiii, xix, xxiv, 22, 27, 88, 159, 274, 346, 348, 349, 350, 352, 354, 356, 357, 358, 360, 361, 362, 386, 443, 455, 457, 475, 652, 654, 658, 660, 661, 662, 679, 701, 817, 820, 843, 867, 892, 1013, 1014, 1027, 1065, 1071, 1074, 1075, 1076, 1077, 1089, 1091, 1097 signals, 28, 48, 50, 53, 58, 64, 103, 185, 232, 250, 257, 275, 286, 296, 312, 327, 331, 335, 346, 351, 362, 384, 385, 399, 412, 418, 424,
Index 456, 496, 526, 544, 550, 570, 647, 662, 685, 693, 699, 728, 821, 827, 831, 835, 844, 868, 878, 913, 978, 999, 1009, 1058, 1064, 1078, 1083, 1094, 1096 signs, 289, 653, 717, 741, 815, 1045 silicon, 1075 silver, 871 similarity, xi, xxiv, 63, 76, 78, 94, 95, 139, 140, 182, 257, 640, 651, 947, 1060 simulation, 6, 325 Singapore, 579, 593 sinuses, 877 siRNA, 100 sites, xii, xiii, xxxiv, 10, 11, 30, 32, 47, 50, 51, 58, 61, 75, 78, 94, 96, 97, 98, 101, 117, 121, 125, 126, 129, 131, 133, 142, 144, 145, 156, 159, 163, 167, 169, 172, 173, 175, 177, 178, 181, 186, 190, 199, 208, 210, 212, 213, 249, 256, 261, 269, 309, 311, 312, 313, 320, 321, 323, 325, 326, 328, 333, 356, 357, 378, 385, 396, 401, 403, 413, 433, 449, 452, 454, 460, 462, 489, 524, 539, 541, 559, 564, 588, 609, 628, 629, 655, 658, 665, 690, 692, 740, 741, 742, 743, 748, 754, 755, 816, 842, 887, 892, 961, 965, 971, 1015, 1016, 1055, 1056, 1078, 1083, 1086, 1087, 1088, 1094 Sjögren, xxx, 849, 851, 852, 853, 858 skeletal muscle, 53, 237, 387, 390, 465, 1086 skin, 663, 888, 905, 906, 917, 1003, 1053, 1061, 1064, 1093 sleep spindle, 906 small intestine, 179 social behavior, 435, 1083 sodium, 27, 118, 119, 497, 653, 923, 1010 software, 328 solid state, 698 solid tumors, xxxv, 634, 859, 967, 982, 1011, 1041, 1044, 1051, 1053 solubility, 215, 259, 266, 267, 334, 462, 493, 604, 695, 702, 754, 786, 954 solvation, 244 somata, 368 somatic cell, 52 somatic mutations, 877, 888, 1092 somatomotor, 667 somatosensory, 659 spastic, 1082 spasticity, 1082 spatial learning, 385 spatial memory, 435, 508
Index specialization, 384, 387 specialized cells, 496, 1068, 1071 species, xiv, xix, xxvii, 10, 12, 54, 60, 139, 141, 143, 144, 188, 189, 207, 215, 217, 229, 242, 266, 276, 291, 318, 386, 444, 445, 462, 466, 469, 493, 497, 514, 523, 549, 554, 558, 559, 565, 566, 575, 576, 580, 588, 596, 600, 608, 609, 610, 620, 629, 672, 674, 675, 678, 679, 682, 684, 685, 686, 690, 709, 716, 718, 720, 743, 746, 747, 748, 749, 752, 761, 762, 764, 765, 766, 771, 799, 817, 818, 822, 823, 834, 841, 922, 931, 951, 955, 956, 994, 999, 1003, 1017, 1066, 1088 specificity, x, xvi, 12, 31, 39, 41, 43, 47, 50, 59, 70, 78, 88, 100, 122, 125, 131, 133, 160, 167, 181, 189, 235, 249, 253, 301, 307, 311, 312, 313, 327, 328, 351, 357, 359, 363, 384, 417, 422, 480, 487, 497, 498, 499, 504, 509, 541, 557, 564, 641, 694, 703, 712, 791, 798, 855, 856, 857, 858, 880, 889, 900, 904, 906, 917, 925, 939, 945, 966, 975, 1017, 1066, 1070 spectroscopy, 673 spectrum, 34, 106, 242, 253, 255, 734, 831, 842, 856, 858, 906 speculation, xxix, 422, 603, 613, 614, 814 speech, 901, 1056, 1064, 1083 speed, xviii, 352, 394, 628 sperm, 234, 239, 300, 770 spermatogenesis, 456, 484 spin, 624 spinal cord, xxiv, 127, 135, 228, 230, 374, 375, 376, 394, 398, 473, 564, 635, 645, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 661, 662, 665, 666, 667, 668, 705, 707, 720, 812, 927, 999, 1008, 1014, 1045, 1057, 1061, 1062, 1066, 1072, 1084 spinal cord injury, 999, 1008 spindle, 182, 201, 233, 282, 299, 785, 791, 878, 1076, 1092 spine, 412, 431, 441, 652, 655 spleen, 124, 133, 134, 176 sporadic tumors, xxxi, 875, 877 Sprague-Dawley rats, 926 sprouting, 648 squamous cell, 290, 596 squamous cell carcinoma, 290, 596 stability, xi, xxvi, xxvii, 2, 8, 17, 20, 21, 22, 37, 48, 71, 72, 79, 80, 81, 90, 98, 102, 110, 145, 148, 177, 192, 196, 244, 246, 247, 259, 264, 315, 316, 320, 322, 388, 400, 412, 414, 418,
1149 428, 433, 436, 454, 479, 482, 486, 540, 589, 638, 658, 673, 676, 735, 738, 739, 741, 744, 762, 769, 869, 873, 879, 885, 894, 904, 920, 927, 935, 936, 946, 952, 966, 968, 1013, 1015, 1075, 1077, 1090, 1092 stabilization, xiv, 34, 54, 81, 86, 90, 100, 207, 243, 244, 259, 261, 264, 287, 417, 427, 446, 447, 477, 524, 538, 540, 584, 722, 769, 823, 869, 885, 927, 928, 949, 994, 1022, 1047, 1071 stabilizers, 508 stages, xiii, xvi, xx, xxxi, 18, 160, 268, 291, 308, 343, 345, 370, 407, 430, 431, 469, 513, 519, 528, 546, 559, 591, 630, 648, 685, 688, 806, 818, 819, 828, 853, 865, 868, 875, 906, 923, 948, 1000, 1007, 1023 standard error, 657 standards, 265, 291 starch, 917 starvation, 20, 53, 61, 63, 68, 231, 682, 684, 704, 782, 931, 1058 status epilepticus, 902, 906 stem cell therapy, 998 sterile, 770 steroid hormone, 24, 449, 454, 455, 496, 498, 499, 507, 511, 589, 885, 1068, 1071 steroid hormones, 496, 507, 589, 885 stimulus, 413, 420, 424, 455, 496, 518, 521, 552, 655, 656, 841, 1082 stoichiometry, 236, 427, 475, 869 storage, 175, 195, 248, 273, 302, 381, 435, 448, 531, 703 strain, 398, 429, 614, 632 strategies, xviii, xx, xxiv, xxvi, xxix, xxxi, xxxv, 4, 248, 273, 304, 317, 394, 395, 527, 537, 540, 580, 596, 617, 625, 628, 652, 682, 695, 701, 716, 833, 843, 847, 861, 864, 883, 923, 947, 981, 984, 991, 993, 1000, 1004, 1005, 1011 strength, xxiv, 10, 119, 320, 362, 381, 417, 425, 678 stress, ix, xv, xviii, xx, xxi, xxii, xxvi, xxxi, xxxiii, 3, 8, 21, 53, 54, 61, 68, 72, 105, 111, 112, 114, 127, 135, 136, 178, 215, 223, 250, 272, 273, 274, 278, 279, 280, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 300, 301, 302, 304, 318, 322, 334, 336, 429, 440, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 456, 457, 458, 459, 460, 465, 466, 469, 470, 475, 476, 477, 478, 479, 483, 485, 486, 489, 491, 492, 493, 495,
1150 496, 497, 501, 502, 507, 522, 523, 524, 525, 535, 538, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 561, 562, 565, 566, 568, 571, 573, 575, 576, 579, 580, 582, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 603, 604, 606, 608, 611, 612, 613, 614, 615, 616, 617, 620, 622, 625, 633, 680, 682, 684, 688, 689, 691, 698, 700, 703, 706, 709, 716, 717, 718, 719, 721, 722, 723, 725, 726, 727, 728, 750, 759, 763, 772, 773, 777, 783, 786, 789, 797, 803, 811, 817, 828, 841, 847, 863, 864, 870, 921, 924, 926, 927, 928, 930, 931, 934, 935, 938, 944, 948, 950, 953, 954, 959, 967, 983, 992, 1014, 1062, 1067, 1071, 1073, 1080, 1090 stress factors, 447, 448 stressors, 283, 924, 991 striatum, xx, xxii, 386, 430, 513, 599, 600, 603, 605, 609, 623, 634, 635, 763, 768, 956, 999 stroke, xxvi, xxxiii, xxxiv, xxxv, xxxvi, 8, 13, 287, 288, 289, 290, 304, 334, 386, 516, 523, 525, 527, 581, 594, 596, 635, 645, 698, 700, 716, 835, 847, 861, 921, 922, 923, 925, 927, 933, 935, 939, 941, 942, 943, 944, 945, 946, 947, 951, 955, 958, 961, 964, 969, 979, 984, 992, 993, 994, 1000, 1004, 1005, 1006, 1007, 1009, 1011, 1014, 1016, 1017, 1018, 1019, 1021, 1022, 1026, 1029, 1030, 1032, 1080 stromal cells, 841, 962, 967, 1031, 1061 strong interaction, 768, 770 structural changes, xiv, 6, 207, 273, 447, 561, 717, 926 structural protein, 16, 180, 197, 937 structural transformations, 625 subacute, 623, 1081 subcortical structures, 600 subdomains, 232 subgroups, 315, 677 subnetworks, 237 substantia nigra, xxii, xxvii, 8, 84, 135, 279, 288, 445, 469, 472, 492, 517, 525, 564, 565, 568, 577, 582, 594, 599, 600, 601, 603, 605, 606, 607, 608, 609, 610, 612, 613, 614, 621, 622, 623, 624, 635, 677, 686, 689, 717, 761, 762, 763, 764, 769, 774, 777, 778, 1073, 1081 substantia nigra pars compacta, 135, 492, 582, 594, 600, 621, 624, 686, 762, 763, 1081 substitutes, 992 substitution, 84, 331, 473, 869
Index substrates, xii, xiii, xv, xvi, xviii, xix, xxi, xxvii, xxxii, 4, 5, 6, 10, 11, 12, 14, 17, 19, 20, 24, 25, 28, 29, 30, 32, 34, 38, 43, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 66, 77, 79, 80, 86, 96, 97, 98, 100, 101, 102, 103, 105, 113, 118, 119, 120, 121, 123, 125, 126, 128, 131, 133, 135, 138, 142, 143, 145, 146, 148, 149, 150, 156, 159, 160, 161, 164, 165, 166, 167, 169, 171, 172, 178, 179, 180, 181, 182, 183, 184, 185, 190, 199, 200, 202, 208, 209, 210, 211, 212, 213, 214, 215, 218, 221, 222, 224, 231, 232, 233, 238, 241, 242, 249, 252, 253, 254, 255, 256, 257, 259, 261, 262, 263, 271, 275, 276, 277, 278, 279, 281, 284, 287, 294, 295, 296, 308, 309, 310, 314, 318, 320, 321, 323, 326, 327, 328, 329, 331, 332, 333, 340, 345, 348, 351, 356, 360, 363, 383, 394, 398, 401, 403, 404, 405, 409, 414, 416, 417, 420, 421, 423, 425, 426, 428, 429, 430, 444, 446, 448, 452, 454, 455, 458, 460, 461, 463, 464, 465, 466, 479, 486, 488, 489, 500, 518, 519, 520, 522, 523, 529, 537, 538, 540, 542, 555, 556, 557, 561, 562, 563, 569, 570, 581, 586, 603, 604, 612, 633, 675, 684, 685, 687, 689, 690, 692, 693, 699, 717, 721, 722, 723, 724, 731, 737, 740, 761, 764, 767, 768, 769, 771, 779, 818, 821, 837, 844, 865, 868, 880, 883, 893, 898, 900, 901, 904, 911, 912, 913, 924, 926, 934, 936, 937, 945, 951, 956, 963, 975, 1005, 1013, 1017, 1024, 1028, 1056, 1063, 1064, 1065, 1067, 1070, 1071, 1072, 1073, 1076, 1079, 1080, 1081, 1086, 1087, 1089, 1092, 1094 sucrose, 239 suffering, 101, 102, 216 sugar, 296, 973 suicide, 88, 476, 1007 sulfate, 27, 301 sulfur, 36, 79, 160 Sun, v, 69, 71, 92, 196, 198, 199, 220, 303, 337, 340, 484, 491, 505, 531, 533, 575, 595, 641, 643, 670, 696, 702, 708, 810, 811, 872, 873, 892, 946, 954, 957, 985, 1029 supply, 11, 23, 36, 104, 150, 288, 523, 600, 640, 923 suppression, 85, 87, 90, 136, 216, 304, 334, 335, 337, 429, 491, 492, 493, 517, 534, 597, 613, 695, 696, 698, 708, 756, 798, 808, 809, 858, 869, 910, 918, 929, 954, 966, 973, 1023 surface region, 434
Index surfactant, 256, 268 surprise, 515 surveillance, xiv, 203, 241, 248, 250, 251, 275, 318, 334, 338, 390, 691, 697, 709, 811, 837, 864, 868 survival, x, xvi, xviii, xxi, xxiii, xxv, 2, 41, 53, 81, 113, 223, 275, 300, 340, 343, 377, 385, 386, 393, 395, 396, 398, 399, 405, 406, 429, 446, 447, 473, 474, 476, 478, 488, 518, 521, 535, 538, 547, 548, 560, 567, 569, 571, 579, 582, 594, 595, 596, 616, 627, 628, 629, 633, 638, 639, 641, 648, 674, 682, 684, 712, 715, 726, 727, 733, 748, 749, 756, 758, 763, 768, 771, 774, 776, 842, 843, 887, 924, 928, 933, 935, 936, 942, 944, 945, 952, 955, 967, 994, 1003, 1013, 1015, 1028, 1060, 1061, 1068, 1086, 1091 survival rate, 595 Survivin, 239 susceptibility, xxxii, xxxvi, 10, 12, 36, 92, 127, 177, 257, 338, 543, 565, 568, 575, 589, 620, 632, 634, 643, 687, 704, 706, 711, 722, 771, 779, 781, 782, 808, 820, 821, 889, 890, 897, 898, 899, 900, 901, 905, 909, 910, 911, 912, 913, 915, 918, 919, 935, 949, 1037, 1046, 1049 susceptibility genes, 899 sweat, 1093 swelling, 282, 629, 634, 635, 770, 787, 1045, 1059, 1072, 1097 switching, 179, 903 symmetry, 120, 139 symptom, 1019, 1056 symptomology, 430, 432 symptoms, xxvi, xxvii, xxviii, xxxvi, 9, 101, 104, 428, 431, 469, 547, 601, 623, 632, 735, 737, 761, 763, 769, 813, 815, 820, 902, 903, 906, 915, 998, 1020, 1037, 1045, 1046, 1064, 1065, 1068, 1074, 1083, 1085 synapse, ix, xviii, xxiii, xxiv, 2, 6, 69, 191, 192, 350, 358, 360, 361, 362, 366, 370, 371, 374, 377, 378, 379, 380, 381, 385, 387, 388, 406, 411, 412, 414, 416, 417, 419, 422, 423, 424, 426, 429, 433, 435, 436, 438, 500, 570, 589, 627, 629, 632, 636, 640, 648, 649, 651, 653, 655, 659, 660, 664, 816, 919, 1054, 1063, 1084 synaptic plasticity, xvi, xvii, xviii, xxiv, 55, 171, 343, 358, 376, 377, 378, 380, 386, 388, 389, 391, 393, 398, 411, 412, 413, 415, 422, 423,
1151 425, 426, 432, 435, 439, 504, 589, 646, 651, 653, 654, 655, 659, 662, 667, 668, 669, 680, 843, 857, 912, 920, 1062 synaptic strength, xxiv, 193, 362, 371, 381, 389, 418, 435, 652, 654, 659 synaptic transmission, 55, 85, 98, 104, 378, 379, 385, 386, 475, 523, 632, 667, 668, 669, 680, 827, 842, 858, 912, 936, 992 synaptic vesicles, 7, 84, 104, 378, 424, 610, 766 synaptogenesis, xvi, 343, 345, 347, 356, 358, 359, 360, 361, 362, 366, 370, 387, 919, 1048, 1060, 1066, 1070 synchronization, xxxii, 897 syndrome, xxx, xxxii, 9, 81, 84, 102, 112, 246, 260, 382, 389, 428, 439, 500, 507, 532, 622, 623, 695, 765, 849, 851, 852, 853, 858, 860, 861, 877, 881, 888, 898, 900, 901, 903, 914, 915, 916, 920, 1025, 1061, 1062, 1064, 1067, 1085, 1093 synthesis, ix, xv, xvi, xxi, 1, 17, 19, 23, 28, 36, 82, 105, 106, 114, 126, 133, 213, 214, 236, 259, 272, 273, 274, 283, 285, 289, 302, 343, 352, 353, 354, 355, 365, 366, 369, 370, 374, 380, 383, 387, 388, 397, 406, 413, 414, 425, 427, 433, 435, 445, 446, 447, 448, 458, 478, 479, 492, 579, 581, 582, 586, 589, 590, 597, 674, 693, 718, 758, 768, 778, 784, 785, 799, 816, 826, 827, 837, 839, 856, 879, 886, 889, 916, 923, 924, 926, 929, 932, 936, 965, 968, 970, 976, 985, 1006, 1066, 1067, 1071, 1072, 1079, 1096 systemic immune response, xxx, 850 systemic lupus erythematosus, xxx, 234, 849, 851, 859, 860, 861, 1057 systemic sclerosis, 856 systems, xvii, xx, xxii, xxv, 7, 13, 28, 31, 64, 66, 85, 96, 136, 155, 165, 200, 251, 253, 256, 265, 273, 275, 287, 290, 293, 305, 311, 317, 327, 355, 360, 377, 378, 393, 397, 405, 431, 464, 468, 474, 476, 502, 503, 528, 535, 537, 539, 540, 565, 580, 581, 596, 672, 675, 676, 678, 680, 682, 693, 701, 703, 728, 731, 733, 738, 752, 774, 856, 919, 932, 933, 944, 952, 994, 1000, 1033, 1063, 1081
T T cell, xxix, xxx, 54, 250, 295, 297, 300, 330, 331, 506, 833, 834, 835, 838, 844, 856, 857, 972, 985, 1009, 1032, 1034, 1074
1152 T lymphocyte, xxx, 194, 330, 331, 383, 834, 835, 843, 845, 846, 849, 869 T lymphocytes, xxx, 331, 383, 835, 846, 849, 869 tactile stimuli, 663 tandem mass spectrometry, 323 tandem repeats, 754, 1059 tangles, xxvi, 8, 55, 98, 228, 235, 315, 316, 445, 467, 471, 650, 693, 746, 748, 752, 756, 1077 targets, ix, xvi, xxiv, xxx, xxxiii, xxxvi, 2, 9, 12, 25, 33, 34, 54, 60, 62, 63, 65, 81, 94, 97, 100, 101, 105, 112, 166, 167, 182, 185, 187, 209, 213, 216, 221, 235, 257, 269, 285, 344, 346, 349, 350, 351, 352, 355, 356, 357, 360, 364, 371, 378, 388, 395, 396, 400, 415, 420, 435, 456, 458, 460, 465, 487, 518, 519, 525, 526, 565, 566, 567, 568, 634, 652, 654, 656, 658, 661, 669, 670, 686, 689, 719, 722, 723, 724, 729, 749, 768, 783, 791, 802, 834, 840, 850, 856, 887, 889, 893, 899, 904, 906, 911, 917, 922, 946, 953, 955, 956, 957, 963, 967, 976, 986, 993, 1037, 1059, 1063, 1074, 1075, 1086 tau, xxvi, 8, 103, 113, 127, 135, 136, 216, 223, 267, 269, 316, 317, 335, 462, 463, 465, 467, 468, 469, 471, 488, 491, 517, 552, 558, 559, 571, 572, 591, 595, 618, 633, 650, 677, 680, 684, 693, 699, 701, 713, 717, 725, 733, 735, 736, 737, 738, 739, 740, 741, 742, 743, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 842, 926, 933, 935, 952, 955, 1068, 1069, 1075, 1077, 1083, 1085, 1090, 1091, 1092 tau pathology, xxvi, 467, 735, 746, 752 taxanes, 1043 Tay-Sachs disease, 246 TBP, 387 T-cells, 972, 1001, 1003 TCP, 254, 268, 444, 449, 455, 490, 1092 TCR, 280, 834, 835 technical assistance, 128, 617 technology, 1063 teens, 1074 tellurium, 399, 407 telomere, 82, 91 telophase, 233 temperature, 29, 37, 39, 160, 178, 348, 445, 448, 479, 485, 1045, 1046 temporal distribution, 233, 717 temporal lobe, 1056, 1070, 1083 tendon, 1042
Index terminals, 7, 273, 359, 375, 397, 603, 608, 634, 640, 642, 648, 653, 766, 772, 1048 ternary complex, 49, 103 TGF, 345, 360, 888, 890 thalamus, 653 thapsigargin, 283 theory, xxviii, 127, 184, 259, 432, 559, 794, 1074 therapeutic agents, 945, 963, 979, 981, 1017 therapeutic approaches, 405, 475, 835, 1053 therapeutic benefits, 585 therapeutic interventions, 993, 1026 therapeutic targets, 12, 861, 980, 1008 therapeutics, xxxiv, 12, 601, 694, 962, 975, 976 therapy, ix, xxxiv, xxxvi, 2, 9, 248, 290, 304, 455, 483, 502, 503, 510, 527, 534, 571, 596, 601, 617, 644, 763, 842, 847, 853, 860, 861, 868, 884, 893, 895, 947, 962, 967, 969, 979, 984, 993, 998, 1000, 1005, 1006, 1007, 1022, 1025, 1027, 1028, 1029, 1030, 1037, 1038, 1039, 1040, 1042, 1043, 1046, 1047, 1049, 1052, 1053, 1088 thermodynamic properties, 244, 245 thermodynamic stability, 821, 831 thermostability, 543 thiamin, 63 thinking, 9, 345, 360, 361, 371, 1097 threat, xxi, 317, 445, 579, 682 threonine, xii, 48, 117, 120, 123, 131, 132, 361, 428, 716, 773, 778, 803, 873, 891, 892, 964, 967, 982, 984, 1006, 1016, 1018, 1028, 1030, 1056, 1061, 1064, 1069, 1078, 1079, 1085, 1086, 1087, 1091 threshold, xviii, xxii, 318, 443, 473, 492, 545, 550, 580, 652, 657, 681, 711, 746, 795, 858, 910, 911, 1082, 1093 threshold level, xxii, 580 thresholds, 909, 910, 911 thrombocytopenia, 1022, 1025, 1040, 1043 thrombolytic therapy, 943, 1019, 1022 thrombosis, 288, 943, 966, 981, 1022 thymocytes, 501, 516, 530, 1024 thymus, 38, 133, 179 thyroid, 517, 529, 852, 860, 885, 887, 895, 1064 thyroid cancer, 887 thyroiditis, 860 time, x, xii, xix, 2, 3, 16, 19, 21, 24, 25, 28, 31, 41, 47, 74, 96, 119, 127, 137, 149, 223, 243, 276, 277, 280, 287, 310, 312, 323, 324, 325, 326, 332, 345, 356, 368, 382, 404, 432, 444, 502, 518, 520, 525, 540, 543, 545, 582, 586,
Index 588, 601, 629, 648, 652, 656, 658, 688, 745, 768, 774, 776, 778, 808, 816, 824, 837, 841, 842, 851, 853, 856, 857, 869, 905, 909, 931, 932, 933, 934, 943, 944, 968, 993, 996, 997, 1002, 1008, 1019, 1022, 1024, 1025, 1046, 1047, 1048, 1084, 1091, 1097 timing, 160, 276, 285, 286, 557, 641, 719, 830, 998 tin, 1042, 1045 tissue, xx, xxix, xxx, xxxv, 13, 16, 23, 104, 105, 129, 173, 189, 289, 302, 315, 330, 355, 374, 375, 376, 383, 385, 387, 395, 423, 431, 465, 467, 495, 497, 498, 499, 501, 523, 526, 549, 587, 597, 606, 620, 672, 677, 692, 717, 718, 724, 729, 814, 818, 820, 825, 826, 835, 850, 854, 860, 864, 869, 870, 877, 880, 881, 882, 888, 904, 911, 922, 923, 937, 942, 943, 958, 966, 968, 984, 993, 994, 995, 996, 998, 1008, 1009, 1012, 1017, 1019, 1022, 1030, 1032, 1057, 1061, 1062, 1068, 1071, 1072, 1074, 1075, 1084, 1086, 1088 tissue plasminogen activator, 922, 943, 958, 968, 984, 993, 994, 995, 998, 1008, 1030, 1032 TMC, 965, 971, 985 TNF, xxxv, 75, 81, 82, 90, 308, 311, 374, 384, 385, 386, 391, 449, 534, 652, 662, 679, 716, 719, 725, 834, 839, 841, 842, 864, 868, 873, 895, 938, 941, 957, 962, 967, 973, 976, 990, 995, 1001, 1012, 1014, 1017, 1057, 1078, 1091 TNF-alpha, 534 TNF-α, xxxv, 81, 308, 311, 652, 662, 679, 719, 967, 973, 976, 990, 1001 tolls, 292 tonic, 902, 905 tonic-clonic seizures, 905 topology, 78, 120, 287, 294, 826, 831 toxic effect, xxix, 10, 319, 462, 521, 558, 603, 687, 744, 752, 799, 813, 820, 824, 825, 967, 972 toxic substances, 614 toxicity, xiv, xix, xxviii, xxix, 10, 12, 85, 89, 195, 207, 216, 224, 258, 269, 273, 304, 317, 318, 319, 322, 335, 336, 337, 338, 339, 340, 429, 444, 462, 468, 469, 472, 474, 475, 476, 487, 492, 493, 506, 516, 517, 525, 528, 543, 549, 564, 581, 605, 607, 609, 611, 613, 615, 618, 620, 635, 673, 674, 677, 678, 681, 682, 684, 687, 688, 689, 691, 692, 695, 696, 697, 699, 700, 704, 708, 710, 711, 716, 726, 734,
1153 743, 747, 768, 770, 786, 794, 795, 797, 800, 801, 807, 808, 810, 812, 813, 820, 832, 931, 934, 935, 946, 956, 997, 1019, 1022, 1038, 1040, 1041, 1045, 1049 toxin, 187, 204, 281, 298, 568, 609, 610, 623, 720, 770, 966, 1035 traffic, 50, 251, 642, 785 training, 381, 427, 913 traits, 247, 260, 1067 transcranial magnetic stimulation, 509 transcription, x, xi, xvii, xix, xxii, xxviii, xxx, xxxi, 3, 15, 17, 28, 31, 38, 42, 48, 57, 58, 62, 68, 71, 81, 82, 94, 100, 102, 106, 112, 153, 171, 180, 183, 190, 248, 250, 264, 272, 284, 285, 286, 289, 299, 301, 318, 322, 339, 345, 360, 361, 373, 378, 384, 386, 389, 391, 406, 414, 424, 427, 431, 439, 443, 446, 449, 451, 454, 456, 457, 458, 467, 483, 484, 485, 486, 487, 498, 510, 517, 518, 523, 538, 540, 543, 544, 545, 546, 547, 548, 550, 551, 552, 560, 561, 579, 580, 582, 586, 590, 591, 592, 593, 594, 595, 598, 636, 638, 658, 661, 670, 678, 684, 689, 691, 699, 706, 710, 719, 729, 767, 769, 770, 771, 793, 795, 797, 806, 808, 823, 825, 834, 837, 840, 842, 843, 844, 846, 847, 863, 864, 866, 867, 868, 878, 879, 881, 882, 885, 894, 922, 924, 927, 928, 930, 937, 938, 945, 949, 957, 959, 994, 999, 1001, 1013, 1014, 1015, 1039, 1059, 1060, 1069, 1071, 1072, 1073, 1078, 1079, 1087, 1092, 1095 transcription factors, x, xxviii, xxx, 15, 28, 48, 58, 62, 68, 180, 248, 250, 301, 318, 322, 345, 406, 424, 427, 454, 456, 483, 484, 485, 498, 518, 546, 550, 552, 586, 590, 591, 594, 598, 670, 678, 684, 689, 691, 729, 767, 771, 793, 795, 797, 825, 834, 840, 843, 878, 879, 924, 928, 937, 994, 1013, 1014, 1059, 1060, 1069, 1072, 1073, 1079 transcriptional upregulation, 286 transcripts, 11, 86, 580, 586 transducer, 285, 389, 460, 834, 960 transduction, x, xi, xx, 2, 3, 17, 31, 42, 48, 56, 62, 64, 68, 71, 72, 81, 161, 198, 378, 384, 428, 445, 449, 452, 454, 455, 537, 596, 597, 664, 791, 827, 872, 933 transection, 639, 994, 1067 transfection, 223, 282, 501, 586, 748, 795 transfer RNA, 272, 286
1154 transformation, 83, 181, 290, 305, 483, 492, 818, 819, 864, 866, 888, 893, 941, 995, 996, 1019, 1068, 1087 transformations, 348 transforming growth factor, 360, 888, 1060 transgene, 319, 430, 511, 631, 645, 688 transition, 19, 134, 161, 162, 164, 166, 167, 191, 215, 480, 528, 535, 551, 679, 747, 818, 865, 878, 879, 880, 881, 896, 931, 952, 965, 1033, 1062, 1092 transition metal, 679 transition metal ions, 679 transitions, ix, 1, 163, 165, 362, 524, 993 translation, xv, 42, 94, 103, 153, 171, 180, 201, 260, 272, 285, 301, 378, 380, 414, 446, 449, 451, 454, 474, 517, 538, 540, 556, 751, 837, 844, 869, 932, 1057, 1089 translocation, xii, xxix, 52, 59, 64, 79, 80, 137, 144, 145, 148, 149, 150, 164, 181, 184, 208, 210, 212, 220, 232, 237, 238, 249, 252, 265, 266, 273, 278, 291, 292, 293, 294, 295, 310, 326, 340, 384, 408, 425, 448, 449, 451, 507, 525, 548, 550, 556, 587, 657, 661, 675, 723, 724, 772, 774, 785, 814, 816, 817, 823, 824, 832, 865, 868, 927, 937, 975, 978, 999, 1008, 1088 transmembrane region, 252, 816 transmission, 55, 85, 98, 104, 375, 378, 379, 385, 386, 387, 412, 438, 504, 653, 657, 659, 667, 669, 672, 766, 767, 769, 771, 773, 830, 937 transplantation, 835, 844, 972 transport, xiv, xv, xvi, xxiii, xxvi, 3, 7, 11, 21, 24, 66, 91, 170, 175, 191, 202, 204, 222, 224, 231, 232, 239, 241, 251, 252, 254, 260, 264, 265, 266, 273, 274, 282, 286, 291, 295, 298, 299, 303, 308, 314, 319, 321, 323, 324, 326, 327, 328, 329, 332, 336, 337, 352, 380, 385, 388, 434, 448, 449, 450, 451, 482, 532, 551, 563, 611, 628, 629, 633, 637, 638, 639, 641, 645, 680, 694, 701, 707, 708, 735, 739, 740, 767, 769, 782, 784, 814, 825, 831, 836, 840, 841, 923, 927, 936, 948, 1024, 1048, 1056, 1059, 1066, 1073, 1074, 1076, 1086, 1087, 1090, 1092 transport processes, xiv, 170 transportation, xvi, 10, 307, 314, 322, 329, 769, 933 trauma, xxvi, xxxii, xxxiii, 447, 650, 653, 716, 851, 921, 1007, 1091
Index traumatic brain injury, 526, 635, 645, 946, 992, 999, 1008 trees, 181 tremor, 85, 763, 966, 1081, 1082 trend, 189 trial, xxxvi, 381, 510, 889, 982, 993, 1012, 1019, 1025, 1028, 1050, 1051, 1052, 1053 tricarboxylic acid, 469 tricarboxylic acid cycle, 469 tricyclic antidepressant, 502, 653 tricyclic antidepressants, 653 triggers, 8, 59, 92, 103, 122, 145, 162, 163, 278, 283, 289, 302, 378, 452, 466, 540, 575, 586, 639, 658, 684, 707, 759, 882, 886, 887, 908, 937, 939, 952, 1014, 1017 trimer, 456, 545, 550, 551 trisomy, 765 trisomy 21, 765 trypsin, xii, 43, 74, 117, 118, 123, 132, 153, 167, 170, 172, 223, 249, 333, 384, 519, 605, 869, 870, 922, 930, 940, 962, 963, 1012, 1016, 1055, 1091 tryptophan, 48, 314, 345, 348, 349, 900, 1069, 1096 tumor, xi, xviii, xxxi, xxxvi, 31, 35, 54, 71, 74, 75, 80, 81, 90, 102, 105, 112, 113, 178, 215, 223, 234, 236, 250, 264, 267, 279, 296, 305, 308, 311, 394, 399, 407, 408, 455, 465, 488, 592, 716, 725, 834, 841, 859, 864, 872, 873, 875, 876, 877, 879, 882, 883, 884, 886, 887, 888, 889, 893, 895, 902, 904, 911, 953, 962, 964, 967, 968, 970, 972, 976, 978, 979, 985, 986, 1016, 1017, 1021, 1028, 1029, 1032, 1034, 1037, 1039, 1040, 1042, 1043, 1044, 1051, 1058, 1062, 1067, 1075, 1078, 1080, 1085, 1088, 1089, 1092, 1093 tumor cells, 234, 859, 884, 889, 895, 967, 1016, 1022, 1032, 1092 tumor growth, 972, 976 tumor necrosis factor, 81, 90, 308, 311, 716, 725, 834, 864, 872, 873, 962, 967, 1029, 1078, 1089 tumor progression, 886, 887 tumorigenesis, xxxi, 66, 780, 869, 875, 877, 879, 884, 887, 888, 893, 992, 1056, 1057 tumors, xviii, xxxi, xxxv, 12, 394, 399, 407, 547, 875, 877, 879, 881, 882, 883, 884, 887, 888, 889, 891, 898, 927, 967, 975, 979, 1016, 1043, 1052, 1061, 1064, 1068, 1075 tumour growth, 851
Index tumour suppressor genes, 866 tumours, 279, 297, 851, 857, 868, 871, 872, 890, 891, 892, 893, 894 turnover, ix, xvii, xviii, xxiii, xxxi, xxxiii, 2, 3, 16, 23, 39, 57, 67, 84, 102, 128, 154, 181, 191, 199, 209, 212, 213, 216, 222, 231, 250, 265, 279, 296, 339, 351, 357, 371, 373, 378, 394, 400, 402, 403, 404, 406, 407, 413, 420, 427, 432, 438, 449, 458, 465, 489, 496, 500, 503, 625, 627, 628, 633, 682, 684, 686, 694, 708, 709, 776, 831, 836, 863, 885, 921, 927, 932, 944, 990, 1015, 1024, 1054 twins, 763, 778, 899, 914 two-state model, 126, 244 type 1 diabetes, 860 tyramine, 786 tyrosine, xxxii, 16, 24, 35, 37, 48, 63, 83, 230, 232, 300, 417, 426, 472, 483, 517, 605, 665, 725, 760, 773, 788, 876, 886, 887, 917, 922, 1012, 1066, 1067, 1070, 1071, 1083, 1089, 1091 Tyrosine, 313, 607, 736, 741, 1014 tyrosine hydroxylase, 472, 517, 605
U Ubiquitin Carboxyl-terminal Hydroxylase L1 (UCH-L1), xxvii, 761 ubiquitin-proteasome system (UPS), ix, xxi, xxxi, 3, 553, 850, 863, 864 UG, 981 UK, 236, 363, 390, 483, 631, 651, 873, 875 ultraviolet irradiation, 530 uncertainty, 229, 673 underlying mechanisms, x, xxx, 24, 31, 42, 581, 834 Unfolded Protein Response (UPR), xxvii, 283, 762 uniform, 139, 230, 376, 377, 448 United Kingdom, 627, 735 United States, 14, 993, 1053 universality, 701 unmasking, 92, 575, 665 untranslated regions, 837 updating, 13 urine, 16, 246 UV, 96, 449, 522, 815, 893 UV radiation, 522 UV-radiation, 96, 815
1155
V vacuole, 21, 31, 33, 83, 682, 774, 778, 779, 780, 781, 783, 784 vagus, 601, 609 validity, 562, 608 values, 213, 311, 324, 656, 910, 940, 968, 976, 1025, 1039, 1045 variability, 97, 151, 229, 603, 899, 1043 variable, xiv, 44, 63, 227, 228, 229, 333, 619, 653, 719, 754, 938, 1009, 1015, 1059, 1082 variation, 260, 770, 788, 821, 853, 1016 vascular cell adhesion molecule, 662, 967 vascular endothelial growth factor (VEGF), 927, 967 vascular occlusion, 932, 993 vasculature, 349, 523, 945 vasodilation, 927 VAT, 145, 217, 224 VCAM, 941, 967, 973, 1012, 1014 vector, 440, 544, 823 VEGF, 922 vehicles, 976 vein, 361, 499, 788 Velcade, xxxiv, xxxv, xxxvi, 12, 13, 305, 516, 517, 525, 634, 641, 644, 776, 889, 940, 943, 958, 961, 966, 982, 990, 1011, 1016, 1021, 1025, 1031, 1032, 1037, 1038, 1051, 1053 velo-cardio-facial syndrome, 520 venlafaxine, 502, 508 ventricle, 516 versatility, 452, 701 vertebrates, 268, 312, 346, 349, 355, 357, 358, 380, 457, 1074 vesicle, 5, 20, 59, 101, 111, 151, 182, 203, 291, 362, 378, 389, 398, 412, 413, 421, 424, 426, 428, 435, 438, 440, 492, 573, 604, 619, 659, 661, 711, 765, 766, 774, 775, 781, 782, 783 vessels, 375, 382, 526, 1029, 1067 victims, 993 vimentin, 98, 110, 237, 254, 257, 822, 824 vincristine, 663, 670, 1045 viral infection, xxviii, xxix, 63, 106, 382, 653, 813, 833 viruses, 59, 87, 128, 180, 242, 257, 836, 842, 978, 1088 vision, xvii, 121, 291, 374 visual system, 350, 365, 645 visualization, 871 visuospatial function, 1083
Index
1156 vitamin D, 885, 889 vomiting, 1025 vulnerability, 584, 595, 605, 722, 730, 785, 811, 828, 829, 850, 857, 983
W walking, 358 Wallerian degeneration, xviii, xxiii, 191, 345, 358, 393, 398, 407, 516, 627, 628, 629, 633, 636, 637, 638, 639, 640, 641, 645, 646, 647, 648, 649, 650, 933, 952, 994, 1096 waste disposal, 192, 1027 weakness, 401, 1025, 1042, 1097 wealth, 274, 911, 992 wear, 16 weight gain, 995, 1083 weight loss, 1019 wheat, 174, 448 white matter, 375, 510, 635, 645, 649, 1073, 1088 wild type, xviii, 61, 187, 251, 256, 287, 386, 394, 399, 402, 403, 454, 473, 648, 743, 746, 866, 867, 1000, 1093 wild type p53, 867 wild-type allele, 763 wind, 658, 665 winning, 664 withdrawal, 407, 523, 647, 649, 652, 656, 657, 659, 725, 957, 1082 women, 510 workers, 22, 28, 29, 76, 96, 186, 320, 501, 688, 967, 970, 971, 972, 978 worms, 474 writing, 1083 WW domains, 348, 900 WWW, 332
X X chromosome, 1082 xenobiotics, 718 xenografts, 483, 1034 xeroderma pigmentosum, 108 X-ray analysis, 452 X-ray diffraction, 123, 698
Y yang, 944 yeast, xiii, xv, xxxv, 21, 27, 32, 39, 44, 48, 50, 51, 53, 58, 60, 61, 62, 63, 67, 68, 70, 72, 74, 75, 76, 77, 79, 80, 82, 83, 85, 87, 89, 90, 91, 96, 97, 98, 104, 108, 109, 110, 113, 114, 120, 122, 123, 129, 131, 140, 141, 144, 147, 149, 152, 153, 155, 157, 159, 160, 161, 165, 167, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 193, 194, 196, 200, 201, 204, 211, 214, 216, 221, 222, 223, 224, 230, 232, 233, 236, 237, 238, 247, 251, 252, 261, 272, 274, 275, 276, 278, 279, 280, 282, 285, 290, 292, 293, 294, 295, 296, 297, 298, 300, 309, 310, 312, 314, 329, 332, 340, 350, 356, 357, 359, 360, 397, 429, 449, 454, 457, 463, 474, 476, 477, 485, 486, 490, 520, 532, 582, 584, 595, 618, 632, 639, 647, 682, 693, 766, 774, 775, 779, 780, 781, 782, 783, 784, 785, 790, 822, 832, 904, 954, 967, 971, 1011, 1070, 1096, 1097, 1101 yield, 47, 149, 310, 816, 904, 911, 913 yin, 944
Z zinc, 75, 77, 78, 79, 82, 89, 90, 296, 367, 498, 535, 584, 589, 770, 900, 1059, 1060, 1078, 1079, 1088, 1094 zippers, 699