Recent Advances in Transthyretin Evolution, Structure and Biological Functions

Recent Advances in Transthyretin Evolution, Structure and Biological Functions Samantha J. Richardson l Vivian Cody...

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Recent Advances in Transthyretin Evolution, Structure and Biological Functions

Samantha J. Richardson

l

Vivian Cody

Recent Advances in Transthyretin Evolution, Structure and Biological Functions

Dr. Samantha J. Richardson RMIT University School of Medical Sciences Bundoora VIC 3083 Bundoora West Campus Australia [email protected]

Dr. Vivian Cody Hauptman-Woodward Medical Research Institute 700 Elliott Street Buffalo NY 14203 USA [email protected]

ISBN 978-3-642-00645-6 e-ISBN 978-3-642-00646-3 DOI: 10.1007/978-3-642-00646-3 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009926025 # Springer-Verlag Berlin Heidelberg 2009 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Since its first description in 1942 in both serum and cerebrospinal fluid, transthyretin (TTR) has had an eventful history, including changes in name from “prealbumin” to “thyroxine-binding prealbumin” to “transthyretin” as knowledge increased about its functions. TTR is synthesised in a wide range of tissues in humans and other eutherian mammals: the liver, choroid plexus (blood- cerebrospinal fluid barrier), retinal pigment epithelium of the eye, pancreas, intestine and meninges. However, its sites of synthesis are more restricted in other vertebrates. This implies that the number of tissues synthesising TTR during vertebrate evolution has increased, and raises questions about the selection pressures governing TTR synthesis. TTR is most widely known as a distributor of thyroid hormones. In addition, TTR binds retinol-binding protein, which binds retinol. In this way, TTR is also involved with retinoid distribution. More recently, TTR has been demonstrated to bind a wide variety of endocrine disruptors including drugs, pollutants, industrial compounds, heavy metals, and some naturally occurring plant flavonoids. These not only interfere with thyroid hormone delivery in the body, but also transport such endocrine disruptors into the brain, where they have the potential to accumulate. The X-ray crystal structure of TTR from vertebrates (fish, chicken, rat, mouse and human) has not changed in its overall structure. Despite this, TTRs in fish, amphibians, reptiles and birds bind T3 with higher affinity than T4, whereas in mammals, while maintaining the same three-dimensional structure in the binding site, TTR binds T4 with higher affinity than T3. The high conservation of quaternary, tertiary, secondary and primary structure, in combination with the sequencing of many non-vertebrate genomes, has allowed the identification of genes coding for TTR-Like Proteins (TLPs) in microbes, plants and non-vertebrate animals. TLPs studied to date do not bind thyroid hormones, but have 5-hydroxyisourate hydrolase activity. The change in function from 5-HIUase in bacteria to distributor of T3 in fish, amphibians, reptiles and birds, to distributor of T4 in mammals, yet maintaining the same overall three-dimensional structure, renders TLP/TTR an excellent model for the study of protein evolution. The function of TTR is still considered controversial by some researchers, as TTR null mice were originally reported to be without an overt phenotype. There exists the possibility that the lack of phenotype is due to redundancies in thyroid v

vi

Preface

hormone and retinoid metabolism, and also that mice living in animal houses in laboratories are not subjected to the same stresses as those in the wild; thus the phenotype is not seen under laboratory conditions. Intriguingly, there are no reports of humans lacking TTR. However, more recent studies have revealed a variety of phenotypes in TTR null mice, shedding light on previously unknown roles of TTR in development and neurobiology. TTR is also widely implicated in human health and disease. TTR is used as a nutritional marker, and also as an indicator of recovery following some diseases and surgery. Perhaps the most prolific area of TTR research concerns human TTR amyloid formation. There are two main types of TTR amyloid: familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited mutation in the TTR gene causing polyneuropathy, whereas senile systemic amyloidosis (SSA; also known as senile cardiac amyloidosis, SCA) is age-dependent and the amyloid fibrils contain wild type TTR. Of the 127 amino acids in the polypeptide, there are at least 100 point mutations that result in FAP. Therefore, there is an urgent need for continued research into the mechanisms of TTR amyloid formation, and for the development of therapeutics including drugs, gene therapy and organ transplants. Thus, progress in medical research into TTR is fundamental to human health. TTR is a fascinating protein from the stand point of protein evolution, and also in medicine. Thus, this is an exciting time for experts in TTR research to come together to write this monograph that covers both the basic and the clinical research in TTR. This monograph describes each of the above-mentioned aspects of TTR and brings the reader up to date on the latest developments and discoveries.

March 2009

Samantha J. Richardson Vivian Cody

Contents

1

2

Mechanisms of Molecular Recognition: Structural Characteristics of Transthyretin Ligand Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vivian Cody and Andrzej Wojtczak

1

Transthyretin Synthesis During Development and Evolution: What the Marsupials Revealed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samantha J. Richardson

23

3

Evolution of Transthyretin Gene Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . Porntip Prapunpoj

45

4

Evolutionary Insights from Fish Transthyretin . . . . . . . . . . . . . . . . . . . . . . . Deborah M. Power, Isabel Morgado, and Joaõ C.R. Cardoso

59

5

The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sarah Hennebry

6

Vertebrate 5-Hydroxyisourate Hydrolase Identification, Function, Structure, and Evolutionary Relationship with Transthyretin . . . . . . Giuseppe Zanotti, Ileana Ramazzina, Laura Cendron, Claudia Folli, Riccardo Percudani, and Rodolfo Berni

77

95

7

Transthyretin-Related and Transthyretin-like Proteins . . . . . . . . . . . . . . 109 A. Elisabeth Sauer-Eriksson, Anna Linusson, and Erik Lundberg

8

The Transthyretin–Retinol-Binding Protein Complex . . . . . . . . . . . . . . . 123 Hugo L. Monaco

9

Transthyretin and Retinol-Binding Protein: Implications in Fish Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Sancia Gaetani and Diana Bellovino

vii

viii

Contents

10

Transthyretin and Endocrine Disruptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Kiyoshi Yamauchi and Akinori Ishihara

11

Genetics: Clinical Implications of Transthyretin Amyloidosis . . . . . . . 173 Merrill D. Benson

12

Molecular Pathogenesis Associated with Familial Amyloidotic Polyneuropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Maria Joaõ Saraiva

13

Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Klaus Altland and Samantha J. Richardson

14

New Therapeutic Approaches for Familial Amyloidotic Polyneuropathy (FAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Yukio Ando, Masaaki Nakamura, Mistuharu Ueda, and Hirofumi Jono

15

Liver Transplantation for Transthyretin Amyloidosis . . . . . . . . . . . . . . . 239 Bo-Goran Ericzon, Erik Lundgren, and Ole B. Suhr

16

Mouse Models of Transthyretin Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . 261 Sadahiro Itoand Shuichiro Maeda

17

What Have We Learned from Transthyretin-Null Mice: Novel Functions for Transthyretin? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Joaõ Carlos Sousa and Joana Almeida Palha

18

Transthyretin Null Mice: Developmental Phenotypes . . . . . . . . . . . . . . . . 297 Julie A. Monk and Samantha J. Richardson

19

Transthyretin Null Mice as a Model to Study the Involvement of Transthyretin in Neurobiology: From Neuropeptide Processing to Nerve Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 Carolina Estima Fleming, Ana Filipa Nunes, Ma´rcia Almeida Liz, and Mońica Mendes Sousa

20

Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Yves Ingenbleek

Index

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Contributors

Klaus Altland Justus-Liebig-University, Institute of Human Genetics Schlangenzahl 14, 35392 Giessen, Germany [email protected] Yukio Ando Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan [email protected] Diana Bellovino National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy [email protected] Merrill D. Benson Indiana University School of Medicine, 635 Barnhill Drive, MS-128, Indianapolis, IN 46202-5126, USA [email protected] Rodolfo Berni Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy [email protected] Joaõ C.R. Cardoso Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005139 Faro, Portugal [email protected] Laura Cendron Department of Chemistry, University of Padua, and ICB-CNR, Section of Padua, Via Marzolo 1, 35131 Padova, Italy and Venetian Institute of Molecular Medicine, Via Orus 2, 35127 Padua, Italy [email protected] Vivian Cody Structural Biology Department, Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA

ix

x

Contributors

Bo-Goran Ericzon Head Division of Transplantation Surgery, CLINTEC, Karolinska Institutet, Karolinska University Hospital Huddinge, F82, 141 86 Stockholm, Sweden [email protected] Carolina Estima Fleming Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal [email protected] Claudia Folli Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy [email protected] Sancia Gaetani National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy [email protected] Sarah Hennebry Human Neurotransmitters Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia [email protected] Yves Ingenbleek Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur, Strasbourg 1, France [email protected] and 15 bis, rue de la Vise, 34540 Balaruc-le-Vieux, France [email protected] Akinori Ishihara Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan [email protected] Sadahiro Ito Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan and Center for Life Science Research, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan [email protected] Hirofumi Jono Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan [email protected]

Contributors

xi

Anna Linusson Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden Ma´rcia Almeida Liz Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal [email protected] Erik Lundgren Department of Molecular Biology, Umea˚ University, 901 87 Umea˚, Sweden [email protected] and Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden Shuichiro Maeda Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan [email protected] Hugo L. Monaco Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy [email protected] Julie A. Monk ProScribe Medical Communications, 481 Gilbert Rd., Preston, VIC 3072, Australia [email protected], url: www.proscribe.com.au Isabel Morgado Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005139 Faro, Portugal and Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany [email protected] Masaaki Nakamura Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan [email protected] Ana Filipa Nunes Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal [email protected]

xii

Contributors

Joana Almeida Palha Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal [email protected] Riccardo Percudani Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy [email protected] Deborah M. Power Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005-139 Faro, Portugal [email protected] Porntip Prapunpoj Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand [email protected] Ileana Ramazzina Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy [email protected] Samantha J. Richardson School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora, 3083 VIC, Australia [email protected] Maria Joaõ Saraiva Molecular Neurobiology Group, IBMC – Instituto de Biologia Molecular e Celular, R Campo Alegre 823, 4150 Porto, Portugal [email protected] and ICBAS – Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Porto, Portugal A. Elisabeth Sauer-Eriksson Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden [email protected] Joaõ Carlos Sousa Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal [email protected] Mońica Mendes Sousa Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal [email protected]

Contributors

xiii

Ole B. Suhr Department of Medicine, Section for Gastroenterology and Hepatology, Umea˚ University Hospital, 901 85 Umea˚, Sweden [email protected] Mistuharu Ueda Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan [email protected] Andrzej Wojtczak Chemistry Department, N. Copernicus University, Torun, Poland [email protected] Kiyoshi Yamauchi Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan [email protected] Giuseppe Zanotti Department of Chemistry, University of Padua, and ICB-CNR, Section of Padua, Via Marzolo 1, 35131 Padova, Italy and Venetian Institute of Molecular Medicine, Via Orus 2, 35127 Padua, Italy [email protected]

Chapter 1

Mechanisms of Molecular Recognition: Structural Characteristics of Transthyretin Ligand Interactions Vivian Cody and Andrzej Wojtczak

Abstract Transthyretin (TTR) is a homotetrameric serum protein responsible for the transport through the general circulation of thyroid hormones and their metabolites, and also binds retinol-binding protein (RBP) that transports retinol. Structure-activity data show that many pharmacological agents can compete for the binding of thyroid hormone to TTR and that this competition can interfere with their normal physiological functions. This review surveys more than 100 TTR crystal structures currently (October 2008) reported in the Protein Data Bank, representing only five species of vertebrates. Despite the highly conserved nature of protein sequences, TTR can bind a wide range of compounds with a high degree of flexibility. TTR is composed of four identical monomeric subunits that assemble around a central channel such that the tetramer possesses molecular 222-symmetry with two hormone-binding sites per tetramer. At physiological conditions, only one domain is occupied; however, negative cooperativity in binding to the second domain causes the second hormone to bind with a lower affinity. To date, more than 100 variants at 67 amino acid sites of the 127 residues of the human TTR monomer have been implicated in amyloid fibril formation that gives rise to familial amyloid polyneuropathy (FAP). Although the mechanism underlying TTR tetramer stability is not well understood, it has been shown that tight binding ligands can reduce the propensity for amyloid fibril formation. Structural data of TTR ligand complexes provide new insight into the design of novel inhibitors that can stabilize specific mutants and thereby delay the onset of fibril formation. Keywords Transthyretin, Conformational flexibility, Active site topology, Proteinprotein interactions

V. Cody (*) Structural Biology Department, Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, New York 14203, USA

S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_1, # Springer‐Verlag Berlin Heidelberg 2009

1

2

V. Cody and A. Wojtczak

1.1

Introduction

Transthyretin (TTR) is one of the three thyroid hormone-binding proteins found in the blood of vertebrates (Robbins 1996; Cody 2005). Produced in the liver, it binds retinol-binding protein that carries retinol (vitamin A) and thyroxine (3,5,30 ,50 tetraiodo-L-thyronine, T4) and differs from thyroxine-binding globulin (TBG) and serum albumin (SA) that has several nonspecific hormone-binding sites. Human TTR is a 55 kDa homotetramer composed of four identical 127 residue subunits that form an extensive b-sheet structure. The homotetramer assembles to form an internal channel in which two T4 molecules are bound per tetramer (Fig. 1.1). Biochemical data revealed that in the general circulation only one site is filled and that there is negative cooperativity in the binding of the second T4 (Cheng et al. 1977). Structure activity data revealed that the thyroid hormones and their metabolites have different binding affinities for TTR, depending on their substituent patterns, that is, the number of iodine atoms present or the type of side chain (propionic or acetic acid) (Table 1.1) (Cody 1980). In addition, many pharmacologic agents and natural products, such as plant flavonoids, nonsteroidal analgesic drugs, and inotropic bipyridines, are strong competitors for T4 binding to TTR and have binding affinities greater than T4 (Robbins 1996; Cody 2002). Recent studies have shown that TTR is one of about 20 human proteins that form fibrils in vivo, and to date more than 100 point mutations in TTR have been associated with the formation of fibrils, highly organized filamentous structures that form by self assembly of aggregated polypeptides. The propensity of TTR variants to form fibrils is the basis of familial amyloidotic polyneuropathy (FAP), a fatal genetic disease that is characterized by specific organ damage that targets the

a

b

Fig. 1.1 (a) TTR assembles as a tetramer with four identical subunits that form a channel with two hormone binding sites. The tetramer is generated by a twofold symmetry axis along the channel with monomers A-A0 (green, violet) and B-B0 (Cyan, gold). (b) Independent monomers of TTR illustrating forward and reverse binding of T4 acetic acid (T4A, Fig. 1.2)

1 Mechanisms of Molecular Recognition

3

Table 1.1 Relative binding affinities and biologic potencies of selected thyroid hormone analogues (Cody 1980) Compound TGB (%) TTR (%) Membrane (%) Nuclear (%) Potencya (%) L-Thyroxine

100.0

D-Thyroxine

54.0

39.3 0.95

96.6

12.5

63.0

–

18.1 3.0

3.6 76.4 23.0 – 3.0 T4-propionic acid 1.7 100.0 30.3 – 0.25 T4-acetic acid L-T3 (3,5,30 -triiodo) 9.0 1.4 100.0 100.0 100.0 38.0 3.1 67.5 0.1 99% is bound to THDPs, this increase in TH concentration would require additional TH distribution capacity. This could be the selection pressure for the developmentally regulated hepatic synthesis of TTR in species where TTR is not synthesised by adult liver. In these species, albumin is the only THDP in adult blood. TTR has higher affinity for THs than for albumin, and would contribute to the distribution of elevated TH levels in blood during these crucial stages of TH-regulated development. Investigations into marsupials were fundamental in developing the hypothesis that an augmented THDP network is required at the time of elevated TH levels in blood during development: (1) Polyprotodont marsupials only synthesise TTR in the liver during development, probably during the elevated TH levels in blood (Richardson et al. 2005). (2) Diprotodont marsupials only synthesise TBG in liver when there are elevated levels of TH in the blood, which boosts their capacity for TH distribution (above that of albumin and TTR, which are synthesised in the liver throughout life) (Richardson et al. 2002, 2005).

2.4.3

Choroid Plexus

The choroid plexus has the highest concentration of TTR mRNA per gram tissue weight in the body. The adult rat choroid plexus has 4.4 mg TTR mRNA per gram wet weight tissue compared to only 0.39 mg TTR mRNA per gram wet weight liver (Schreiber et al. 1990). In chickens, the adult choroid plexus has 7.2 mg TTR mRNA per gram wet weight, while the liver has only 0.33 mg TTR mRNA per gram wet weight (Duan et al. 1991). The pattern of TTR synthesis by the choroid plexus during development differs between precocial (relatively independent soon after birth) and altricial (very dependent on their mothers after birth) animals. At the time of birth, the brain of precocial animals (e.g. sheep) is significantly further developed compared with the brains of altricial animals (e.g. rats). Therefore, it was of interest to compare TTR gene expression in the choroid plexus of altricial with precocial animals. In rats (altricial), TTR mRNA was detected in the choroid plexus primordia from E12.5 (i.e. 12.5 days post conception). The proportion of TTR mRNA in total RNA increased 40-fold from E12.5 until birth. The maximum of TTR mRNA as a proportion of total brain mRNA (about 140% level in adults) occurred 2 days before birth, just prior to the period of fastest brain growth. By 8 days after birth, TTR mRNA levels had decreased to adult levels (Fung et al. 1988).

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S.J. Richardson

In sheep (precocial), TTR mRNA was detected in choroid plexus from embryos just a couple of days after gestation. At E40 (40 days post conception), the proportion of TTR mRNA in total mRNA was 34% of that for adult choroid plexus. By E90, this had increased to 70% of the adult value and remained constant throughout the rest of gestation (Tu et al. 1990). In sheep (precocial), the maximal increase in brain weight occurred 50 days before birth (Tu et al. 1990), while in rats (altricial), the maximum increase in brain weight occurred about 9 days after birth (Fung et al. 1988). For both species, the choroid plexus had its maximal growth rate prior to that of the rest of the brain. The critical period of brain development that is dependent on the THs is earlier for precocial animals than for altricial animals (Fisher and Polk 1989). The maximum TTR mRNA level in the choroid plexus of sheep occurs during the first half of gestation, while in rats, it occurs 2 days prior to birth. If TTR is involved in transporting THs across the blood–CSF barrier into the brain and acts as a THDP in the CSF, in each of these species, the timing of the maximal expression of the TTR gene is highly appropriate. Given that (1) THs have a profound effects on brain development; (2) the blood– brain barrier starts to develop as soon as the first blood vessel grows into the brain (Saunders et al. 1999); (3) the choroid plexus develops more rapidly than other parts of the brain; (4) the choroid plexus has an important role in regulating the composition of the CSF; (5) the timing of maximal TTR mRNA in the choroid plexus of altricial and precocial animals is just prior to the maximal growth rate of the brain and (6) TTR null mice have reduced TH levels in the subventricular zone (Richardson et al. 2007), it would follow that the synthesis of TTR from the stage of the choroid plexus primordia would appear to have an important regulatory role in determining the delivery of THs to the brain.

2.4.4

Visceral Yolk Sac and Placenta

TTR synthesis has been documented in both the placenta and VYS of the rat. Levels of TTR mRNA in the VYS were higher than those in the fetal liver throughout gestation. As stated previously, TTR synthesised by the VYS is involved in the transfer of THs from the maternal circulation to fetus, and also provides TTR to the fetal circulation before the fetal liver has reached full TTR synthesis rates. TTRRBP secreted by the VYS facilitates retinol transport to the fetus. More recent papers have confirmed these findings, added quantitative data and implicated specific transcription factors in the regulation of TTR gene expression in rodents (see Richardson 2007). A recent study reported the synthesis of TTR by trophoblasts in 38–40 weeks gestation in human placentas (McKinnon et al. 2005). However, earlier time points, including those where sufficient TH levels are crucial for normal fetal development, particularly during first trimester, have not been reported.

2 TTR Synthesis During Development and Evolution: What the Marsupials Revealed

2.4.5

33

Other Tissues

Scant data are available on TTR or its mRNA levels in other tissues during development. TTR mRNA in the RPE of rats was first detected at E16 and increased gradually until E21. There was a surge in TTR mRNA levels in the RPE at P0 followed by an increase to adult levels by P7. This surge was suggested to be a consequence of an external stimulus response, such as incident light (Mizuno et al. 1992). TTR mRNA has been reported in human fetuses at 12 weeks gestation in the islets of Langerhans (Gray et al. 1985) and in mid-term fetuses in the pancreatic endocrine A-cells (Jacobsson 1989). At 20–23 weeks gestation, over-expression of the TTR gene in the intestine was reported in human trisomy 18 (Edward’s Syndrome) (Loughna et al. 1995). TTR mRNA was also detected at E9 in the rat foregut endoderm (Makover et al. 1989).

2.5

TTR Synthesis During Evolution (Adult Animals)

2.5.1

Liver

2.5.1.1

Albumin is the Oldest THDP in Vertebrates

For a comprehensive analysis, serum from adult individuals from about 150 species was analysed for the presence of THDPs. All species studied were found to have albumin, and in some species (e.g. fish, amphibians, reptiles and some mammals) albumin was the only THDP (e.g. Richardson et al. 1994; Schreiber and Richardson 1997). Therefore, it was concluded that albumin is the phylogenetically oldest THDP in adult vertebrates. 2.5.1.2

No Clear Phylogeny for TBG in Vertebrates

By contrast, TBG-like proteins were detected in serum from several eutherian, marsupial and monotreme species, but no clear phylogeny was apparent (Richardson et al. 1994). Furthermore, many of these TBG-like proteins were not unambiguously identified, and could feasibly include TBG, lipoproteins that bind 125I-T4, or other 125I-T4 binding proteins that migrate as globulins. 2.5.1.3

Hepatic TTR Synthesis in Adult Birds, Diprotodont Marsupials and Eutherians

Birds and eutherians had TTR in addition to albumin. An intriguing situation became apparent among the Australian marsupial species: some had albumin as their only THDP, while others had TTR in addition to albumin. Those that had TTR

34

S.J. Richardson

in their serum belonged to the Order Diprotodonta, while those without TTR in their serum belonged to the Order Polyprotodonta (Richardson et al. 1993, 1994; Fig. 2.1). As stated previously, this classification is not currently favoured by taxonomists, but we have used this classification because it fits exactly with the serum TTR data for the 67 species of adult Australian marsupials studied. Thus, it appears that hepatic TTR synthesis remains ‘turned on’ in the adult in the lineages leading to eutherians, birds and diprotodont marsupials, and is ‘turned off’ after development in the lineages leading to polyprotodont marsupials, reptiles, amphibians, fish and possibly monotremes. Although the hypothesis for hepatic TTR synthesis during development is related to the transient increased levels of THs in blood, there are two current hypotheses concerning possible selection pressures for maintaining hepatic TTR synthesis during adult life. 2.5.1.4

Hepatic TTR Synthesis and the Increase in Lipid Pool to Body Mass Ratio: Consider Marsupials

A study by Hulbert and Else (1989) compared many physiological parameters of an adult reptile and an adult eutherian of similar body mass. Amongst other data, they showed that the eutherian had larger sizes of internal organs (including liver, brain, kidney and heart, which are among the metabolically most active), and that these tissues contained about 50% more phospholipid than the corresponding tissues in the reptile. As THs are lipophilic and preferentially partition into the lipid phase rather than the aqueous phase (Dickson et al. 1987; Hillier 1970), the increase in the relative size of the lipid pool could have been a selection pressure for maintaining hepatic TTR synthesis during adult life of eutherians compared with reptiles (Richardson et al. 1994). As TTR has higher affinity for THs than albumin has, the presence of TTR in the blood would augment the pool of circulating THs, thereby counteracting the increased lipid pool (sink) into which THs could partition. Another example that supports this hypothesis is that of the Australian marsupials. The diprotodont marsupials have hepatic TTR synthesis as adults, but the polyprotodont marsupials do not. The digestive tracts of herbivorous (diprotodont) marsupials are longer than those of carnivorous (polyprotodont) marsupials (Hume 1982). The intestines have a substantial lipid content and are the extra-thyroidal tissue with the highest TH content (Nguyen et al. 1993), and it has been suggested that the THDPs may be responsible for the regulation of delivery of THs to the intestines (Distefano et al. 1993). Thus, the diprotodont marsupials with longer digestive tracts have TTR in their blood, while the polyprotodont marsupials with shorter intestines do not. 2.5.1.5

Hepatic TTR Synthesis and Homeothermy: Marsupials Fall between the Cracks

Most adult animals that synthesise TTR in their livers throughout life are homeothermic (maintain their body temperature at or near 37 C by metabolic means).


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TTR was found in serum from all studied species of birds and eutherians, which are known homeotherms. However, TTR was not detected in the serum from adult fish, amphibians, reptiles, which are ectotherms (body temperature is determined by behaviour and the environment) (see Richardson 2007; Fig. 2.1). As THs are involved in the regulation of oxygen consumption, basal metabolic rates and homeothermy, it is conceivable that there is a link between the increase in the size of the most metabolically active organs and increase in the TH distribution capacity in blood (TTR) and homeothermy. Thus, a selection pressure for maintaining hepatic TTR synthesis throughout life could have been to enable appropriate distribution of THs throughout the body to maintain homeothermy. Marsupials are ‘poor homeotherms’ and there has been no evidence that the presence of TTR in marsupial blood is correlated with better control of body temperature.

2.5.1.6

The TTR–RBP Complex: Marsupials Raise a Question

TTR binds many ligands, including naturally occurring compounds, pollutants and non-steroidal anti-inflammatory drugs (see chapter ‘TTR and Endocrine Disruptors’). The only protein ligand known to be bound by TTR is RBP, which itself binds retinol (Kanai et al. 1968). It was suggested that the TTR–RBP/retinol complex (80 kDa) or the retinol/RBP–TTR–RBP/retinol complex (100 kDa) prevented loss of RBP/retinol (21 kDa) through glomerular filtration of the kidneys (Raz and Goodman 1969). This hypothesis may hold true for eutherians, but it is not immediately convincing when considering adult marsupials. Diprotodonta have TTR in their blood while Polyprotodonta do not (Richardson et al. 1994). This raises the questions as to whether there is a difference in glomerular filtration size cut-off in diprotodont marsupials compared with that of polyprotodont marsupials, or if glomerular filtration has a lower molecular weight cut-off in marsupials than in eutherians, or if a plasma protein other than TTR binds to RBP in all marsupials or in Polyprotodonta.

2.5.2

Choroid Plexus

TTR synthesis was not detected in the choroid plexus from fish or amphibians, but was the major protein synthesised and secreted by the choroid plexus from reptiles, birds, monotremes, marsupials and eutherians (see Richardson 2005). It appears that the TTR gene in the choroid plexus was turned on once at the stage of the stemreptiles, the closest common ancestor to reptiles, birds and mammals, but not of amphibians and fish (see Fig. 2.1). The stem-reptiles were the first to develop traces of a cerebral neocortex (Kent 1987), resulting in an increase in brain volume. As THs are lipophilic and readily partition into cell membranes, the increase in brain volume may have been a selection pressure for ‘turning on’ the TTR gene in the choroid plexus. This resulted in TTR assisting movement of THs from the blood

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across the blood–CSF barrier into the brain, and also acting as a THDP in the CSF (Schreiber and Richardson 1997). It is most probable that the stem-reptiles had the TTR gene in their genomes, possibly expressed in the liver during development. A change in the specificity of transcription factors could have been all that was required to activate expression of the TTR gene in the choroid plexus. The major protein synthesised and secreted by the choroid plexus of juvenile and adult amphibians is the lipocalin prostaglandin D synthetase (PGDS) (Achen et al. 1992), also known as beta-trace (Beuckmann et al. 1999) and Cpl1 (Lepperdinger 2000). PGDS is a monomeric 20 kDa protein that belongs to the lipocalin superfamily. Lipocalins have a calyx (cup) structure and are specialized in binding small molecules (Godovac-Zimmermann 1988). This raises the question as to whether PGDS was the functional evolutionary precursor to TTR in the choroid plexus.

2.5.3

Other Tissues

The majority of data in this section is derived from the studies of eutherian mammals. Therefore, to obtain a more global picture, investigations in species from other classes of vertebrates in both juveniles and adults are required. In adult eutherian mammals, TTR synthesis has been reported in the placenta, RPE, CPE, intestine, pancreas and meninges (in addition to the liver and choroid plexus). In adult reptiles, TTR mRNA has been identified in the choroid plexus from adult animals of four species: stumpy-tailed lizards (Tiliqua rugosa) (Achen et al. 1993), red-eared slider turtle (Trachemys scripta), the common snapping turtle (Chelydra serpentine) (Richardson et al. 1997) and in whole eyes and choroid plexus of the salt-water crocodile (Crocodylus porosus) (Prapunpoj et al. 2002). One study of an adult fish species (Sparus aurata, sea bream) detected TTR mRNA by RT-PCR (i.e. higher levels of sensitivity compared to the above mentioned studies) in the liver, intestine, brain, skin, heart, skeletal muscle, kidney, testis, gills and pituitary (Santos and Power 1999). Thus, given the paucity of data from noneutherian species, a generalized picture of TTR synthesis in vertebrate’s tissues other than the liver and choroid plexus is not yet possible.

2.6

Evolution of TTR Structure and Function: Marsupials are a ‘Missing Link’

In earlier studies of 125I-T4 binding to TTRs from various species, it had been noted that chicken TTR appeared to have a lower affinity for T4 than human TTR did (S. Richardson, unpublished observations). However, the amino acids in the TH binding sites are identical in human and chicken TTRs (Duan et al. 1991). As the


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N-termini had been shown to be located in the vicinity of the opening to the THbinding channel of TTR (Hamilton et al. 1993), this raised the question as to whether the N-terminal regions of TTR might be affecting the entry of THs into the binding sites. The X-ray crystal structure of chicken TTR was determined (Sunde et al. 1996). It did not show diffraction for the N-terminal regions, implying that the four N-terminal regions were moving freely in solution. However, it did confirm that the TH binding sites were identical in chicken and human TTRs (Sunde et al. 1996). This was surprising, as a difference in binding affinity was expected to be due to a difference in ligand binding site. To further investigate the possibility that the N-terminal regions influenced TH affinity, TTRs were purified from several species of eutherians (short hydrophilic N-terminal regions), birds (long hydrophobic N-terminal regions) and marsupials (intermediate N-terminal regions). The literature revealed that, depending on the method used, Kd values for human TTR and T4 ranged from 0.3 to 128 nM (see Chang et al. 1999). Therefore, a precise, accurate and reproducible method for assaying Kd values of TTRs with THs was developed (Chang et al. 1999). As expected, eutherian TTRs bound T4 with higher affinity than T3. However, avian TTRs bound T3 with higher affinity than T4. Marsupial TTRs had intermediate affinities, but bound T4 with higher affinity than T3 (Chang et al. 1999). However, similar to avian TTRs, reptilian and amphibian TTRs were also found to bind T3 with higher affinity than T4 (Prapunpoj et al. 2002, 2000b; Yamauchi et al. 1998; Table 2.2). Piscine TTRs bind T3 with higher affinity than T4 (Santos and Power 1999; Yamauchi et al. 1999), but specific Kd values are yet to be determined. Thus, shorter hydrophilic N-terminal regions are correlated with higher

Table 2.2 Dissociation constants of T3 and T4 from TTRs from a variety of species Kd T3 (nM) Kd T3/Kd T4 Source of TTR Kd T4 (nM) Eutherians Human 13.6 56.6 4.2 Sheep 11.3 63.5 3.2 Rat 8.0 67.2 8.4 Marsupials Wombat 21.8 97.8 4.5 Possum 15.9 206.1 12.9 Wallaby 13.8 65.3 4.7 Birds Emu 37.4 18.9 0.51 Chicken 28.8 12.3 0.43 Pigeon 25.3 16.1 0.64 Reptile Crocodile 36.7 7.56 0.21 Amphibian Toad 508.0 248.0 0.49 Data are from Chang et al. 1999; Prapunpoj et al. 2000; Prapunpoj et al. 2002. From Richardson 2007

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affinity for T4 binding, and longer more hydrophobic N-terminal regions are correlated with higher affinity for T3 binding. Chimeric TTRs were synthesised, where N-terminal regions from one species were replaced with those from another species (e.g. human/crocodile; Xenopus/ crocodile). Kd values for T3 and T4 were determined (Prapunpoj et al. 2002, 2006). As expected, the N-termini altered the affinity and specificity of T3 and T4 binding to TTRs. More specifically, it appeared that the core of TTR had a major influence on determining the affinity of T3 and the N-terminal regions mainly affected the affinity of T4. (For further details, see chapter ‘Evolution of Transthyretin Gene Structure’.) These data supported the hypothesis that the character of the N-terminal regions of TTR affects the affinities for THs, and revealed that mammalian TTRs are the exception rather than the rule, in that they preferentially bind T4, while TTRs from fish, amphibians, reptiles and birds preferentially bind T3. Marsupial TTRs can be considered as a ‘missing link’ between avian/reptilian TTRs and eutherian TTRs in terms of structure and function. Marsupial TTR N-terminal regions are intermediate in structure (length and hydrophobicity) between those of avian/reptilian TTRs and eutherian TTRs, and provided one of the ‘steps’ in the step-wise change in the position of the intron 1/exon 2 splice site that caused the change in structure from longer and more hydrophobic to shorter and more hydrophilic (see chapter ‘Evolution of Transthyretin Gene Structure’). While marsupial TTRs bind T4 with higher affinity than T3 (similarly to other mammalian TTRs), they have lower affinity for T3 than eutherian, avian or reptilian TTRs and intermediate affinities for T4 between those of eutherian TTRs and avian and reptilian TTRs. This is consistent with the structure of the N-terminal regions of marsupial TTRs being intermediate between those of eutherian and avian/ reptilian TTRs, and the hypothesis that the N-terminal region affects affinity for T4.

2.7

Marsupial Models for Studying TTR Amyloid Formation

The evolutionary changes in TTR are clustered around the N-terminal regions, while the point mutations in human TTR that result in amyloidosis are evenly spread throughout the molecule (see Richardson 2007). In general, the amino acid residues that occur as point mutations in human TTR and result in amyloid formation are not found in other species. One notable exception is amino acid 68. In wild-type human TTR residue 68 is Ile, but if it is mutated to Leu, TTR amyloidosis results. However, Leu is the normal amino acid in this position in several marsupial TTRs (Schreiber and Richardson 1997). These marsupials are apparently without symptoms of amyloidosis. Elucidation of the 3D structure of a marsupial TTR with Leu68 could give us valuable insights into how Leu is accommodated at position 68 in a marsupial TTR, but not in a human TTR. A second example of a marsupial TTR that is of interest for the study of TTR amyloid formation is wallaby TTR. Human TTR is believed to dissociate from


39

tetramers into dimers, and from dimers into monomers before forming amyloid fibrils. All amyloidogenic mutations in human TTR destabilise the tetramer (Altland et al. 2007). Under specific conditions that cause the human TTR dimers to dissociate into monomers (Altland et al. 1999), wallaby TTR dimers remain intact (see chapter ‘Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity’). Again, structural data on key residues in wallaby TTR (e.g. His31) could give insight into the mechanism of TTR amyloid formation.

2.8

Conclusions

1. Taking together the data for TTR synthesis in the liver and in the choroid plexus, during both development and evolution, we arrive at the following profile (Fig. 2.1): (a) TTR synthesis only in the liver, only during development: fish, amphibians Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood. (b)TTR synthesis in the liver during development and in the choroid plexus throughout life: reptiles, polyprotodont marsupials and probably monotremes Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood; and the increase in brain volume required TTRmediated transport of T4 across the choroid plexus and TTR as a THDP in the CSF. (c) TTR synthesis in the liver and in the choroid plexus throughout life: birds, diprotodont marsupials, eutherians Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood; increased lipid volume to body mass ratio and/or homeothermy required an additional THDP in blood; and the increase in brain volume required TTR-mediated transport of T4 across the choroid plexus and TTR as a THDP in the CSF. The complex patterns of tissue-specific TTR synthesis imply that TTR synthesis is tightly regulated during development and during evolution. It also implies a multi-factorial set of selection pressures governing its synthesis. 2. Marsupial TTRs are intermediate in structure and in function, between those of reptilian/avian TTRs and eutherian TTRs. They provide evidence of a step-wise shift in the evolution of TTR structure and function. TTR changed from distributing T3 in fish, amphibians, reptiles and birds to distributing T4 in mammals. A possibility for the selection pressure leading to

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this could be an additional level of regulation of TH gene expression by TH. Instead of the ‘active’ form of the hormone being distributed around the body and brain (i.e. T3), in mammals, TTR distributes the precursor form (i.e. T4). This could require tissue-specific or cell-specific regulation of activation by deiodinases from T3 to T4. This could give tighter control over regulation of TH-responsive gene expression. 3. Marsupials should be considered as models for studying TTR amyloid formation. Why Leu68 can be tolerated in marsupial TTRs but causes amyloid formation in human TTR is highly intriguing and should be investigated. Furthermore, features of wallaby TTR that render it more stable than human TTR under specific conditions should be elucidated.

References Achen, M. G., et al. (1993) Transthyretin gene expression in choroid plexus first evolved in reptiles. American Journal of Physiology 265:R982–R989 Achen, M. G., et al. (1992) Protein synthesis at the blood-brain barrier. The major protein secreted by amphibian choroid plexus is a lipocalin. Journal of Biological Chemistry 267:23170–23174 Alberts, B., et al. (2002) Molecular Biology of the Cell. Garland Science, New York Altland, K., et al. (2007) Genetic microheterogeneity of human transthyretin detected by IEF. Electrophoresis 28:2053–2064 Altland, K., et al. (1999) Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients. Electrophoresis 20:1349–1364 Beuckmann, C. T., et al. (1999) Binding of biliverdin, bilirubin, and thyroid hormones to lipocalintype prostaglandin D synthase. Biochemistry 38:8006–8013 Blay, P., et al. (1993) Transthyretin expression in the rat brain: effect of thyroid functional state and role in thyroxine transport. Brain Research 632:114–120 Bridges, C. D., et al. (1984) Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids. Vision Research 24:1581–1594 Cavallaro, T., et al. (1990) The retinal pigment epithelium is the unique site of transthyretin synthesis in the rat eye. Investigative Ophthalmology and Visual Science 31:497–501 Chang, L., et al. (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. European Journal of Biochemistry 259:534–542 Chanoine, J. P., et al. (1992) Role of transthyretin in the transport of thyroxine from the blood to the choroid plexus, the cerebrospinal fluid, and the brain. Endocrinology 130:933–938 Dickson, P. W., et al., (1985) High prealbumin and transferrin messenger RNA levels in the choroid plexus of rat brain. Biochemical and Biophysical Research Communications 127:890–895 Dickson, P. W., et al. (1987) Thyroxine transport in choroid plexus. Journal of Biological Chemistry 262:13907–13915 Distefano, J. J., et al., (1993) Transfer kinetics of 3,5,30 -triiodothyronine and thyroxine from rat blood to large and small intestines, liver, and kidneys in vivo. Endocrinology 132:1735–1744 Duan, W., et al., (1991) Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates. European Journal of Biochemistry 200:679–687 Fisher, D. A., Polk, D. H. (1989) Development of the thyroid. Bailliere’s Clinical Endocrinology and Metabolism 3:627–657


41

Fung, W. P., et al., (1988) Structure and expression of the rat transthyretin (prealbumin) gene. Journal of Biological Chemistry 263:480–488 Funkenstein, B., et al., (1999) Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution. Molecular and Cellular Endocrinology 157:67–73 Godovac-Zimmermann, J. (1988) The structural motif of beta-lactoglobulin and retinol-binding protein: a basic framework for binding and transport of small hydrophobic molecules? Trends in Biochemical Sciences 13:64–66 Gray, H. D., et al., (1985) Sites of prealbumin production in the human fetus using the indirect immunoperoxidase technique. Virchows Archive A: Pathological Anatomy and Histopathology 406:463–473 Hamilton, J. A., et al., (1993) The x-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30 ! Met variant to 1.7-A resolution. Journal of Biological Chemistry 268:2416–2424 Hillier, A. P. (1970). The binding of thyroid hormones to phospholipid membranes. Journal of Physiology 211:585–597 Hulbert, A. J., Else, P. L. (1989) Evolution of mammalian endothermic metabolism – mitochondrial activity and cell composition. American Journal of Physiology 256:R63–R69 Hume, I. D. (1982) Digestive Physiology and Nutrition of Marsupials. Cambridge University Press, Cambridge Jacobsson, B. (1989) In situ localization of transthyretin-mRNA in the adult human liver, choroid plexus and pancreatic islets and in endocrine tumours of the pancreas and gut. Histochemistry 91:299–304 Kanai, M., et al., (1968) Retinol-binding protein: the transport protein for vitamin A in human plasma. Journal of Clinical Investigation 47:2025–2044 Kato, M., et al., (1985) Plasma and cellular retinoid-binding proteins and transthyretin (prealbumin) are all localized in the islets of Langerhans in the rat. Proceedings of the National Academy of Sciences of the United States of America 82:2488–2492 Kawaji, T., et al., (2005) Transthyretin synthesis in rabbit ciliary pigment epithelium. Experimental Eye Research 81:306–312 Kent, G. C. (1987) Comparative anatomy of the vertebrates. Time Mirror/Mosby College, St Louis. Lemkine, G. F., et al. (2005) Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor. The FASEB Journal 19:863–865 Lepperdinger, G. (2000) Amphibian choroid plexus lipocalin, Cpl1. Biochimica et Biophysica Acta 1482:119–126 Loughna, S., et al., (1995) Molecular analysis of the expression of transthyretin in intestine and liver from trisomy 18 fetuses. Human Genetics 95:89–95 Makover, A., et al., (1989) An in situ-hybridization study of the localization of retinol-binding protein and transthyretin messenger RNAs during fetal development in the rat. Differentiation 40:17–25 McKinnon, B., et al., (2005) Synthesis of thyroid hormone binding proteins transthyretin and albumin by human trophoblast. Journal of Clinical Endocrinology and Metabolism 90:6714–6720 Mendel, C. M., et al., (1989) Thyroxine transport and distribution in Nagase Analbuminemic rats. Journal of Clinical Investigation 83:143–148 Mendel, C. M., et al., (1987) Thyroid hormone binding proteins in plasma facilitate uniform distribution of thyroxine within tissues – A perfused rat liver study. Endocrinology 120:1742– 1749 Meredith, R. W., et al., (2007) A phylogeny and time scale for marsupial evolution based on sequences for five nuclear genes. Journal of Mammalian Evolution 15:1–36 Mizuno, R., et al., (1992) Temporal expression of the transthyretin gene in the developing rat eye. Investigative Ophthalmology and Visual Science 33:341–349

42

S.J. Richardson

Nguyen, T. T., et al., (1993) Steady-state organ distribution and metabolism of thyroxine and 3,5,30 -triiodothyronine in intestines, liver, kidneys, blood, and residual carcass of the rat invivo. Endocrinology 133:2973–2983 Ong, D. E., et al., (1994) Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium. Biochemistry 33:1835–1842 Prapunpoj, P., et al., (2006) Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS Journal 273:4013–4023 Prapunpoj, P., et al., (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia. Molecular Biology and Evolution 17:1199–1209 Prapunpoj, P., et al., (2002) Crocodile transthyretin: structure, function, and evolution. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 283:R885–R896 Prapunpoj, P., et al., (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 279:R2026–R2041 Raz, A., Goodman, D. S. (1969) The interaction of thyroxine with human plasma prealbumin and with the prealbumin-retinol-binding protein complex. Journal of Biological Chemistry 244:3230–3237 Refai, E., et al., (2005) Transthyretin constitutes a functional component in pancreatic beta-cell stimulus-secretion coupling. Proceedings of the National Academy of Sciences of the United States of America 102:17020–17025 Richardson, S. J. (2005) Expression of transthyretin in the choroid plexus: Relationship to brain homeostasis of thyroid hormones. In: W. Zheng, A. Chodobski (eds.), The Blood-Cerebrospinal Fluid Barrier. CRC Press, Boca Raton, pp. 275–304 Richardson, S. J. (2007) Cell and molecular biology of transthyretin and thyroid hormones. International Review of Cytology 258:137–193 Richardson, S. J., et al., (2002) Developmental profile of thyroid hormone distributor proteins in a marsupial, the tammar wallaby Macropus eugenii. General and Comparative Endocrinology 125:92–103 Richardson, S. J., et al., (1993) The expression of the transthyretin gene in liver evolved during the radiation of diprotodont marsupials in Australia. General and Comparative Endocrinology 90:177–182 Richardson, S. J., et al., (1994) Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins. American Journal of Physiology 266:R1359–R1370 Richardson, S. J., et al., (1997) Abundant synthesis of transthyretin in the brain, but not in the liver, of turtles. Comparative Biochemistry and Physiology B – Biochemistry and Molecular Biology 117:421–429 Richardson, S. J., et al., (2007) Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice. Neuroscience Letters 421:234–238 Richardson, S. J., et al., (2005) Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 288:R1264–R1272 Sandler, B., et al., (2004) Thyroxine-thyroid hormone receptor interactions. Journal of Biological Chemistry 279:55801–55808 Santos, C. R. A., Power, D. M. (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140:2430–2433 Saunders, N. R., et al., (1989) Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development. Anatomy and Embryology (Berlin) 180:227–236 Saunders, N. R., et al., (1999) Barrier mechanisms in the brain, II. Immature brain. Clinical and Experimental Pharmacology and Physiology 26:85–91 Schreiber, G. (1987) Synthesis, processing, and secretion of plasma proteins by the liver and other organs and their regulation. In: F. W. Putnam (ed.), The Plasma Proteins. Academic Press, New York, pp. 293–363


43

Schreiber, G., et al., (1990) Thyroxine transport from blood to brain via transthyretin synthesis in choroid plexus. American Journal of Physiology 258:R338–R345 Schreiber, G., Richardson, S. J. (1997) The evolution of gene expression, structure and function of transthyretin. Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology 116:137–160 Setchell, P. J. (1974) Development of thermoregulation and thyroid function in marsupial Macropus eugenii (Desmarest). Comparative Biochemistry and Physiology 47:1115–1121 Sklan, D., Ross, A. C. (1987) Synthesis of retinol-binding protein and transthyretin in yolk sac and fetus in the rat. Journal of Nutrition 117:436–442 Soprano, D. R., et al., (1986) Retinol-binding protein and transthyretin mRNA levels in visceral yolk sac and liver during fetal development in the rat. Proceedings of the National Academy of Sciences of the United States of America 83:7330–7334 Southwell, B. R., et al., (1993) Thyroxine transport to the brain: role of protein synthesis by the choroid plexus. Endocrinology 133:2116–2126 Southwell, B. R., et al., (1991) Ontogenesis of transthyretin gene expression in chicken choroid plexus and liver. Comparative Biochemistry and Physiology B: Comparative Biochemistry 100:329–338 Stauder, A. J., et al., (1986) Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain. Journal of Histochemistry and Cytochemistry 34:949–952 Sunde, M., et al., (1996) The crystal structure of transthyretin from chicken. European Journal of Biochemistry 236:491–499 Talent, J. (1984) Australian biogeography past and present: determinants and implications. In: J. J. Veevers (ed.), Phanerozoic Earth History of Australia. Oxford University Press (Clarendon), London, pp. 57–93 Thomas, T., et al., (1990) Plasma protein synthesis and secretion in the visceral yolk sac of the fetal rat: gene expression, protein synthesis and secretion. Placenta 11:413–430 Tu, G. F., et al., (1990) Expression of the genes for transthyretin, cystatin C and beta A4 amyloid precursor protein in sheep choroid plexus during development. Brain Research Developmental Brain Research 55:203–208 Tyndale-Biscoe, H. (1973). Life of Marsupials. Arnold, London. Tyndale-Biscoe, H. C., Janssens, P. A. (1988) The Delevoping Marsupial: Models for Biomedical Research. Springer-Verlag, Berlin van Rheede, T., et al., (2006) The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians. Molecular Biology and Evolution 23:587–597 Woodburne, M. O., Zinsmeister, W. J. (1982) Fossil Land Mammal from Antarctica. Science 218:284–286 Yamauchi, K., et al., (1999). Purification and characterization of thyroid-hormone-binding protein from masu salmon serum – A homolog of higher-vertebrate transthyretin. European Journal of Biochemistry 265:944–949 Yamauchi, K., et al., (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA – comparison of its pattern of expression during metamorphosis with that of lipocalin. European Journal of Biochemistry 256:287–296 Yamauchi, K., et al., (1993) Purification and characterization of a 3,5,30 -L-triiodothyroninespecific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132, 2254–2261.

Chapter 3

Evolution of Transthyretin Gene Structure Porntip Prapunpoj

Abstract Transthyretin (TTR) is one of the three major thyroid hormone (TH)binding proteins in plasma and cerebrospinal fluid (CSF) of vertebrates. Binding of TTR to THs is necessary to counteract the loss of these hormones from the vascular and interstitial compartments, resulting from high lipid solubility and from the tendency to partition into and accumulate in the cell membranes. TTR and the other two components of the TH-binding protein system (albumin and thyroxine-binding globulin) function as a network. The loss of one of the proteins could be compensated by increased binding to the remaining plasma proteins. The gene structure of TTR at genomic and mRNA levels from many animals have been characterized over the 20 years since 1985. The structure of the TTR gene from three vertebrate species, that is, human, mouse, and rat, has been characterized, and the entire TTR genomic sequences are highly conserved among these species. They have two intronic open reading frames (ORFs) but neither is productively expressed as an independent polypeptide. The proximal and distal promoters with several regulatory elements necessary for transcription and tissue-specific expression of the TTR gene are located at the 50 -flanking region of the transcription initiation site. The polypeptide coding region of TTR has been studied for a variety of species from fish to human. TTR cDNA from up to 24 vertebrates have been isolated and analyzed to date. The nucleotide sequence and primary structure of TTR are highly conserved, in particular among eutherian, marsupial, bird, reptile, amphibian and fish species. All positions in the central channel, in particular, those involved in the binding interaction with THs have not been altered since at least 400 million years ago. However, mutations of TTR mostly occur in the 50 -terminal region of the TTR gene, which corresponds to the N-terminal segment of TTR polypeptide, during evolution of vertebrates. Shortening and change in hydropathy of the N-terminal regions of TTR occurred and have been demonstrated to influence the binding affinities to THs. The molecular mechanism underlying these phenomena is a series of single

P. Prapunpoj Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand e-mail: [email protected]


45

46

P. Prapunpoj

base mutations that leads to a stepwise progressive movement of the 30 splice site, but not the 50 splice site, of the TTR intron 1. It is apparent that a rare molecular mechanism of positive Neo-Darwinian evolution has occurred during the evolution of TTR gene. Keywords cDNA, Evolution, Gene, Structure, Transthyretin

3.1 3.1.1

Genomic Structure of the Transthyretin Gene General

The transthyretin (TTR) gene is located on different chromosomes in different species. While the TTR genes from human and rat are located on chromosome 18 (Wallace et al. 1985; Remmers et al. 1993), those from mouse are located at the C6-D1 region of chromosome 4 (Qiu et al. 1992) and those from Sus scrofa and Bos taurus are on chromosome 6 (Arachibald et al. 1996) and 24 (Larsen et al. 1996), respectively. The TTR genes are varied in length spanning from 6.9 kb (in human) to 7.3 kb (in rat), and are composed of four exons and three introns. Until now, the genomic structure of the TTR gene from only three vertebrate species, that is, human, mouse and rat, has been characterized by nucleotide sequences of the 50 and 30 flanking regions, intron–exon borders, and part of introns being sequenced. The entire sequences of all exons, the 50 proximal region, and the flanking region of the exon–intron borders are highly conserved between human and rodent TTR genes (Tsuzuki et al. 1985; Wakasugi et al. 1985, 1986). The 50 -flanking region of the human and mouse TTR genes and the 30 end region of the first intron of the mouse TTR gene contain sequences homologous to the glucocorticoid receptor (GCR)binding site sequences (Sasaki et al. 1985; Tsuzuki et al. 1985). Moreover, the latter TTR gene also contains an enhancer sequence that is present in the immunoglobulin kappa-chain joining-constant kappa intron (Wakasugi et al. 1986).

3.1.2

Genomic Structure of Human TTR

Each human TTR monomer is encoded by a single-copy gene (Sparkes et al. 1987) that was located by Southern hybridization to the long arm of chromosome 18 (Wallace et al. 1985), in particular at the 18p11.1-q12.3 region, and most likely at the cen-q12.3 (Jinno et al. 1986). The TTR gene spans about 6.9–7.0 kilobases and contains four exons and three introns (Sasaki et al. 1985; Tsuzuki et al. 1985). Exon 1 encodes 23 amino acid residues, which comprises 20 residues of a signal peptide and the first three residues of the mature protein; exons 2, 3, and 4 encode for 44, 45, and 35 amino acid residues, respectively, of the mature protein. The 50 -untranslated region (50 -UTR) (26 bases) and the 30 -untranslated region (30 -UTR) (139 bases) are located in exon 1 and exon 4, respectively. There are three introns of 934, 2,090,

3 Evolution of Transthyretin Gene Structure

47

and 3,308 bp, and the GT/AG splicing consensus sequence was identified at all exon/intron borders of the gene (Sasaki et al. 1985). Two independent ORFs, ORF1 and ORF2, with putative regulatory sequences for transcription in the same transcription direction as that of TTR locate in the first and the third intron of the TTR gene (Tsuzuki et al. 1985). The nucleotide sequence of ORF1 is conserved among vertebrate species and codes for polypeptides of 60 and 37 amino acid residues. ORF2 also has two putative initiation codons and codes for a polypeptide of 49 amino acid residues (Tsuzuki et al. 1985). The transcriptional process under these TTR intronic ORFs occurs in liver, pancreas, and brain. However, these transcripts were produced as part of larger transcripts rather than as gene-specific mRNAs (Soares et al. 2003). Two copies of the Alu sequence with opposite directions, which are possibly involved in TTR gene regulation, were found in introns 2 and 3. Several regulatory signals including the Goldberg–Hogness sequence, CAAT boxlike sequence (GTCAAT), two glucocorticoid responsive elements (GRE), and liver-specific nuclear factors HNF-1, 3, 4, and C/E BP locate in the 50 -flanking region of human TTR gene (Sasaki et al. 1985, 1989). The cis-elements that are required for developmental tissue-specificity and for level of expression were also demonstrated at 0.6 to 6.0 kb of the gene (Nagata et al. 1995). The 30 -UTR of the human TTR gene contains polyadenylation signal located at 123 bp downstream from the coding region or at 23 bp upstream from the polyadenylation site (Tsuzuki et al. 1985). In addition, three enhancer core sequences were found in introns and 30 -UTR (Sasaki et al. 1985).

3.1.3

Genomic Structure of TTR from Rodents

The chromosomal structures of the TTR genes from other vertebrates that have been characterized till now are from mouse (Wakasugi et al. 1986) and rat (Fung et al. 1988). These rodent TTR genes are single copies, and the nucleotide sequences of the genes are highly conserved in comparison to human TTR. The genes contain four exons, three introns, TATA-box, and CAAT-box sequences, and the highly conserved regions are in the exons, 50 -flanking region, and 30 end region of intron 1, where only one ORF rather than two as identified in human TTR is present (Wakasugi et al. 1986). Introns of the mouse TTR gene are 0.9, 3.4, and 3.6 kb in length, but have low homology (up to 49% homology) to the corresponding human TTR introns. Two GCR-binding sites and one JkCk enhancer, which are found in immunoglobulin kappa chain gene, are located in the 30 end region of intron 1, similar to that found in the human TTR gene. In addition, two mouse mammary tumor virus (MMTV) GCR-binding site sequences are in the 50 -flanking region (Wakasugi et al. 1986). The proximal promoter (108 to 150 nucleotides) and the distal enhancer element (1.6 to 2.15 kb) of mouse TTR gene possess several binding sites for nuclear factors (Costa et al. 1986). Of the three binding sites present in the enhancer, two are responsible for the cell-specificity and the third is required for the general activation mechanism (Costa et al. 1988). Several nuclear

48

P. Prapunpoj

factors have been identified so far, including the activation protein one (AP-1), HNF-1 (binding site is 130 to 116 bp), HNF-3 (strong binding site is 106 to 94 bp while weak binding site is 140 to 131 bp), HNF-4 (binding site is 151 to 140 bp), HNF-6 (binding site is 106 to 94 bp), and CCAAT/enhancer binding protein (C/EBP; binding sites are 195 to 177 bp and 135 to 112 bp) (Costa et al. 1989, 1990; Grayson et al. 1989; Samadani and Costa 1996; Samadani et al. 1996). Cooperation between these factors to the TTR promoter and enhancer regions is required for the tissue-specific expression of the gene (Costa and Grayson 1991), such as nuclear factors NHF-1, 3, and 4 and C/EBP are required for TTR gene expression in the hepatocytes (Costa and Grayson 1991). The rat TTR gene spans to 7.3 kb (Fung et al. 1988). The nucleotide sequence of the proximal 50 -flanking region (position 51 to 89 bp), which corresponds to the binding sites for hepatocyte nuclear factors in mouse TTR (Costa et al. 1986), is highly conserved in comparison to human (93% identity) and mouse (97% identity) TTR genes. However, the homology significantly decreases with the nucleotide sequence further upstream from this region (Fung et al. 1988). The rat TTR gene has dual promoters, similar to those human and mouse TTR genes (Motojima and Goto 1989, 1990). A major proximal promoter is mainly used in liver and a minor distal promoter is mainly used in kidney and significantly in brain. Both the TATA-box like sequence (TATATAA sequence; at 30 to 24 bp) and the CAAT-box like sequence (GTCAAT; at 101 to 96 bp) are in the upstream region of the proximal promoter; however, neither is in the distal promoter. An additional transcriptional start site for the specific expression of rat TTR gene in the brain was located at least 65 bp upstream of the normal start site (Motojima and Goto 1989). The AATAAT sequence is present upstream of the region and this region is expressed only in the brain tissue.

3.2

Evolution of the TTR Gene Structure and the Stepwise Shift of the Splicing Mechanism

In the evolution of the TTR gene structure, two types of mutation patterns can be distinguished. The first type consists of random mutations found throughout the polypeptide chain of the human TTR subunit, and more than 80 positions of this mutation type are related to the medical disorder called amyloidosis (see details in chapters on TTR amyloid mutations). The second type consists of mutations occurring near the N-terminal region of the TTR subunit, which lead to changes in length and hydropathy of the N-terminal regions during evolution. Up to date, the primary structures of TTRs from many vertebrate species have been characterized. Although some were obtained directly from the amino acid sequencing, most were derived from nucleotide sequencing of the TTR cDNAs. Up to 24 vertebrate species of TTR cDNAs have been isolated and characterized. These include eutherians (human, hedgehog, shrew, rat, mouse, pig, cow, sheep, and rabbit), metatherians (wallaby, kangaroo, dunnart, opossum, and sugar glider),


49

bird (chicken), reptiles (crocodile and lizard), amphibians (bullfrog and xenopus), and fishes (sea bream, pacific bluefin tuna, and two genera of lamprey, Petromyzon marinus and Lampetra appendix). Messenger RNA of the vertebrate TTR subunit is generally composed of a 50 -UTR, a coding region, and a 30 -UTR preceding the poly (A) tail, similar to other mRNAs of eukaryotes. The nucleotide sequence in the coding region of these TTR genes is highly conserved, in particular, among eutherians, marsupials, birds, reptiles, and amphibians. This coding region corresponds to TTR presegment, 18–20 amino acid residues, and the functional subunit, which is varied in length depending on animal species, that is, from 125 amino acid residues in hedgehog TTR to 136 amino acid residues in lamprey TTR (Fig. 3.1). Alignment of the TTR primary structures, either obtained directly from amino acid sequencing or derived from the nucleotide sequence of TTR cDNA, from 28 vertebrates including mammals (small eutherians, insectivores, marsupials), birds, reptilians, amphibians, and fish shows that residues in all positions in the central channel of TTR including those involved in the binding interaction with thyroid hormones (THs) (Blake and Oatley 1977; Blake 1981; Wojtczak et al. 1996) are conserved and have not evolved since primitive fish (Fig. 3.1). Homology of the amino acid sequences is high, in particular, among eutherians, marsupials, birds, and reptiles (Table 3.1). On the contrary, most mutations of TTR that occurred during evolution are located only in the 50 -terminal region of the TTR gene that corresponds to the first ten amino acids (based on the amino acid numbering of human TTR) from the N-terminal region of the TTR subunit. A systematic change of the N-terminal region of the TTR subunit from longer and relatively hydrophobic to shorter and more hydrophilic is observed in the evolution of TTRs from eutherians from their common ancestors. Analysis and systemic comparisons between nucleotide sequences of TTR cDNAs and genomic DNAs isolated from eutherian, marsupial, and birds revealed that the shortening of the TTR polypeptide chain at the N-terminus occurred at the border between exon 1 and exon 2 (Aldred et al. 1997). Comparing the nucleotide sequences of genomic DNA and cDNA at the border between exon 1 and exon 2 showed that the position of the 30 splice site of exon 1 and the 50 splice site of intron 1 of all studied TTRs remained unchanged. The consensus recognition sequences for splicing at the 50 end of the intron are typical but well agreed with that found in other vertebrate genes (Moore et al. 1993), that is, the nucleotides U or C rather than A is preferred for the consensus sequence in the exon 1 region flanking the splice site. However, all changes occurred in the region of 30 end of intron 1 and the 50 end of exon 2. Position of the 30 splice site of the TTR intron 1 shifted to the 30 direction in successive steps during evolution of eutherians from their ancestral TTR genes similar to that in fish, leading to a successive shortening of the 50 end of exon 2 (Fig. 3.2). The mechanism underlying this splice site shift possibly occurred by a series of single base mutations that converted codons for amino acids into a new splice recognition site. According to the fact that the splice site recognition sequence at the 30 end of introns is relatively short (Moore et al. 1993), codons for amino acids, in particular, glutamine, CAA, and histidine, CAC or CAU, can be converted into the 30 splice site recognition sequence CAG by a single base change,

P. Prapunpoj

50

-------

Presegment-----

Mature Protein

------>

-------------disordered---------------

Human Hedgehog Shrew Rhesus Monkey 1 Rhesus Monkey 2 Pi g Sheep Bovine Rabbit Rat Mouse Tammar Wallaby Grey Kangaroo Brush-tail Possum Sugar Glider Wombat Stripe-faced Dunnart Grey Opossum Virginia Opossum Chicken Pigeon Emu Ostrich Crocodile Lizard Bullfrog Xenopus Sea Bream Lamprey

MASHRLLLLC LAGLVFVSEA GPT ******F*** ******M*** *** ***R****** *****L*T** *** *** *** ***Y****** ********** **A ***F****** ********** S*A ***F**F*** ********** *SV **V ***L**F*** ****I*A*** **G ***L**F*** ********** **A **F*S***** ****A****T AAV **F*S***** ****A****T AAV V **F*S***** ****L***** **V A*E **F*S***** ****L*L*** **V **F*S****G **S*L***D* A*V A*V **F*ST**VF ******L*** A*L A*L A*L A*L **F*SM**VF ******LT** A*L *G*SS***V* ***M*YLT** A*L **YYNT*A*L TIFIFSGAFH RAQ ***FKSF** **L*AI**** A*P *LQPLHC**L ASAVLCNTAP T** *--RF*C**V *VASSSLLCR ADD

-20

-1 +1 3

-10 -strand ----E-----

Human Hedgehog Shrew P ig Sheep Bovine Rabbit Rat Mouse Tammar Wallaby Grey Kangaroo Sugar Glider Stripe-faced Dunnart Grey Opossum Chicken Crocodile Lizard Bullfrog Xenopus Sea Bream Lamprey

V V V V V V G G H A D D H K S H E S -

- -

-

-

-

-

H H P A V A I T S T S S S S T S K H

H H X H H H H H H H H H H H H H H E

- -

EEEFVEGIYK D*K****V** D*K*****I* D*K** ** *** *DK****L** *DK****L** S*K****V** D *K * T * * V * R D*K****V*R DDK*G**L** DDK*G**L** DDK*G**L** DDQ*G**L** D*K*G**L** **Q****V*R D*K******R D*Q**Q*L** **Q******* D*Q*T**V** *QQ*PA*V*R DKD*T**T**

VEIDTKSYWK **L******* **L***T*** **L******* **L******* **L******* **L******* **L** ***** **L******* **F**I**** **F**I**** **F**I**** **F**V**** **F**F***N **F**S**** **F**S**** **F**S**** L*FA**RF*S I*FA**AF*G **F***A**T *RFE*QQ**T

ALGISPFHEH T********Y ******S**Y ** * ****** Y S********Y S********F *********Y *********Y T********F ***V*****Y ***V*****Y ***V*****Y TF*******Y ***V*****Y G**L*****Y ***L*****Y ***V*****Y K**LT****Y K**L*****Y NQ*ST****V KT**T****A

70

80

90

AE V* V* ** ** ** ** ** *D *D *D *D *D *D *D *D *D VD VD ** **

+4

-strand -----A----

-strand -----B----

PLMVKVLDAV ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** ********** **** ********** ******* ********S* ********** ******* ********** ***** ******* ******* ********** ********** ********** ********** *****I**** *****AI*S*

RGSPAINVAV *****V**** Q****V**** * * *****V**G* *****A**G* *****A**G* *****VD*S* *****VD*** *****VD*** **R**V**D* **R**V**D*

-strand ------C------

-strand D

HVFRKAADDT WEPFASGK K**K****E* ******** R**K****E* ********

TS ESGELHGLTT ** ********** ** *F********

K**K****G* K**K****E* K**K****E* ***K****E* K**K*T**GS K**K*TSEGS K**K*TEEQ* K**K*TEEQ*

** ** ** ** *A *A *N *N

*****L** ******** ******** ******** ******** ******** **L**A** **L**A**

*F******** D********* ********** KT******** ********** ********** DN**I*E*** DN**I*E***

**R**V**D* K**K*TEKQ* **L*****

*N DN**I*E**S

*****V**D* K**K*TEEQ* **L***** *****V**N* K**K*SEEQ* *****T**

*N NN**I*E**S *N DY**I*E**N

*****A**** K**K****G* *QD**T**

*T *F**I*E***

*****A***I **R**TSI** **I**AKLP* **I**A*LL* K*T**GS**L Q*K**AG*KL

K**K*TS*GD K*SKMSEEGD K**KQNE*KS N****TNSGK K*SQ*T**GG S*MK*QD*AS

*T *N ** *T *D *G

*F**V*E**S *F**I*E*** SD**I*N*A* SD**I*N*** AT****N*I* KT**S*H*IS

30

40

50

60

20

10 -strand -----G----

-strand -----F----

-- -helix-

GTGESKC *Q*** ***Q*** *VD**** *ID**** *A***** *A***** *A**P** ***D*** *A***** *A***** EGEH*** ESEH*** *GEH**X *GED*** *GDDX*X *AED*** *AED*** *AEDX*X *SVD*** *SVD*** *SVD**X *SVD**X *SID*** *SID*** *EAD*** *EAD*** *GSDTR* *GVKDS*

*QE**A** *KE**N** *DLIS**T **QIT*** *TQI*T*V *KEV*T*V

-strand ----H---

VVFTANDS ******** ******** ** ** ** ** ******** ******** ******** ******** ******** *******A *******A *******A *******A ***K***A ******** ******** ***S**** *******A *******A ***D*HPE ***M*HGA

GPRRYTIAAL *Q******** *K******** * R* H*** * ** *L*H****** ***H****** *H*S****** * H *H *** ** * *H*H****** *H*H****** *H*H*****Q *H*H*****Q *H*H*****Q *H*H****** *H*H****** *H*H****** *H*H****** *H*H**T*V* *H*Q****V* *HGH**L*L* *HKH*H*PM*

LSPYSYSTTA ********** ********** ********** ********** ********** ***F****** **** **** ** ********** *****F**** *****F**** *****F**** ***F*F**** ********** ***F****** ***F****** ***F****** *T***F**** *T***F*S** ***F**T*** ****FFA*G*

VVTNPKE L*SD*** L*SD*** L *SS***GAL L*SS*** L*SS**A **S**Q* **S**QN **S**QN I*S**T* I*S**T* I*S**T* **S***D **S***D **SD*Q* **SD*Q* **SD*** **SDV**AHV I*SE*HDDL **SSVH* I*VDG*GH

100

110

120

130

Fig. 3.1 Comparison of the amino acid sequences of vertebrate TTRs and derived amino acid sequences from 28 animal species (20 complete and 8 N-terminal sequences) are aligned. Those residues in other species with identical amino acids to those in human TTR are indicated by asterisks. First residues in the mature polypeptide are in bold. Features of secondary structure of human TTR are indicated above the sequences. The numbering of residues is based on human TTR: negative numbers, residues in the presegment; positive numbers, residues of the mature protein; a, b, g, d, e, z, Z, y; and i, positions of residues in noneutherian species. Residues in the core and the central channel of the human TTR subunit, according to Blake et al. (1974, 1978), are singly and doubly underlined, respectively. Gray shading, amino acids in the central channel those are completely conserved; arrows, positions of exon borders. Sources of TTR sequences: Human (Mita et al. 1984; Tsuzuki et al. 1985); Hedgehog and Shrew (Prapunpoj et al. 2000a); Rhesus monkey 1 and Rhesus monkey 2 (van Jaarsveld et al. 1973); Pig (Duan et al. 1995a); Sheep (Tu et al. 1989); Bovine (Schreiber et al. 1993); Rabbit (Sundelin et al. 1985); Rat (Dickson et al. 1985; Duan et al. 1989; Fung et al. 1988); Mouse (Wakasugi et al. 1985, 1986); Tammar Wallaby (Brack et al. 1995); Grey Kangaroo (Aldred et al. 1997); Brush-tail Possum, Sugar Glider, and Wombat (Richardson et al. 1994); Stripe-faced Dunnart and Grey Opossum (Duan et al. 1995b); Virginia Opossum (Richardson et al. 1996); Chicken (Duan et al. 1991); Pigeon, Emu, and Ostrich (Chang et al. 1999); Crocodile (Prapunpoj et al. 2002); Lizard (Achen et al. 1993); Bullfrog (Yamauchi et al. 1993); Xenopus (Prapunpoj et al. 2000b); Sea Bream (Santos and Power 1999); Lamprey: Petromyzon marinus (Manzon et al. 2007)

Sea bream 45 Xenopus 36 46 Lizard 38 51 56 Crocodile 40 51 58 80 Chicken 40 54 60 79 90 Opossum 45 48 58 72 73 74 Dunnart 41 48 59 72 74 75 82 Sugar 45 48 60 71 74 73 84 89 Glider Kangaroo 40 47 58 71 73 73 82 84 92 Wallaby 41 48 59 71 72 73 83 83 92 98 Mouse 42 47 57 70 75 75 74 75 74 72 72 Rat 44 49 61 68 74 75 73 73 74 71 72 93 Sheep 44 46 57 68 71 75 74 74 75 73 74 82 82 Shrew 42 44 53 68 71 71 69 68 70 67 68 78 79 83 Hedgehog 40 45 60 68 73 76 72 73 73 71 72 85 86 88 90 Human 42 45 53 67 71 74 71 71 72 69 70 81 82 85 85 87 Identity (%) of amino acid sequences were calculated using multiple sequence alignment of ClustalW program (Thompson et al. 1994). Accession number for the sequences: Human, AAA73473; Hedgehog, AAF70058; Shrew, O46375; Sheep, P12303; Rat, P02767; Mouse, P07309; Wallaby, P42204; Kangaroo, Q29616; Sugar Glider, P49142; Dunnart, P49143; Opossum, P49141; Chicken, P27731; Crocodile, O55245; Lizard, P30623; Xenopus, BAA77579; Sea Bream, AAC26108; Lamprey, ABI93605

Table 3.1 Comparison of amino acid sequences of TTRs from 17 vertebrate species Lamprey Sea bream Xenopus Lizard Crocodile Chicken Opossum Dunnart Sugar glider Kangaroo Wallaby Mouse Rat Sheep Shrew Hedgehog

52

P. Prapunpoj ---- Presegment----

Mature Protein------> ---------disordered---------------

Human Hedgehog Shrew Rat Mouse Tammar Wallaby Grey Kangaroo Sripe-faced Dunnart Grey Opossum Chicken Crocodile Lizard Xenopus Lamprey

MASHRLLLLC ******F*** ***R****** ***L**F*** ***L**F*** **F*S***** **F*S***** **F*S***** **F*S****G **F*ST**VF **F*SM**VF *G*SS***V* ***FKSF** *--RFLC**V

-20

-10

LAGLVFVSEA ******M*** *****L*T** ****I*A*** ********** ****A****T ****A****T ****L*L*** **S*L***D* ******L*** ******LT** ***M*YLT** **L*AI**** *VASSSLLCR

GPT *** *** **G **A AAV AAV **V A*V A*L A*L A*L A*P ADD

-1 +1 3

V V V G H A D H K S H E S

H H A I S S S S H

H H H H H H H H E

GTGESKC *Q*** ***Q*** *A***** *A***** EGEH*** ESEH*** *AED*** *AED*** *SVD*** *SID*** *SID*** *EAD*** *GVKDS*

- - - - - - - - - +4

10

-strand ----B---

-strand -----A---

PLMVKVLDAV ********** ********** ********** ********** ********** ********** ********S* ********** ********** ********** ********** ********** *****AI*S*

RGSPAINVAV *****V**** Q****V**** *****VD*** *****VD*** **R**V**D* **R**V**D* *****V**D* *****V**N* *****A**** *****A***I **R**TSI** **I**A*LL* Q*K**AG*KL

HVFRKAADDT K**K****E* R**K****E* K**K*T**GS K**K*TSEGS K**K*TEEQ* K**K*TEEQ* K**K*TEEQ* K**K*SEEQ* K**K****G* K**K*TS*GD K*SKMSEEGD N****TNSGK S*MK*QD*AS

20

30

40

Fig. 3.2 Comparison of intron 1 splice sites of TTRs from 14 vertebrate species The sequences of the first 40 amino acid residues including the presegment of TTR from 13 species are aligned with that of human TTR. An arrow indicates the position of the splice site at the exon 1/exon 2 border. Bold letters are the first amino acid at the N-terminus of the mature TTR polypeptide. Sources of splice site data: Human (Tsuzuki et al. 1985); Rat (Fung et al. 1988); Tammar Wallaby, Grey Kangaroo, Stripe-faced Dunnart, Grey Opossum, Chicken, and Lizard (Aldred et al. 1997); Hedgehog, Shrew, Mouse, Crocodile, Xenopus, and Lamprey (as referenced in Fig. 3.1)

that is, from A, C, or U to G. This conversion apparently has occurred at the 50 end of exon 2 of several TTR genes during evolution of eutherians. The histidine codon CAU in marsupials is possibly converted into the consensus recognition sequence CAG by a single base change from U to G, and, at least one other single base substitution, G to U or C, has occurred to inactivate the former 30 splice site recognition sequence operating in marsupial. A valine codon in position g apparently changed into the 30 splice site recognition sequence CAG, by either three base changes or deletion of the valine codon, during the evolution of the TTR gene in marsupial from its ancestor gene similar to that in bird and reptile. In addition, the conversion of the histidine codon CAU (in position e) or the histidine codon CAC (in position i) to the consensus sequence CAG occurred during evolution of bird and reptile TTR from the amphibian-like ancestor and that of amphibian from the fish-like ancestor TTR, respectively (Fig. 3.3b). These changes during evolution of the 50 end of exon 2 of the TTR gene resulted in a progressive movement of the 30 splice site, but not the 50 splice site of intron 1 (Fig. 3.3a), in the 30 direction and successive steps from fish to amphibian, reptilian and avian, marsupial and, finally, eutherian species, resulting in a progressive shortening of the 50 end of exon 2 and increase in hydrophilicity of the N-terminal region of the TTR subunit.

3.3

Influence of Changes in Gene Structure on Function of TTR

The binding affinities of THs to TTRs from several animal species were revealed (Chang et al.1999; Yamauchi et al. 1998 Prapunpoj et al. 2000b, 2002; Morgado et al. 2008). Affinity to T4 increased while that to T3 decreased during evolution of eutherians from their ancestors. The crystal structure of TTR tetramer revealed that


a

AG -8

-7

-6

-5

-4

-3

-2

-1

+1

2

53

guaagu g

3

Human

GGA CUG GUA UUU GUG UCU GAG GCU GGC CCU ACG Gly Leu Val Phe Val Ser Glu Ala Gly Pro Thr

Hedgehog

GGA CUG GUA UUU AUG UCU GAA GCU GGC CCU ACG Gly Leu Val Phe Met Ser Glu Ala Gly Pro Thr

**********gau**uau**aaucc*agu*cca*gucuccu*u*uaccucagcug*aguag*gggu

Shrew

GGA CUG GUG CUC GUC ACC GAG GCA GGC CCA ACG Gly Leu Val Leu Val Thr Glu Ala Gly Pro Thr

*********aaau*cugugaagucc*u*c***cc*uuuccugacuag*cga*ccag**gugacu**

gugaguguuucugugacaucccauuccuacauuuaagauucacgcuaaaugaaguagaagugacuc

Rat

GGA CUG AUA UUU GCG UCU GAA GCU GGC CCU GGG Gly Leu Ile Phe Ala Ser Glu Ala Gly Pro Gly

*******g*c******guga**caga*auggcagguagac*uuagau**agg***gccucccu*ca

Mouse

GGA CUG GUA UUU GUG UCU GAA GCU GGC CCC GCG Gly Leu Val Phe Val Ser Glu Ala Gly Pro Ala

*******a*c******gcgau*caga*auggcaguuagac*uuagau**a****a**ugccuu**u

Wallaby

GGC CUG GCG UUU GUG UCU GAG ACU GCA GCU GUG GLY Leu Ala Phe Val Ser Glu Thr Ala Ala Val

**a***u*ggu*acugaaagua**c*u*uug*gguuagc*u**ucau*ucc*uagu*g*caagg*a

Kangaroo

GGC CUG GCG UUU GUG UCU GAG ACU GCA GCU GUG Gly Leu Ala Phe Val Ser Glu Thr Ala Ala Val

**a***u*ggu*acuga*aguag*c*u*uug*gguuagc*ucuca**uucau***g**caa*guaa

Dunnart

GGC CUG GUG UUC CUG UCU GAG GCU GGA CCU GUG Gly Leu Val Phe Leu Ser Glu Ala Gly Pro Val

******u*ggu*acuga*aguag***u*cug*ggu*agccucuca**uccau***u**caa*guaa

Opossum

AGC CUG CUG UUU GUG UCU GAU GCU GCU CCU GUG Ser Leu Leu Phe Val Ser Asp Ala Ala Pro Val

******u*ggu*ccuga*aaguca*u**uuug*gguugacucuca**u**aucccuagcagacaag

Chicken

GGA CUG GUA UUU CUC UCC GAA GCU GCA CCA CUG Gly Leu Val Phe Leu Ser Glu Ala Ala Pro Leu

******c***uaaa

Crocodile

GGA CUG GUA UUU CUG ACU GAA GCU GCC CCA CUG Gly Leu Val Phe Leu Thr Glu Ala Ala Pro Leu

**a**c*c*****cauuuagggggcaga*uu*ga*g**c*guaaaau*uaau*aa**u*agc*uaa

Lizard

GGA AUG GUG UAC CUC ACU GAA GCU GCA CCA CUG Gly Met Val Tyr Leu Thr Glu Ala Ala Pro Leu

**a**ca**g*ac**g**aua**g*ggcua*c*agga*aaa*aaugc*g**cucacac*uga*gca

Xenopus

CUA CUG GCA UUU GUC UCA GAG GCU GCA CCA CCG Leu Leu Ala Phe Val Ser Glu Ala Ala Pro Pro

******a*g*ug*g*gu*c*uguaa*aacuca*gcuuuccuuuuaauug*agcacua*g*cuga*g

Lamprey

GCA UCC AGC UCG CUA CUC UGC AGG GCU GAC GAU Ala Ser Ser Ser Leu Leu Cys Arg Ala Asp Asp

**a****caca*caucgucau***cgucg

b

cncuga u u a

c

polypyrimidine tract -

Human

accacaaagaauaaauccuuucacucugaucaauuuuguuaa

-

-

-

cuu cuc acg ugu

-

-

cuu cuc

cag u uac

-

-

G 4

5

6

7

8

9

10

acc cag

↓GGC ACC GGU GAA UCC AAG UGU Gly Thr Gly Glu Ser Lys Cys

Hedgehog

*aaga**ga**a*****a***g*****ugc********c*g*

*** g** *uu ***

**c ***

**u

g** ***

↓--- --- GGU CAA UCC AAG UGU Gly Gln Ser Lys Cys

Shrew

cuag**cu*c*a*g*auaga*guuaaga*cuugccaau**ug

u*g acu u*u ca*

u*g **u

cuu

ug* ***

↓GGC ACU GGU CAG UCC AAG UGU Gly Thr Gly Gln Ser Lys Cys

Rat

***gg******c**gc**a**u*******c*c*c**cac*g*

*a* *** u*u ***

**c ***

**u

g** ***

↓GGU GCU GGA GAA UCC AAG UGU Gly Ala Gly Glu Ser Lys Cys

Mouse

***gg******c**gc*gg**u*******c*c****cac*g*

*a* u** u*u ***

**c ***

*gu

g** ***

↓GGU GCU GGA GAA UCC AAA UGU Gly Ala Gly Glu Ser Lys Cys

Wallaby

g*acuugccu**cuca*u*cag*u*ucc*a**ccccac*g*g

a** u** ugu **c

*** *ca

**g

↓CAC CAU GAA GGU GAG CAU UCC AAG UGC His His Glu Gly Glu His Ser Lys Cys

Kangaroo

ggacuuggcu**cuca*u*cag*u*ucc*a**ccccac*g*g

a** u** ugu **c

*** *ca

c*g

↓CAC CAU GAA AGU GAG CAU UCC AAG UGC His His Glu Ser Glu His Ser Lys Cys

Dunnart

uaacuugccc*gcuca*u**agcuaucc*au*ccccgc*g*g

u** u** ugu **c

*** *ca

c*g

↓GCC CAU GGA GCU GAG GAU UCC AAA UGC Ala His Gly Ala Glu Asp Ser Lys Cys

Opossum

**ucaugccug*cuca*u*cag*ugucc*a**uccccc*g*g

*** u** ugu **c

*** *ca

c*g

↓AUC CAU GGA GCU GAA GAU UCC AAA UGC Ile His Gly Ala Glu Asp Ser Lys Cys

Chicken

gag*agguuuu*gc*aggc*agugaacagcucc***acaacc

uc* *cu cgu *uc

gc* *ag

↓GUC UCC CAU Val Ser His

GGC UCU GUU GAU UCC AAA UGC Gly Ser Val Asp Ser Lys Cys

Crocodile

gauguggu*****gcc**agcgcg*u*ac*gc***ag*cau*

u** u** cu* cu*

ucc aag

↓GUU UCC CAU Val Ser His

GGU UCU AUU GAU UCC AAA UGC Gly Ser Ile Asp Ser Lys Cys

Lizard

uuu*ug*uc**g****ggg**uuag*a*gcauu****ccccc

*cc *c* cac cu*

a** aag

↓GUU UCA CAU Val Ser His

Xenopus

uaaua*u*au*accuaa***c**gca*c**u*ucca*cc*cu

u** g** u*a *ag

↓GGA CAU GCU Gly His Ala

Lamprey

*u*ggugcucgcu**cagccagcgcg**cg*cccacgcac*g

↓GAC CAC AAG AGC CAC GAA TCC CAC GAG GGA GGC GTC AAG GAC TCG TGC Asp His Lys Ser His Glu Ser His Glu Gly Gly Val Lys Asp Ser Cys

UCC CAU Ser His

GGC UCC AUU GAU UCC AAG UGU Gly Ser Ile Asp Ser Lys Cys GGA GAA GCC GAC UCC AAG UGU Gly Glu Ala Asp Ser Lys Cys

Fig. 3.3 Comparison of nucleotide and amino acid sequences of TTRs at the (a) exon 1/intron 1 border and (b) intron 1/exon 2 border. The 50 and 30 splice sites of intron 1 of TTR precursor mRNAs from 13 vertebrate species are aligned with those from human. The splice sites are indicated by arrows. The consensus recognition sequences for splicing (Moore et al. 1993) are indicated in bold above the position of the splice sites in human TTR precursor mRNA. Nucleotides identical with those in the consensus sequence for the 30 splice site branch point are underlined. Nucleotides in exons are in upper case; those in introns are in lower case. The amino acid residues at the N-terminus, determined by Edman degradation of the mature proteins or of the recombinant TTR, and their corresponding codons are represented in bold. For longer nucleotide sequences of intron 1, see Prapunpoj et al. (2002)

54

P. Prapunpoj

amino acid residues that are components in the binding cavity and involved in the binding with THs (Blake et al. 1974, 1978) are conserved. In addition, no amino acid residues of the N-terminal segment of TTR subunit is involved with or is a part of the binding sites. Instead, the N-terminal segment forms a radon-coil structure with high conformational flexibility (Hamilton et al. 1993; Wilce et al. 1995) on the surface of each TTR monomer, located at the entrance of the tunnel containing the TH-binding sites of the TTR tetramer. The direct involvement of these segments in the binding of THs had been, therefore, neglected. However, an indirect involvement has nevertheless been postulated. The influence on the binding of THs perhaps by interfering with the access of THs to the binding sites in the interior of the TTR molecule was demonstrated using a series of chimeric TTRs. By using the heterologous expression system of Pichia pastoris, a methylotrophic yeast, the recombinant native TTRs and the chimeric TTRs in which the N-terminal region was altered or truncated were successfully synthesized (Prapunpoj et al. 2002, 2006). Binding affinities to T4 and T3 of the recombinant TTRs including two native TTRs, that is, from human and saltwater crocodile (Crocodylus porosus), and three C. porosus chimeric TTRs, that is, one with the N-terminal sequence replaced by that of Xenopus laevis TTR (xeno/crocTTR), one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), and the other with the N-terminal segment removed (truncated crocTTR), were determined using a highly reproducible and sensitive method (Chang et al. 1999). Changes in the affinities to T3 and T4 of C. porosus TTR were clearly observed when the nucleotide sequence at the 50 -terminal region of the TTR gene was either altered or truncated (Prapunpoj et al. 2006). The Kd values for T3 and T4 of the recombinant human TTR were 53.26 3.97 and 19.73 0.13 nM, respectively, while those of the recombinant C. porosus TTR were 33.03 0.42 and 55.69 7.69 nM, respectively (Prapunpoj et al. 2002). However, the affinity of human/ crocTTR for T4 (Kd was 22.75 1.89 nM) was higher than that of both human and C. porosus TTRs for T4, but the affinity to T3 (Kd was 5.40 0.25 nM) was similar for C. porosus TTR, which led to a Kd T3/T4 ratio of 0.24. In addition, the truncated crocTTR had a similar affinity for both T3 (Kd was 57.78 5.65 nM) and T4 (Kd was 59.72 3.38 nM), with a Kd T3/T4 ratio of 0.97. These led to the postulation that the N-terminal region has a role in determining the affinities of T3 and T4 to TTR. This hypothesis has been further confirmed by experiments with fish TTR. The affinity for T4 of the sea bream TTR decreased when the first six amino acid residues were removed (Morgado et al. 2008), suggesting a possibility that some residues in the N-terminal segment influence the binding to T4.

3.4

Conclusion

THs are involved in many regulatory aspects of metabolism in vertebrates. Because of its high lipid solubility, binding to TH-binding proteins is a necessity to prevent accumulation of THs in membranes and to ensure the appropriate extracellular and


55

intracellular distribution of the hormones. One of the most important and widely known functions of TTR, which acts in a network with other plasma TH-binding proteins, is to ensure an even distribution of THs (synthesized in and secreted from the thyroid gland) to target tissues throughout the body. Among vertebrates, the requirements of THs (in particular, the biologically active form, T3) are varied depending on many factors, including complexity of organs and a particular period of life, for example, during the developmental period, that requires higher concentrations of the hormones circulating in the blood. Differences in the action of THs are principally regulated via the conversion of the more biologically active T3 from the less active T4 by the deiodinases in the specific target tissues. This could also be achieved via influencing distribution of THs during transport in the blood-stream. Although the basic molecular structures of TTRs at both the genomic and the protein levels in all studied vertebrates are similar, for example, all TTR genes comprise four exons and three introns, TTR monomers are rich in b-pleated sheet structure and four monomers form the tetrameric protein. The affinities in binding to T3 and T4 differ quite dramatically during evolution of the vertebrates, that is, the affinity to T4 increased while the affinity to T3 decreased during the evolution of mammalian TTRs from common ancestoral TTRs. These changes in function are influenced by the N-terminal region of the TTR subunit that changed from longer and relatively hydrophobic to shorter and more hydrophilic during evolution. The stepwise shift of the 30 splice site of the TTR intron 1 in the 30 direction has been proven to be the underlying mechanism that led to the systemic change of the N-terminal region of each TTR subunit. The organization of the genome into exons and introns could speed evolution, as changes in one or a few bases occurring near splice sites could result in the deletion or addition of whole sequences of amino acids (Gilbert 1978). Therefore, with the splicing mechanism, novel or functionally improved proteins could be generated during evolution. The single base mutations near splicing sites that can lead to stepwise directional gradual changes in a protein have been demonstrated in TTR gene. It is most likely that during evolution, selection pressure has operated on the length and composition of the 50 end of exon 2 of the TTR gene, leading to the shorter and more hydrophilic N-termini during the evolution of eutherian TTRs from their ancestors. Such shifts could produce the stepwise changes in the functional properties of TTRs, in particular, the changing affinities observed for THs. This stepwise ‘‘improvement’’ of protein function could be the point of operation of selection pressure and it is an example of a molecular mechanism of positive Neo-Darwinian evolution.

References Achen MG, Duan W, Pettersson TM, Harms PJ, Richardson SJ, Lawrence MC, Wettenhall REH, Aldred AR, Schreiber G (1993) Transthyretin gene expression in choroid plexus first evolved in reptiles. Am J Physiol Regulat Integr Comp Physiol 265:R982–R989.

56

P. Prapunpoj

Aldred AR, Prapunpoj P, Schreiber G (1997) Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 39 splice site of intron 1). Eur J Biochem 246:401–409. Arachibald AL, Couperwhite S, Jianq ZH (1996) The porcine TTR locus maps to chromosome 6q. Anim Genet 27:351–353. Blake CCF, Oatley SJ (1977) Protein-DNA and protein-hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor? Nature 268:115–120. Blake CCF, Geisow MJ, Oatley SJ, Re´rat B, Re´rat C (1978) Structure of prealbumin: secondary, ˚ . J Mol Biol tertiary and quaternary interactions determined by Fourier refinement at 1.8 A 121:339–356. Blake CC (1981) Prealbumin and the thyroid hormone nuclear receptor. Proc R Soc Lond B Biol Sci 211:413–431. Brack CM, Duan W, Hulbert AJ, Schreiber G (1995) Wallaby transthyretin. Comp Biochem Physiol 110B:523–529. Blake CCF, Geisow MJ, Swan ID, Rrat C, Rrat B (1974) Structure of human plasma prealbumin at 2.5 A resolution. A preliminary report on the polypeptide chain conformation, quaternary structure and thyroxine binding. J Mol Biol 88:1–12. Chang L, Munro SLA, Richardson SJ, Schreiber G (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. Eur J Biochem 259:534–542. Costa RH, Grayson DR (1991) Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region. Nucleic Acids Res 19:4139–4145. Costa RH, Lai E, Darnell JE (1986) Transcriptional control of the mouse prealbumin (transthyretin) gene: Both promoter sequences and a distal enhancer are cell specific. Mol Cell Biol 6:4697–4708. Costa RH, Lai E, Grayson DR, Darnell JE (1988) The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor (s) at two other sites. Mol Cell Biol 8(1):81–90. Costa RH, Grayson DR, Darnell JE (1989) Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and a1-antitrypsin genes. Mol Cell Biol 9:1415–1425. Costa RH, van Dyke TA, Yan C, Kuo F, Darnell JE (1990) Similarities in transthyretin gene expression and differences in transcription factors: live rand yolk sac compared to choroid plexus. Proc Natl Acad Sci USA 87:6589–6593. Dickson PW, Howlett GJ, Schreiber G (1985) Rat transthyretin (prealbumin): molecular cloning, nucleotide sequence, and gene expression in liver and brain. J Biol Chem 260:8214–8219. Duan W, Cole T, Schreiber G (1989) Cloning and nucleotide sequencing of transthyretin (prealbumin) cDNA from rat choroids plexus and liver. Nucleic Acids Res 17:3979. Duan W, Achen MG, Richardson SJ, Lawrence MC, Wettenhall REH, Jaworowski A, Schreiber G (1991) Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates. Eur J Biochem 200:679–687. Duan W, Richardson SJ, Ko¨hrle J, Chang L, Southwell BR, Harms PJ, Brack CM, Pettersson TM, Schreiber G (1995a) Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties. Eur J Biochem 230:977–896. Duan W, Richardson SJ, Babon JJ, Heyes RJ, Southwell BR, Harms PJ, Wettenhall REH, Dziegielewska KM, Selwood L, Bradley AJ, Brack CM, Schreiber G (1995b) Evolution transthyretin in marsupials. Eur J Biochem 227:396–406. Fung WP, Thomas T, Dickson PW, Aldred AR, Milland J, Dziadek M, Power B, Hudson P, Schreiber G (1988) Structure and expression of the rat transthyretin (prealbumin) gene. J Biol Chem 263:480–488. Gilbert W (1978) Why genes in pieces? Nature 27:501. Grayson DR, Costa RH, Darnell JE (1989) Regulation of hepatocyte-specific gene expression. Ann NY Acad Sci 557:243–256.


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Hamilton JA, Steinrauf LK, Braden BC, Liepnieks JJ, Benson MD, Holmgren G, Sandgren O, Steen L (1993) The X-ray crystal structure refinements of normal human transthyretin and the ˚ resolution. J Biol Chem 268:2416–2424. amyloidogenic Val30! Met variant to 1.7 A Jinno Y, Matsumoto T, Kamei T, Kondoh T, Maeda S, Araki S, Shimada K, Nukawa N (1986) Localization of the human prealbumin gene to 18p11.1-q12.3 by gene dose effect study of southern blot hybridization. Jpn J Human Genet 31:243–248. Larsen NJ, Womack JE, Kirkpatrick BW (1996) Seven genes from human chromosome 18 map to chromosome 24 in the bovine. Cytogenet Cell Genet 73:184–188. Manzon RG, Neuls TM, Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151:55–65. Mita S, Maeda S, Shimada K, Araki S (1984) Cloning and sequence analysis of cDNA for human prealbumin. Biochem Biophys Res Commun 124:558–564. Moore MJ, Query CC, Sharp PA (1993) Splicing of precursors to mRNA by the spliceosome. In: Gesteland RF, Atkins JF (eds) The RNA World. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp 303–357. Morgado I, Melo EP, Lundberg E, Estrela NL, Sauer-Eriksson AE, Power DM (2008) Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal. Mol Cell Endocrinol 295(1–2):48–58. Motojima K, Goto S (1989) Brain-specific expression of transthyretin mRNA as revealed by cDNA cloning from brain. FEBS Lett 258:103–105. Motojima K, Goto S (1990) Dual promoters and tissue-specific expression of rat transthyretin gene. Biochem Biophys Res Commun 173:323–330. Nagata Y, Tashiro F, Yi S, Murakama T, Maeda S, Takahashi K (1995) A 6-kb upstream region of the human transthyretin gene can direct development, tissue-specific, and quantitatively normal expression in transgenic mouse. J Biochem 117:169–175. Prapunpoj P, Richardson SJ, Fumagalli L, Schreiber G (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order Insectivora, class Mammalia. Mol Biol Evol 17:1199–1209. Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol 279:R2026–R2041. Prapunpoj P, Richardson SJ, Schreiber G (2002) Crocodile transthyretin: structure, function, and evolution. Am J Physiol 283:R885–R896. Prapunpoj P, Leelawatwatana L, Schreiber G, Richardson SJ (2006) Change in structure of the Nterminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS J 273:4013–4023. Qiu H, Shimada K, Cheng Z (1992) Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization. Cytogenet Cell Genet 61:186–188. Remmers EF, Goldmuntz EA, Zha H, Crofford LJ, Cash JM, Mathern P, Du Y, Wilder RL (1993) Linkage map of seven polymorphic markers on rat chromosome 18. Mamm Genome 4:265–270. Richardson SJ, Bradley AJ, Duan W, Wettenhall REH, Harms PJ, Babon JJ, Southwell BR, Nicol S, Donnellan SC, Schreiber G (1994) Evolution of marsupial and other vertebrate thyroxinebinding plasma proteins. Am J Physiol Regulat Integr Comp Physiol 266:R1359–R1360. Richardson SJ, Wettenhall REH, Schreiber G (1996). Evolution of transthyretin gene expression in the liver of Didelphis virginiana and other American marsupials. Endocrinology 137:3507–3512. Samadani U, Costa RH (1996) The transcriptional activator hepatocyte nuclear factor 6 regulates liver gene expression. Mol Cell Biol 16:6273–6284. Samadani U, Qian X, Costa RH (1996) Indentification of a transthyretin enhancer site that selectively binds the hepatocyte nuclear factor-3 beta isoform. Gene Expr 6:23–33. Santos CRA, Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140:2430–2433.

58

P. Prapunpoj

Sasaki H, Yoshioka N, Takagi Y, Sasaki Y (1985) Structure of the chromosomal gene for human serum prealbumin. Gene 37:191–197. Sasaki Y, Yoshioka K, Tanahashi H, Furuya H, Sasaki H (1989) Human transthyretin (prealbumin) gene and molecular genetics of familial amyloidotic polyneuropathy. Mol Biol Med 6:161–168. Schreiber G, Pettersson TM, Southwell BR, Aldred AR, Harms PJ, Richardson SJ, Wettenhall REH, Duan W, Nicol SC (1993) Transthyretin expression evolved more recently in liver than in brain. Comp Biochem Physiol 105B:317–325. Soares ML, Centola M, Chae J, Saraiva MJ, Kastner DL (2003) Human transthyretin intronic open reading frames are not independently expressed in vivo or part of functional transcripts. Biochim Biophys Acta 1626:65–74. Sparkes RS, Sasaki H, Mohandas T, Yoshioka K, Klisak I, Sakaki Y, Heinzmann C, Simon M (1987) Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2-q12.1. Hum Genet 75:151–154. Sundelin J, Melhus H, Das S, Eriksson U, Link P, Tra¨gargh L, Peterson PA, Rask L (1985) The primary structure of rabbit and rat prealbumin and a comparison with the tertiary structure of human prealbumin. J Biol Chem 260:6481–6487. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. Tsuzuki T, Mita S, Maeda S, Araki S, Shimada K (1985) Structure of the human prealbumin gene. J Biol Chem 260:12224–12227. Tu G-F, Cole T, Duan W, Schreiber G (1989) The nucleotide sequence of transthyretin cDNA isolated from a sheep choroid plexus cDNA library. Nucleic Acids Res 17:6384. van Jaarsveld PP, Edelhoch H, Goodman DS, Robbins J (1973) The interaction of human plasma retinol-binding protein and prealbumin. J Biol Chem 248:4698–4705. Wakasugi S, Maeda S, Shimada K, Nakashima H, Migita S (1985) Structure comparisons between mouse and human prealbumin. J Biochem (Tokyo) 98:1707–1714. Wakasugi S, Maeda S, Shimada K (1986) Structure and expression of the mouse prealbumin gene. J Biochem (Tokyo) 100:49–58. Wallace MR, Naylor SL, Kluve-Beckerman B, Long GL, McDonald L, Shows TB, Benson MD (1985) Localization of the human prealbumin gene to chromosome 18. Biochem Biophys Res Comm 129:753–758. Wilce JA, Craik DJ, Ede N, Jackson DC, Schreiber G (1995) 1H NMR studies of peptide fragments from the N-terminus of chicken and human transthyretin. Biochem Mol Biol Int 36:1153–1159. Wojtczak A, Cody V, Luft JR, Paugborn W (1996) Structure of human transthyretin complexed ˚ resolution. ˚ resolution and 30 , 50 -dinitro-N-acetyl-L-thyronine at 2.2 A with thyroxine at 2.0 A Acta Crystallogr D52:758–765. Yamauchi K, Kasahara T, Hayashi H, Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132:2254–2261. Yamauchi K, Takeuchi H, Overall M, Dziadek M, Munro SLA, Schreiber G (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA: comparison of its pattern of expression during metamorphosis with that of lipocalin. Eur J Biochem 256:287–296.

Chapter 4

Evolutionary Insights from Fish Transthyretin Deborah M. Power, Isabel Morgado, and Joaõ C.R. Cardoso

Abstract Thyroid hormones (THs) synthesised and released by the thyroid follicles in fish circulate bound to TH distributor proteins (THDPs). Transthyretin (TTR) is a tetrameric THDP with a conserved structure in vertebrates and functions as a high affinity T4 binder in tetrapods and as a dual T3/T4 binder in fish. The difference in TH affinity appears to be linked to the longer N-terminal region in piscine TTR. Relatively few studies exist on gene regulation in fish, but those that do indicate that it may be similar to those described in mammals. Recently, a family of transthyretin-like proteins was identified and shown to correspond to the enzyme 5-hydroxyisourate hydrolase (HIUase) of purine metabolism in zebra fish. A combination of in silico and laboratory-based approaches indicate that TTR has a widespread distribution but is most abundant in liver, while HIUase expression seems to be mainly in the liver. Gene transcripts encoding TTR and HIUase genes are described in representatives of the orders Cypriniformes, Salmoniformes, Pleuronectiformes, Perciformes, Tetraodontiformes, Beloniformes, Gasterosteiformes, Gadiformes and Petromyzontiformes. The evolution of TTR and HIUase genes in fish is discussed. Keywords Evolution, Gene structure, Gene linkage, Regulation, Tissue distribution

Abbreviations RBP THs

Retinol binding protein Thyroid hormones

Deborah M. Power (*) Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005-139 Faro, Portugal e-mail: dpower @ualg.pt


59

60

THDP T4 TBG TSH TTR TLP T3

4.1

Deborah M. Power et al.

Thyroid hormone distributor protein Thyroxine Thyroxine-binding globulin Thyroid stimulating hormone Transthyretin Transthyretin-like proteins Triiodothyronine

Introduction

Fish are the most successful group of extant vertebrates with an estimated 25,000 species, which may increase in number when molecular methods for classification are applied. Fish are part of the subphylum Vertebrata (possessing a cranium and vertebrae) and include the ‘ancient’ jawless Agnatha and modern jawed fish such as the cartilaginous Chondrichthyes (which includes sharks and rays) and the bony Osteichthyes (which includes ray-finned fish, Actinopterygii and lung fish). The fish are of considerable scientific interest because of their basal position in the evolution of vertebrates the existence of a variety of genome sizes and ploidy levels, the specific genome duplication proposed to have occurred in the teleost lineage (Ohno 1970; Jaillon et al. 2004; Taylor et al. 2003), their adaptation to a wide range of aquatic habitats throughout the World, their extreme biological diversity and the numerous examples of active speciation. In fact, fish have been crucial in establishing current models of vertebrate genome evolution (Jaillon et al. 2004; Ventakesh 2003). Despite their diversity and even taking into account the very limited number of species studied, there appears to have been a general conservation of an important regulatory network, the endocrine system. The thyroid system is no exception and most of the elements of this endocrine axis are conserved from fish to mammals. The present review will focus on one of the thyroid hormone distributor proteins (THDP), transthyretin (TTR), in fish. The recently identified family of genes encoding TTR-like proteins (TLP) will be touched upon in the context of TTR evolution in fish, as these recently identified proteins are dealt with in detail in other chapters of the present book. A number of reviews exist about TTR in vertebrates and provide an integrated picture of evolution and function of this protein across diverse classes of animals (Richardson 2007; Schreiber and Richardson 1997), and so this will only briefly be considered. The thyroid axis will be overviewed for readers unfamiliar with fish endocrinology and an update of the status of TTR in fish, which was first reviewed in 2000 will be given (Power et al. 2000). Some unpublished data will be introduced to highlight new and pertinent information about piscine TTR gene evolution and function.

4 Evolutionary Insights from Fish Transthyretin

4.2

61

Thyroid Axis

The thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), are produced by thyroid follicles, which in most fish are not encapsulated in a gland but instead are scattered throughout the connective tissue of the lower jaw usually aggregated around the ventral aorta (reviewed by Bentley 1998; Leatherland 1994). The basic structural unit of the thyroid is the follicle, which is composed of a single layer of epithelial cells surrounding a central colloid-filled cavity. The thyroid appears to have the longest phylogenetic history of any endocrine gland and its basic follicular unit has been conserved in all vertebrates and homologous tissues have been identified in protochordates, including amphioxus (Cephalochordata) and ascidians (Urochordata) (Barrington 1962). The central components of the thyroid axis have largely been conserved across vertebrates, and in fish, thyroid function from TH biosynthesis to TH action seem to fit the mammalian model. Thyroid stimulating hormone (TSH) stimulates T4 secretion from the thyroid, which is converted in the periphery to T3 by deiodination. A family of selenoprotein enzymes, the deiodinases (I, II and III), activate and inactivate the THs in fish, including agnathans, chondrichthyes and teleosts (Orozco and Valverde-R 2005). THs bring about their action by binding to nuclear thyroid receptors (TRs) and activate or repress gene expression (Chin and Yen 1997; Zhang and Lazer 2000). The cytosolic TH-binding proteins and plasma membrane TH transporters identified in mammals still remain to be characterised in fish. In contrast to mammals, where plasma T4 largely exceeds T3 levels (50–100:1), plasma T3 levels in teleosts may exceed that of T4, suggesting a strong 50 -monodeiodination activity, and peripheral deiodination may be the primary control of teleost thyroid function (reviewed in Eales and Brown 1993; Power et al. 2008). In teleosts, as in mammals, more than 99% of the THs do not circulate in the free form (Eales and Shostak 1985; Weirich et al. 1987) but are bound to plasma proteins including lipoproteins (Babin 1992). In fish, TH transport by plasma proteins is poorly explored and so far only two of the three THDPs present in the blood of mammals have been reported, albumin (Richardson et al. 1994) and TTR (Kawakami et al. 2006; Santos and Power 1999; Yamauchi et al. 1999; Manzon et al. 2007).

4.3

Thyroid Hormones Distributor Proteins

The relative abundance and hormone binding properties of THDPs are not conserved in vertebrates. In a series of studies, reviewed by Richardson (2002), the presence of THDPs in serum samples from representatives of different vertebrates (mammals, birds, reptiles, amphibians and also fish, (Richardson et al. 2005)) was analysed. As albumin is the only THDP identified in all species studied, it is

62


proposed to be the evolutionarily oldest TH carrier protein. The same studies suggest an apparent absence of TTR in the plasma of adult amphibians, reptiles and fishes, although the presence of gene transcripts and protein is well established for the early stages of development (Funkenstein et al. 1999; Kawakami et al. 2006; Richardson et al. 2005; Santos and Power 1996, 1999; Yamauchi et al. 1993, 1998, 1999), which are characterised by a surge in TH blood levels (Hulbert 2000). The recent development of specific anti-sera for piscine TTR its presence by revealed by both western blot and ELISA blood of juvenile as a tetramer in juvenile (5 months), young adult (13 months) and adult (26 months) sea bream (Sparus aurata, Morgado et al. 2007b) and several other perciform species (authors’ observations). These data raise questions about why TTR was not detected in adult blood in the previous studies that used binding assays followed by native electrophoresis (Richardson et al. 2005). In the study by Morgado et al. (2007b), a specific ELISA was developed and revealed that the concentration of TTR in sea bream plasma is 4 mg ml1, which is 100 times lower than that in human plasma (0.3 mg ml1) (Ingenbleek et al 1972; Purkey et al. 2001; Sachs and Bernstein 1986), and may explain the failure to detect it in binding assays. In fact, it has been estimated that in humans the concentration of plasma TTR largely exceeds that of plasma THs (0.05-0.12 mg ml1), and less than 1% is estimated to be involved in TH transport (Robbins and Bartalena 1986). In this context, it has been proposed that the large excess of plasma TTR is associated with its second important function, to bind and transport all-trans-retinol complexed with retinol binding protein (RBP) and thus reduce glomerular filtration of retinol (Kanai et al. 1968; Monaco et al. 1995; Peterson 1971; Van Jaarsfeld et al. 1973). In the 1970s, RBP was isolated from the plasma of several fish species (Shidoji and Muto 1977) and rainbow trout (Oncorhynchus mykiss) and was shown to interact weakly with mammalian TTR (Berni et al. 1992). The subsequent cloning and isolation of TTR (Santos and Power 1996, 1999; Yamauchi et al. 1999; Kawakami et al. 2006; Manzon et al. 2007) and RBP (Bellovino et al. 2001; Sammar et al. 2001) from fish permitted binding studies with the piscine proteins, which revealed that TTR has a very low affinity for RBP (Folli et al. 2003). More recently, it has been clearly demonstrated that they do not form a complex (Zanotti et al. 2008). We hypothesise that the high circulating levels of plasma TTR and its tendency to form a TTR-RBP complex in mammals and birds (Heller 1976; Kopelman et al. 1976; Blaner 1989; Blomhoff et al. 1990), but not in fish, may indicate that the role of TTR in retinol transport may be an innovation in tetrapods. Further studies will be required to clarify when TTR emerged as an RBP binder in vertebrates and the evolutionary pressure that favoured the molecular modifications in the gene(s) which led to this function. Confirmation of whether RBP exists or not in lamprey (Petromyzon marinus, Lampetra appendix) and studies of its interaction with recently cloned lamprey TTR (Manzon et al. 2007) should contribute to resolve this issue. Nonetheless, preliminary analysis of the amino acids in TTR important for the stabilization of the TTR-RBP complex in humans (Folli et al. 2003)


63

reveals that they are more conserved in lamprey (5 out of 8 amino acids) than in sea bream (2 out of 8 amino acids).

4.4

Structure and Hormone Binding Characteristics

Cloning of sea bream TTR cDNA coupled with homology modelling using human (Hamilton et al. 1993) (PDB ID: 1tta), rat (Wojtczak et al. 2001) (PDB ID: 1gke) and chicken (Sunde et al. 1996) (PDB ID: 1tfp) TTR predicted that the overall topology of sea bream TTR is preserved, although its surface potential, especially at the TH binding site, is much more electronegative in chicken and sea bream (Power et al. 2000). Subsequent crystallographic studies confirmed the conserved structure of sea bream TTR with that of mammalian and avian TTRs (Folli et al. 2003; Eneqvist et al. 2004) and revealed that it is a tetramer that contains two high affinity sites to which L-T4 may bind. The TH binding site is highly conserved, although Ser 117 (human sequence) is substituted by Thr in sea bream TTR (Folli et al. 2003). The affinity of fish TTR for THs is ambiguous and initial qualitative binding studies indicated that T3 appeared to have greater affinity than T4 for sea bream TTR. However, the development of a specific binding assay using recombinant sea bream TTR revealed that it binds T4 and T3 with a similar affinity (9.8 0.97 and 10.6 1.7 nM, respectively; Morgado et al 2008). Interestingly, truncation of the N-terminal region of the protein modified its affinity for T4 but did not significantly affect its affinity for T3 (Morgado et al. 2008). The results of this study substantiate the idea that the N-terminus of TTR plays an important role in determining hormone affinity (Prapunpoj et al. 2006). Recent studies have revealed that in common with what is observed in other vertebrates (Brouwer et al. 1999; Cheek et al. 1999; Ishihara et al. 2003a, b; Lans et al. 1993; McKinney and Waller 1994; Rickenbacher et al. 1986; Yamauchi et al. 2000), piscine TTR has a high affinity for a number of pollutants commonly found in the environment (Morgado et al. 2007a). Interestingly, however, despite the relatively high overall sequence and structural conservation of vertebrate TTR, the binding profile of chemical contaminants varies, for example, diethylstilbestrol (DES) and pentachlorophenol are powerful inhibitors of [125I] T3 binding to chicken, bullfrog, Xenopus, Rana and Japanese quail TTR, but have poor affinity for piscine TTR (Ishihara et al. 2003a, b; Morgado et al. 2007a; Yamauchi et al. 2000). It will be of interest in the future to establish the structural basis of this differing affinity and such studies may help to better define tetramer/ligand interactions. Moreover, understanding the biological consequences for the organism of the high affinity of some pollutants for TTR represents a significant challenge because of the importance of THs at different stages of the life cycle. The tendency of pollutants to accumulate in aquatic ecosystems (from industrial residues, land run-offs and acid rains) and the consequent continuous exposure of aquatic organisms to such pollutants make studies in aquatic vertebrates such as fish pertinent.

64

4.5 4.5.1


Evolution TTR Tissue Distribution and Function

TTR has been identified in most vertebrate classes, including placental mammals, marsupials, birds, reptiles, amphibians and fish (Dickson et al. 1985; Duan et al. 1991, 1995; Larsson et al. 1985; Manzon et al. 2007; Mita et al. 1984; Prapunpoj et al. 2000a, 2002; Richardson et al. 1994, 1997; Santos and Power 1996, 1999; Tsuzuki et al. 1985; Yamauchi et al. 1993, 1999), and their overall amino acid sequence similarity is relatively high. TTR tissue expression and secretion is variable in the vertebrates in which it has been studied, and on the basis of the existing data, its specific expression in the liver or choroid plexus has been proposed to be class- and stage-dependent (reviewed in Richardson 2007). To explain life-stage-specific variations in the expression of TTR and other THDPs, it has been hypothesised that an augmented THDP network occurs at times of elevated TH in blood, such as, during development (Richardson et al. 2005). As mentioned earlier, fish are extremely diverse, and representatives from only a fraction of the superorders have been studied. However, considerably more examples from fish are required before a clear picture of the evolution of TTR, and its tissue-specific expression or life-stage-specific variations can be established. In Cyclostomata (e.g. lamprey), RT-PCR has revealed moderate TTR expression in the liver and heart and low expression in the brain gills, gut, kidney and blood of larvae (Manzon et al. 2007). Analysis of TTR was restricted to the liver and brain in adult fish, where the expression appeared to be much lower than that of the larval and juvenile stages. Analysis of TTR expression in other fish is restricted to a salmoniform (masu salmon, Oncorhynchus masu) and two perciformes (sea bream and Pacific bluefin tuna, Thunnus orientalis). In these species, TTR appears to be expressed during ontogeny (Funkenstein et al. 1999; Santos and Power 1996, 1999) and in juveniles, principally in the liver, although in the sea bream it is also detected at appreciable levels in the intestine and heart and to a lesser extent in the kidney, skin and brain (Power et al. 2000). Despite repeated attempts to identify TTR protein by immunohistochemistry (IHC) in the liver of juvenile and adult sea bream, sea bass (Dicentrarchus labrax) and Atlantic halibut (Hippoglossus hippoglossus), it has not been possible (unpublished observations). Western blot of adult sea bream liver extracts also revealed relatively low concentrations of TTR protein in spite of fairly constant circulating concentrations (4 mg ml1) and generally high hepatic transcript abundance. We hypothesise that such observations are consistent with the constitutive release of TTR (Morgado et al. 2007b). In addition, the detection of TTR in adult plasma suggests that it is very important as a THDP during development in fish. Relatively few physiological studies of TTR have been carried out on fish and its regulation and response to changing conditions remain largely unknown. In the sea bream injection of the sex steroid, estradiol-17 (5 mg g1 body weight) causes a reduction in hepatic TTR transcripts (Funkenstein et al. 2000), although it remains


65

to be established if this is a normal physiological response during reproduction. Food restriction for several weeks in sea bream (Power et al. 2000) causes downregulation of liver TTR mRNA expression, suggesting that fish may be similar to mammals in which TTR is a marker of malnutrition or stress (Ingenbleek et al. 1972; Ingenbleek and Young 1994, 2002; Ingenbleek and Berstein 1999; Beck and Rosenthal 2002; Brugler et al. 2002). When plasma T3 or T4 are increased in fish, there is a significant increase in the circulating levels of TTR, although hepatic transcription is unchanged (Morgado et al. 2007b). Further studies of hepatic gene regulation and protein turnover are required to elucidate the way in which TTR contributes to TH homeostasis and whole animal physiology in fish.

4.5.2

TTR and TLP Evolution in Fish

The integration of data about TTR gene sequence, protein structure and hormone affinity across vertebrates has led to a general evolutionary model. TTR is proposed to have changed from a T3 binder in ‘lower vertebrates’ to a T4 binder in eutherians as a result of changes in the length of the N-terminal region. In vertebrates, the TTR gene is composed of four coding exons, and the longer hydrophobic N-terminus in fish and the shorter hydrophilic region in mammals are a consequence of a shift in the intron1/exon2 splice site during evolution (Aldred et al. 1997; Prapunpoj et al. 2000a, b, 2002). In masu salmon and bluefin tuna, the affinity of TTR is greater for T3, and in sea bream, TTR has similar affinity for T3 and T4 and the N-terminus of the fish proteins is several amino acids longer than mammalian TTR (Kawakami et al. 2006; Morgado et al. 2008; Yamauchi et al. 1999). The affinity of lamprey TTR for THs has not yet been determined, but analysis of the 50 region of the genomic DNA is consistent with the proposed evolutionary model and the N-terminus is nine amino acids longer than mammalian TTR (Manzon et al. 2007). The increase in available sequence data for fish in recent years makes evolutionary studies relevant, although a more comprehensive analysis still requires the inclusion of sequences from far more species. A family of transthyretin-like proteins (TLPs) or transthyretin-related proteins (TRPs, Eneqvist et al 2003) has recently been identified in prokaryotes and eukaryotes and shown to be enzymes (Lee et al. 2005; Ramazzina et al. 2006). The enzyme 5-hydroxyisourate hydrolase (HIUase) of purine metabolism in zebra fish (Danio rerio, Q06S87) is a TLP, and duplication of this gene is proposed to have given rise to TTR in vertebrates (Zanotti et al. 2006). To conduct phylogenetic analysis of TTR and TLP in fish, extensive searches in Genebank (www.ncbi.nlm.nih.gov) and Ensembl (www. ensembl.org) (October 2008) were carried out and specific cDNA, ESTs and predicted genes were retrieved (Table 4.1). It was possible to identify gene transcripts encoding TTR and HIUase genes (TLP) in 18 species, which included representatives of the orders Cypriniformes, Salmoniformes, Pleuronectiformes, Perciformes, Tetraodontiformes, Beloniformes, Gasterosteiformes, Gadiformes and Petromyzontiformes. The ESTs encoding TTR were identified in a range of

66


Table 4.1 Accession numbers of the TTR and HIUase (TLPs) proteins, predicted genes and ESTs clones identified by searching public databases (available from NCBI and Ensembl) using the mature precursor sequences of published teleost TTR sequences with the tBlastn algorithm (sequences with e-value smaller than 105 were selected) Accession numbers Observations ACTINOPTERYGII (ray-finned fishes) Beloniformes Oryzias latipes (Medaka) TTR Not identified HIUase1 AM156132 BJ516106, BJ010835, BJ024650, DK059860, DK047918, DK070190, DK164988, DK141970, DK082087, DK145206, DK168247, DK170492, DK147440 BJ910632, DK261436, DK261403, DK242464, DK261679, DK242707, DK242998, DK261968, DK229688, DK248571 DC247390, DC256322 ENSORLG00000009326 HIUase2 ENSORLG00000000779 Gasterosteiformes Gasterosteus aculeatus (Three-spined stickleback) TTR Not identified HIUase1 DN731287, DW668813, DW668812 ENSGACG00000015936 HIUase2 ENSGACG00000008507 Perciformes Sparus aurata (Seabream) TTR CB184881, CB184737, CB184929 AAC26108 HIUase

AM950811, AM952219, AM952201, AM951997 Dicentrarchus labrax (Seabass) TTR CV186278 HIUase FM000902, FM000585 FM019182 Pleuronectiformes Platichthys flesus (Flounder) TTR DW985862 ABU54858 HIUase DV566090, EC378613 Psetta maxima (Turbot) TTR Not identified HIUase EY454781

Neurula Embryo

Liver

Fin Predicted Predicted

Brain Predicted Predicted

Liver and embryos Santos and Power (1999) Liver

Liver Liver Kidney

Liver Goetz et al. (unpublished) Liver

Liver (continued )

4 Evolutionary Insights from Fish Transthyretin Table 4.1 (continued) Accession numbers Tetraodontiformes Tetraodon nigroviridis (Green spotted pufferfish) TTR CR642656, CR635654, CR650478, CR649397, CR636486, CR651447, CR651621, CR652101, CR656042 ENSTNIG00000010473 HIUase SCAFFOLD_14581 Takifugu rubripes (Japanese pufferfish) TTR ENSTRUG00000006085 HIUase1 SCAFFOLD_2288 HIUase2 SCAFFOLD_9318 Cypriniformes Danio rerio (Zebra fish) TTR CN167258 EH532041, EH505861,EH515185, EH488817 AAI64894, XP_001919368 HIUase BC090440, BC097189 Q06S87 ENSDARG00000068644 Cyprinus carpio (Common carp) TTR CA966004, CA964904 CAD66520 HIUase Not identified Carassius auratus (Goldfish) TTR ABY40363 HIUase Not identified Gobiocypris rarus (Rare minnow) TTR ACE74244 HIUase EE394013 Gadiformes Gadus morhua (Cod) TTR ES240035, ES238908, ES239200, ES240301 HIUase ES471358 Salmoniformes Oncorhynchus mykiss (Rainbow trout) TTR BX886606, CX256523,BX878043, BX878043, CU073679, CX256522, CA377780 CX152238,CX148258,CX139266,CX138173, CX138172 CB497711 HIUase CU066583 BJ494178 Salmon salar (Atlantic salmon) TTR CK888905, BE518610, AM397609

67

Observations

Liver

Predicted Predicted Predicted Predicted Predicted

Liver Gut/Internal Organs Predicted Embryo Zanotti et al. (2006) Predicted Liver Apreda et al. (unpublished)

Wiens et al. (unpublished)

Li et al. (unpublished) Liver

Liver Liver

Multi-tissues Juvenile Steelhead Multi-tissues Embryo Liver (continued )

68 Table 4.1 (continued) Accession numbers CK887387 CK895595 CK990828 HIUase EG904021, EG904019 Thymallus thymallus (Grayling) TTR FF846145, FF846144 HIUase Not identified CEPHALASPIDOMORPHI (Lampreys) Petromyzontiformes Petromyzon marinus (Sea lamprey) TTR ABI93605 HIUase Not identified Lampetra appendix (American brook lamprey) TTR ABI93606 HIUase Not identified


Observations Kidney Swim bladder Multi-tissues Multi-tissues Multi-tissues

Manzon et al. (2007)

Manzon et al. (2007)

different piscine tissue libraries, which included liver, gut, kidney, swimbladder, whole juvenile and embryo. In contrast, HIUase cDNAs were principally identified in liver, although they were also identified in whole embryo and trunk kidney cDNA, suggesting this gene may have a more restricted tissue distribution than TTR, although gene expression studies are required to confirm the in silico results. An alignment of piscine TTR and HIUase amino acid sequences (Fig. 4.1) revealed that they are highly conserved. A novel zebra fish HIUase gene (HIUase2) was identified and duplicate HIUase genes were also found in stickleback (Gasterosteus aculeatus), Takifugu (Takifugu rubripes) and medaka (Oryzias latipes) genomes (Fig. 4.1). In common with the gene structure of teleost TTR, four coding exons were also identified for HIUase genes and sequence comparison suggests that in some species N-terminal splicing of the HIUase gene probably occurs and accounts for shorter transcripts lacking exon 1, which are similar in size to the mouse homologue. In the other teleosts for which sequence data exists, a single copy gene for HIUase was identified and the presence of a duplicate copy remains to be established. The teleost TTR and HIUase genes share conserved splice consensus regions and the signature motifs identified in prokaryotes and eukaryotes such as the N-terminal VKILDAV and C-terminal GHRHY in TTR and DGRCPG in TLPs are also present (Hennebry et al. 2006). The C-terminal TLP motif, YRGS, was present in nearly all piscine HIUase, with the exception of stickleback HIUase2 (ENSGACG00000008507, contig_1506) in which R has been substituted by T (YTGS) and in medaka HIUase2 (ENSORLG00000000779, chromosome 6) where the C-terminal YRGS motif is substituted by HRSN. Of particular note was the large number of cDNAs identified for HIUase in the medaka, which were the product of the HIUase1 gene. However, no EST transcripts


SP

TTR Mouse Seabass Seabream Flounder Takifugu* Tetraodon Goldfish* Carp Zebrafish Cod Trout Salmon Grayling* Lamprey SeaLamprey

69

MASLRLFLLCLAGLVFVSEAGPA--------GAGESKC MVK MLQP-LRCLLLTSAVLLCNATPT---PTEKHGGTDTRC TVK MLQP-LHCLLLASAVLLCNTAPT---PTDKHGGSDTRC MVK -IQP-VHCLLLASAVLLCNSSPT---PTEKHGGSDTKC TVK -----------------------------AHGGSDTKC TVK MLPL-LLSLLLVSAALPSHAAPV----LKSHGGSDTTC TVK -------------------SAPV-----GIHGGSDAHC TVK MAKA-VICVLLVSLFACCRSAPV-----GIHGGSDVHC TVK MAKE-VICVLLASLFALCRSAPV-----AFHGGSDAHC TVK MMRP-LLWLLLASISLPCDTAPV-----EKHGESDTTC MVK MDSS-LLCVLLATAVLLCSAAPV-----DRHGESDTHC MVK MDSS-LLCVLLAAAVLLCSAAPV-----DRHGESDTHC MVK ------------------------------------------K MTRFLCLLVLVASSSLLCSADDDHKSHESHEGGVKDSC MVKA -MRFLCLLVLVASSSLLCRADDDHKSHESHEGGVKDSC MVKA

AVR AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK SVR SVQ

S AVD T AGS T AGS T AGS T AGP TAAGP T AGN T AGN T AGN L AAS V AGA V AGA V AGS K AAG K AAG

A A A A A A A A A A A T A K K

K K K K N T D D D K S S S S S

F-KKTSEGS S-QKAADGG S-QKTADGA F-QKAADGA Y-QRTADGG Y-KKAADGG Y-RQEQGGT Y-RQDQGGT F-RQDQGGT S-KKGTDGE S-RRVNGMT S-RRVNEMT S-RRVNGMT M-KKQEDAS M-KKQDDAS

--------------------------------MATESS TTH TAS L AQG ---------MSTFR-LQQLKGHISPENKITTMASLE-S TTH VAM V ASN -------------------------------MSTAEVS TTQ SSD V GAR -------------------------------MAGSA-S TTH TAT V GAN ---------------------------------MMESCT TTH SVD V AAR --------MAAAESSPLTTHVLNTGDGVPAAMAAAESS TTH TGD V AAR ---------MGANR-LQQLKGHILPENKITAMAGSP-S TTH TGM V GSN ---------MSVSR-LQKLKGHILSENKSTAMAGSP-S TTH TAL I ASN -------------------------------MAAAASS TTH TGD I AAK -------------------------------------- TTH TAM I ASN -------------------------------------------------------------------------------------------S TTH TGM I ASN -----------MNR-LQHIRGHIVSADKHINMSATLLS STH IAQ V GAN ------------------------------MSSASDIS STH TGD V AQR -----------MNR-LQHIRGHIVSADKY--MAATLPS TTH TAQ V GAN ---------MSADRRLHNLSNQIVAANPCGAMAQPG-S TTH TAM V GSN ---------MSTSR-LQHIKDHLLDEYTCAEMAAPY-S TTH TGM V GAH ------------------------------MSSTLPNS TTY TGD V GAR ------------MIKFLLFLAIAAATVISNAELAVPTAS SAH ISG S AGG

C T A T A A A A A A A A T T T T A A Q

R SRLEAPCQQ R YRQDPSSKT S HRLDSELVI S HRQDPLTSA S HRLEPPLMV S HRLDSDLMI S YRQDPSTNV S SRQVPSTND S HRLDSELMI H YRQDPSTNA S HRLDSKLMI N YRKDPSNDT V HRLDPVSSA S HRLEPRITV I YRLDPISSV L YKQDSSAAA S HRMDPSTSL S HRLDSLLVI LAFILLN--NG

EPFAS TH AN TQ AT TQ AN TQ AN TQ AN EK AS EK AS EK AS TH AI AQ AS AQ AS AQ AS KE AT KE AT

K AES V AT V AT V DT M AS T AT KV IT KV MT KV MT V ET V LT V LT V LT V GKT V GKT

ELHG EIHD EIHN ESHN EIHN EIHE EVHN EVHN EVHN EVHN EVHN EVHN EVHN ESHH ESHH

78 82 82 81 57 81 62 80 80 80 80 80 44 86 85

RTSY LD NT I AD SV T EA TT I AD SL T ED SV T ED TT T DD IN T DD SI T ED KT T ED SV - ED KT T ED TT I DD TV S ED TT T ED TT I SD TT T DD TV T DD GSQF QDN

RCPG RYPG RCPG RCPG RCSG RCPG RCPG RCPG RCPG RCPG CCPG RCPG RCPG RCPG RCPG RCPG RCPG RCPG RVDW

55 76 56 55 54 79 76 76 56 49 31 50 75 57 73 77 76 57 73

HIUase Mouse Medaka1 Medaka2 Stickleback1 Stickleback2 Seabass2 Seabream1 Flounder1 Turbot2 Takifugu1* Takifugu2* Tetraodon1* Zebrafish1 Zebrafish2 Minnow1 Cod1 Trout1 Salmon2 Celegans

ME QL TM SL SL NM SL TL NM SL NM SL NI SL NL NL NL NL TN

TTR Mouse Seabass Seabream Flounder Takifugu* Tetraodon Goldfish* Carp Zebrafish Cod Trout Salmon Grayling* Lamprey SeaLamprey

TTDEK TEQQ TEQQ TEQQ TEQK PEEA TEQE TEQE AEQE TDQQ SDQD SDQD SEQD GDKD SDKD

VE PA PA SA LP PP TP TP TP PA QS QS QA TE T TE T

ELD E D E D E D D D D D E D E D E D D D E D E D E D R D R E

KS KA KA KS KS KA KS KA LT KA KA KA KA QA QQ

KTL KNE TNQ KNE KNE MNE KAE KAE KTE KSQ KSQ KSQ KSQ TKA TKT

I P S P S P S P SVP N P R P R P R P S P T P T P T P I P I P

HEFAD TANDSGHRH T AA P TAVVSNPQNHEAAD EAHTEGHRH T A -P TAVVTD-H-HEVAE DAHPEGHRH T A P TAVVSSVHEHEAAD EAHAEGHRH T A P - TAVVTDTHQHEVTN EAHSEGHRH T A P TALVTDVQHHEVTN EAHAGGHRH T A P TALVSDVPHHQL-------------------------------------HQLAD EAHAEGHRH T A P TAVVVKAHEHQLAD EAHAEGHRH T A P TAVVVKAHDHEMAE EAHGEGHLH T A P TAVVS-THQHETAE EAHAEGHRH T A P TAVVMKAHEHETAE EAHAEGHRH T A P TAVVMKAHEHETAE EAHAEGHRH T A P TAVVINAHEHEAAE MAHGAGHKH H P P F A GTIVGDAEGH HEAAE MAHGAGHKH H P P F A GAIVVDGKGH

F H R H R R H H R H R H R R R H R R V

ER AQ AS AQ GS GS VQ AQ GS AK GP AQ GK QQ GK DR GQ GQ EP

KER QES YPYVE TITKETQ-K H P P YRGS ASL D S YPYVE TINDPGQ-K H P R YRGS ESRTQ S YPYVE SISEPEQ-S H A P HRSN QSMEE C YPYVE TINDPGK-KHH P R YRGS ESR Q SLHPYVE SISDHHQ-RIH P R IYTGS ETL Q S YPYVE TISEPDQ-RVH P R YRGS ESL E C YPYVE TINNPGQ-K H P R YRGS ASI D S YPYVE TINDPGL-K H P R YRGS ESL Q S HPYVE TISSPEQ-R H P R YRGS ERI E G YPYVE TINDPGQ-K H P P YRGS ESL HIS YPYVE R---------------------------ESM Q S YPYVE IINDPGQ-K H P R YRGS DAL E C YPYVE TITNTSQ-H H P R YRGS ESL Q S YPYVE TITDVDQ-RLH P R YRGS DAL E C YPYVE TITDPSQ-K H P C YRGS GCL EES YPYVE TIRNPVD-K H P R YRGS GSL E S YPYVE TITDHSQ-K H L C R YRGS ESL HNS YPYVE TITNPNE-R H P R YRGS TAKNVES YPYVE NIRNATQ-H H P T P G YRGS

147 151 151 149 126 150 94 149 149 148 149 149 113 156 155

HIUase Mouse Medaka1 Medaka2 Stickleback1 Stickleback2 Seabass2 Seabream1 Flounder1 Turbot2 Takifugu1* Takifugu2* Tetraodon1* Zebrafish1 Zebrafish2 Minnow Cod1 Trout1 Salmon1 Celegans

TPSQIKP T TKEL TA RREA TP TTQM TS AAREELGP T SRRA AA TKET TP TKEL TP SREA IS TPQA TSD PHPD LP TSQM TS TKEN IA SRDA TP KKES VA TTREL TP TRET TPA TTKA TP SPDFTLIP T

D E E D E E E D E E E D E E E D E E I

118 139 119 118 117 142 139 139 119 112 67 113 138 120 136 140 139 120 136

Fig. 4.1 Multiple sequence alignment of the teleost and lamprey TTR and HIUase mature precursors with mouse TTR and HIUase and C.elegans TLP. The order of teleost sequences in the alignment is in agreement with Table 4.1 and their common names are indicated: Medaka (Oryzias latipes), Stickleback (Gasterosteus aculeatus), Seabass (Dicentrarchus labrax), Seabream (Sparus aurata), Flounder (Platichthys flesus), Turbot (Psetta maxima), Takifugu (Takifugu rubripes), Tetraodon (Tetraodon nigroviridis), Goldfish (Carassius auratus), Carp (Cyprinus carpio), Zebra fish (Danio rerio), Minnow (Gobiocypris rarus), Cod (Gadus morhua), Trout (Oncorhynchus mykiss), Salmon (Salmon salar), Grayling (Thymallus thymallus), Lamprey

70


TAKIFUGU Scaffold_401

TTR ZEBRAFISH Chr24

TETRAODONT Scaffold_14628

ECT2

B4GALT6 TTR

RFC4 B4GALT6

B4GALT6

B4GALT6

TTR

TTR

TTR

0,1M

0,03M

3M

0,124M STICKLEBACK Scaffold_348

MEDAKA Chr3

HIUase ZEBRAFISH Chr 18

TCF25

MVD PDCD5

HIUase1

MTHFSD9 TCF25

TCF25 HIUase1

PDCD5

PDCD5

MTHFSD

MVD FOXF1

FOXF1

MTHFSD

HIUase1 0,029M

Chr6 Chr 25

RFWD3 HIUase2 0,1M 0,4M

MOUSE Chr 7 PCDC5

0.1M Scaffold_1056

0,2M

ZNF277P

MOUSE Chr 18

ECT2 RFC4

TNN12

ZNF277P TNN12

TNN13 CACNA2D4 LRTM2

TNN13 CACNA2D4

RFWD3 HIUase2 ZNF277P

HIUase2

HIUase 9M

LRTM2 RFWD3 0.4M

Fig. 4.2 Comparison of the TTR and HIUase (TLPs) gene environments in teleosts with the mouse homologue regions. Genes are presented by open boxes and chromosomes or genome fragments by vertical lines. Only common genes are represented and are named according to HUGO annotation. TTR and HIUase genes are in bold and underlined and their respective linked genes, B4GALT6 and PDCD5 are in bold. Homologue genes were identified based upon their sequence similarity using the BLAST programme and the linkage maps were constructed based on gene annotated available from Mapviewer (http://www.ncbi.nlm.nih.gov/mapview/) and Ensembl (www.ensembl.org) databases. The figure is not drawn to scale and the size of the genome regions analysed is indicated

< Fig. 4.1 (continued) (Lampetra appendix) and Sea lamprey (Petromyzon marinus). The HIUase duplicates identified in teleosts are designated as 1 (most similar to the published zebra fish HIUase sequence) and 2. Vertical arrows indicate the localisation of exon–intron splice sites and the double arrow the homologue region of the human signal peptide sequence (SP). The degree of amino acid conservation between TTR and HIUase is indicated by shading and an asterisk (*) indicates teleost sequences that are incomplete. Specific motifs previously identified by Hennebry et al. (2006) are within open boxes and the region where amino acids are proposed to have been lost at the 50 end of exon 2 is in a dashed box. The conserved exon–intron splice sites are indicated by open arrows and the closed arrow indicates the beginning of the homologue region of the mature human TTR protein. Accession numbers of the predicted genes, ESTs and peptides used to generate the alignment are indicated in Table 4.1. The Minnow TTR sequence was not included as it was very incomplete. The accession numbers for TTR and HIUase from mouse (Mus musculus) are NP_038725 and Q9CRB3, respectively, and transthyretin-like protein (TLP) for C. elegans is ZK697


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for its paralogue or for the predicted stickleback HIUase2 gene were identified, suggesting that they may be low abundance or expressed in tissues for which few ESTs exist in the NCBI database. A further intriguing observation in the medaka and stickleback was the failure to identify a homologue of the teleost TTR gene despite the extensive number of Genebank sequences that exist for both model organisms (22,500 and 19,000, respectively, in the Unigene database, http://www. ncbi.nlm.nih.gov/), and it will be of interest to explore the significance of these observations. It is generally accepted that gene duplication has fuelled organismal complexity, and two major rounds of genome duplications (1R and 2R) are suggested to have occurred at the origin of vertebrates. Such events explain the general increase in gene number in vertebrate genomes and provide a plausible explanation as to how TTR emerged in the vertebrates. Despite the demonstrated sequence and structural conservation of TTR and HIUase (TLP) genes in vertebrates, comparison of the immediate gene environment fails to reveal homologous regions, suggesting that after gene duplication the future TTR gene was transposed to a different genome region (Fig. 4.2). However, some conservation of the flanking genes exist in teleosts and mammals and homologues of B4GALT6 map in close proximity to TTR and homologues of PDCD5 are linked to teleost HIUase1 and mouse HIUase, suggesting that the HIUase2 gene is the duplicated gene form of TLPs, which arose from a specific duplication event. The failure so far to identify a TTR paralogue in teleosts may indicate that it was either eliminated from the genome or arose as a consequence of partial genome duplication.

Acknowledgements Some of the work described was co-financed by POCI 2010 and European Social Funds attributed by the Portuguese National Science Foundation (FCT) to project POCTI/ CVT/38703/2001 and Pluriannual funding to CCMAR.

References Aldred A, Prapunpoj P, Schreiber G (1997) Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3’ splice site of intron 1). Eur J Biochem 246:401–409 Babin PJ (1992) Binding of thyroxine and 3,5,30 -triiodothyronine to trout plasma lipoproteins. Am J Physiol 262:E712–E720 Barrington EJ (1962) Hormones and vertebrate evolution. Experientia 18:201–210 Beck FK, Rosenthal TC (2002) Prealbumin: a marker for nutritional evaluation. Am Fam Phys 65:1575–1578 Bentley PJ (1998) Comparative Vertebrate Endocrinology, 3rd edn. Cambridge University Press, Cambridge Bellovino D, Morimoto T, Mengheri E, et al. (2001) Unique biochemical nature of carp retinolbinding protein: N-linked glycosylation and uncleavable NH2-terminal signal peptide. J Biol Chem 276:13949–13956

72


Berni R, Stoppini M, Zapponi MC (1992) The piscine plasma retinol-binding protein: purification, partial amino acid sequence and interaction with mammalian transthyretin. Eur J Biochem 204:99–106 Blaner WS (1989) Retinol binding protein: the serum transport protein for vitamin A. Endocr Rev 26:1241–1251 Blomhoff R, Green MH, Berg T, Norum KR (1990) Transport and storage of vitamin A. Science 250:399–404 Brouwer A, Longnecker MP, Birnbaum LS, et al. (1999) Characterization of potential endocrinerelated health effects at low-dose levels of exposure to PCBs. Environ Health Perspect 107 (Suppl 4):639–649 Brugler L, Stankovic A, Bernstein L, et al. (2002). The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 40:1360–1369 Cheek AO, Kow K, Chen J, McLachlan JA (1999) Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107:273–278 Chin WW, Yen PN (1997) Molecular mechanisms of nuclear thyroid hormone action. In: Braverman LR (ed) Diseases of the Thyroid. Humana Press, pp. 1–15 Dickson PW, Howlett GJ, Schreiber G (1985) Rat transthyretin (prealbumin). Molecular cloning, nucleotide sequence, and gene expression in liver and brain. J Biol Chem 260:8214–8219 Duan W, Achen MG, Richardson SJ, et al. (1991) Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates. Eur J Biochem 200:679–687 Duan W, Richardson SJ, Babon JJ, et al. (1995) Evolution of transthyretin in marsupials. Eur J Biochem 227:396–406 Eales J, Brown S (1993) Measurements and regulation of thyroidal status in teleost fish Rev. Fish Biol Fish 3:299–347 Eales JG, Shostak S (1985) Free T4 and T3 in relation to total hormone, free hormone indices, and protein in plasma of rainbow trout and arctic charr. Gen Comp Endocrinol 58:291–302 Eneqvist T, Lundberg E, Karlsson A, et al. (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J Biol Chem 279:26411–26416 Eneqvist T, Lundberg E, Nilsson L, et al. (2003) The transthyretin-related protein family. Eur J Biochem 270:518–532 Folli C, Pasquato N, Ramazzina I, et al. (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555:279–284 Funkenstein B, Bowman CJ, Denslow ND, et al. (2000) Contrasting effects of estrogen on transthyretin and vitellogenin expression in males of the marine fish, Sparus aurata. Mol Cell Endocrinol 167:33–41 Funkenstein B, Perrot V, Brown CL (1999) Cloning of putative piscine (Sparus aurata) transthyretin: Developmental, expression and tissue distribution. Mol Cell Endocrinol 157:67–73 Hamilton JA, Steinrauf LK, Braden BC, et al. (1993) The X-ray crystal structure refinements of ˚ resolution. normal human transthyretin and the amyloidogenic Val-30 ! Met variant to 1.7 A J Biol Chem 268:2416–2424 Heller J (1976) Purification and evidence for the identity of chicken plasma and egg yolk retinolbinding protein-prealbumin complex. Dev Biol 51:1–9 Hennerbry SC, Wright HM, Likic VA, et al. (2006) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64:1024–1045 Hulbert AJ (2000) Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc 75:519–631 Ingenbleek Y, Bernstein L (1999) The stressful condition as a nutritionally dependent adaptive dichotomy. Nutrition 15:305–320 Ingenbleek Y, De Visscher M, De Nayer P (1972) Measurement of prealbumin as index of proteincalorie malnutrition. Lancet 2(7768):106–109


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Ingenbleek Y, Young VR (1994) Transthyretin (prealbumin) in health and disease: nutritional implications. Ann Rev Nutr 14:495–533 Ingenbleek Y, Young VR (2002) Significance of transthyretin in protein metabolism. Clin Chem Lab Med 40:1281–1291 Ishihara A, Nishiyama N, Sugiyama SI, Yamauchi K (2003a) The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor. Gen Comp Endocrinol 134:36–43 Ishihara A, Sawatsubashi S, Yamauchi K (2003b) Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors. Mol Cell Endocrinol 199:105–117 Jaillon O, Aury J-M, Brunet F, et al. (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431:946–957 Kanai M, Raz A, Goodman DS (1968) Retinol-binding protein: the transport protein for vitamin A in human plasma. J Clin Invest 47:2025–2044 Kawakami Y, Seoka M, Miyashita S, et al. (2006) Characterization of transthyretin in the Pacific bluefin tuna, Thunnus orientalis. Zoolog Sci 23:443–448 Kopelman M, Cogan U, Mokady S, Shinitzky M (1976) The interaction between retinol binding proteins and prealbumins studied by fluorescence polarization. Biochim Biophys Acta 439: 449–460 Lans MC, Klasson-Wehler E, Willemsen M, et al. (1993) Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem Biol Interact 88:7–21 Larsson M, Petterson T, Carlstrom A (1985) Thyroid-hormone binding in the serum of 15 vertebrate species: isolation of thyroxine binding globuline and prealbumin analogs. Gen Comp Endocrinol 58:360–375 Leatherland J (1994) Reflections on the thyroidology of fishes: from molecules to humankind. Guelph Ichthyology Rev 2:1–67 Lee Y, Lee do H, Kho CW, et al. (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774 McKinney JD, Waller CL (1994) Polychlorinated biphenyls as hormonally active structural analogues. Environ Health Perspect 102:290–297 Manzon RG, Neuls TM, Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151:55–65 Mita S, Maeda S, Shimada K, Araki S (1984) Cloning and sequence analysis of cDNA for human prealbumin. Biochem Biophys Res Com 124:558–564 Morgado I, Hamers T, Van der Ven L, Power DM (2007a) Disruption of thyroid hormone binding to sea bream recombinant transthyretin by ioxinyl and polybrominated diphenyl ethers. Chemosphere 69:155–163 Morgado I, Melo EP, Elisabeth Sauer-Eriksson A, et al. (2008) Piscine transthyretin hormone affinity and fibril formation: the role of the N-terminal. Mol Cell Endocrinol. 295:48–58 Morgado I, Santos CRA, Jacinto R, Power DM (2007b) Regulation of transthyretin by thyroid hormones in fish. Gen Comp Endocrinol 152:189–197 Monaco HL, Rizzi M, Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041 Ohno S (1970) Evolution by Gene Duplication. Springer, Berlin Orozco A, Valverde R-C (2005) Thyroid hormone deiodination in fish. Thyroid 15:799–813 Peterson PA (1971) Studies on the interaction between prealbumin, retinol-binding protein, and vitamin A. J Biol Chem 246:44–49 Power DM, Elias NP, Richardson SJ, et al. (2000) Evolution of the thyroid hormone-binding protein, transthyretin. Gen Comp Endocrinol 119:241–255 Power DM, Silva N, Campinho MA (2008) Metamorphosis. In: Finn RN, Kapoor BG (eds) Fish Larval Physiology. Science Publishers, USA

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Prapunpoj P, Leelawatwatana L, Schreiber G, Richardson SJ (2006) Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS J 273:4013–4023 Prapunpoj P, Richardson SJ, Schreiber G (2002) Crocodile transthyretin: structure, function, and evolution. Am J Physiol 283:R885–R896 Prapunpoj P, Richardson SJ, Fugamalli L, Schreiber G (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order Insectivora, class Mammalia. Mol Biol Evol 17:1199–1209 Prapunpoj P, Yamauchi K, Nishiyama N, et al. (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol 279:R2026–R2041 Purkey HE, Dorrell MI, Kelly JW (2001) Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. PNAS 98:5566–5571 Ramazzina I, Folli C, Secchi A, et al. (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nature Chem Biol 2:144–148 Richardson SJ (2002) The evolution of transthyretin synthesis in vertebrate liver, in primitive eukaryotes and in bacteria. Clin Chem Lab Med 40:1191–1199 Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258:137–193 Richardson SJ, Bradley AJ, Duan W, et al. (1994) Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins. Am J Physiol 266:R1359–R1370 Richardson SJ, Hunt JL, Aldred AR, et al. (1997) Abundant synthesis of transthyretin in the brain, but not in the liver, of turtles. Comp Biochem Physiol B 117:421–429 Richardson SJ, Monk JA, Shepherdley CA, et al. (2005) Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish. Am J Physiol 288:R1264– R1272 Rickenbacher U, McKinney JD, Oatley SJ, Blake CC (1986) Structurally specific binding of halogenated biphenyls to thyroxine transport protein. J Med Chem 29:641–648 Robbins J, Bartalena L (1986) Plasma transport of thyroid hormones. In: Thyroid Hormone Metabolism. Marcel Dekker, New York Sachs E, Bernstein LH (1986) Protein markers of nutrition status as related to sex and age. Clin Chem 32(2):339–341 Sammar M, Babin PJ, Durliat M, et al. (2001) Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues. Gen Comp Endocrinol 123:51–61 Santos CRA, Power DM (1996) Piscine (Sparus aurata) transthyretin. Ann Endocrinol 57:58 Santos CRA, Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinol 140:2430–2433 Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B 116:137–160 Shidoji Y, Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: Isolation and partial characterisation of piscine retinol binding protein. J Lipid Res 18:679–691 Sunde M, Richardson SJ, Chang L, et al. (1996) The crystal structure of transthyretin from chicken. Eur J Biochem 236:491–499 Taylor JS, Braasch I, Frickey T, et al. (2003) Genome duplication, a trait shared by 22,000 species of ray-finned fish. Genome Res 13:382–390 Tsuzuki T, Mita S, Maeda S, et al. (1985) Sturcture of the human prealbumin gene. J Biol Chem 260:12224–12227 Van Jaarsveld PP, Edelhoch H, Goodman DS, Robbins J (1973) The interaction of human plasma retinol-binding protein and prealbumin. J Biol Chem 248:4698–4705 Venkatesh B (2003) Evolution and diversity of fish genomes. Curr Opin Genet Dev 13:588–592 Weirich RT, Schwartz HL, Oppenheimer JH (1987) An analysis of the interrelationship of nuclear and plasma triiodothyronine in the sea lamprey, lake trout, and rat: evolutionary considerations. Endocrinol 120:664–677


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Wojtczak A, Cody V, Luft JR, Pangborn W (2001) Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallog D 57:1061–1070 Yamauchi K, Kasahara T, Hayashi H, Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinol 132:2254–2261 Yamauchi K, Nakajima J-I, Hayashi H, et al. (1999) Purification and characterization of thyroidhormone-binding protein from masu salmon serum: A homolog of higher-vertebrate transthyretin. Eur J Biochem 265:944–949 Yamauchi K, Prapunpoj P, Richardson SJ (2000) Effect of diethylstilbestrol on thyroid hormone binding to amphibian transthyretins. Gen Comp Endocrinol 119:329–339 Yamauchi K, Takeuchi H, Overall M, et al. (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA: comparison of its pattern of expression during metamorphosis with that of lipocalin. Eur J Biochem 256:287–296 Zanotti G, Cendron L, Ramazzina I, et al. (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9 Zanotti G, Folli C, Cendron L, et al. (2008) Structural and mutational analyses of proteinprotein interaction between transthyretin and retinol-binding protein. FEBS Journal 275(23): 5841–5854 Zhang J, Lazar MA (2000) The mechanism of action of thyroid hormones. Ann Rev Physiol 62:439–466

Chapter 5

The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase Sarah Hennebry

Abstract The primary, secondary, tertiary and quaternary structures of TTRs have been highly conserved during the evolution of vertebrates. This suggests that the TTR gene must have evolved prior to the divergence of vertebrates from invertebrates. Open reading frames predicted to encode a TTR homolog, transthyretin-like protein (TLP) were identified in all kingdoms. The Salmonella dublin TLP was selected as a model by which to characterise the structure, function and evolutionary relationships between TTR and TLP. The X-ray crystal structure of the S. dublin ˚ . As the first X-ray crystal structure of a TLP determined, TLP was solved to 2.5 A the results revealed the remarkable conservation of the TLP/TTR fold. The only significant differences in the structures of S. dublin TLP with TTRs were localised to the dimer–dimer interface and indicated that thyroid hormones could not be bound by TLP. Studies by others had indicated that cytoplasmic TLPs functioned in purine metabolism by hydrolysing 5-hydroxyisourate (5-HIU). It was demonstrated that a periplasmic TLP, the S. dublin TLP also hydrolysed 5-HIU. Three residues at the dimer–dimer interface of S. dublin TLP were shown to be essential for chemical catalysis and are 100% conserved in all TLP sequences but absent in TTRs. Therefore it was proposed that following the duplication of the TLP gene in early protochordate evolution, loss of these catalytic residues resulted in the formation of a deep, negatively charged channel which runs through the centre of the TTR tetramer. The results thus demonstrate the remarkable evolution of the TLP/TTR protein from a hydrolytic enzyme to a thyroid hormone distributor protein. Keywords 5-Hydroxyisourate, Periplasm, TLP

S. Hennebry Human Neurotransmitters Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia

S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_5, # Springer-Verlag Berlin Heidelberg 2009

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5.1

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Introduction

Studies investigating the evolution of the structure of TTR have highlighted the significant conservation of the primary, secondary, tertiary and quaternary structures of vertebrate TTRs. Alignments of the TTR subunit sequences from the organisms in which it has been identified, revealed significant sequence homology (for a review, see Power et al. (2000) and previous chapter). The most evolutionarily distant TTR sequences, those from human and sea bream, share 48% identity (67% similarity). Within the group of eutherian TTR sequences, the identity is higher, with 82% sequence identity (92% similarity) between human and rat TTR sequences. The significantly higher degree of sequence similarity among TTR sequences compared to sequence identity, suggests that the overall chemical properties of the protein have been conserved throughout its evolution (Power et al. 2000). In the previous chapter, the X-ray crystal structures of TTR from human, rat, chicken and fish were compared. The secondary, tertiary and quaternary structures of these proteins were shown to be highly conserved. Given the homology of TTR structures throughout vertebrate evolution, it was postulated that the TTR gene arose prior to the divergence of vertebrates from invertebrates. In 2000, Prapunpoj et al. (2000) announced the identification of open reading frames (ORFs) in the recently sequenced genomes of invertebrate organisms. These ORFs were predicted to encode a putative TTR homolog which was named the transthyretin-like protein (TLP). At the time, little was known about TLP other than the fact that it shared approximately 60% sequence similarity with TTR. To further characterise the distribution of TLP genes in nature, a more extensive mining of the available databases was required. Determination of the sequence characteristics of TLP and in particular, the shared sequence characteristics of TLP and TTR was also required to elucidate the evolutionary relationship, if any, between TLP and TTR. This chapter focuses on the initial characterisation of TLP structure and function using bioinformatic tools, and the subsequent in vitro characterisation of a bacterial TLP, the Salmonella sp. TLP.

5.2 5.2.1

The Identification of Non-Vertebrate TTR Homologs The Relative Distributions of TTR and TLP Genes

At the time of the first discovery of TLP ORFs, in 2000, very few genomes had been completely sequenced. The last decade has seen a considerable shift in the manner and speed with which protein phylogenies could be determined as a result of large-scale sequencing of genomes from a wide variety of organisms. Genome sequence data have increased exponentially and have enabled the completion of a more thorough analysis of the distribution of TLP genes by examining the number of organisms with TLP genes (Eneqvist et al. 2003; Hennebry et al. 2006a).

5 The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase

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Phylogenetic analyses have also been used to provide a powerful insight into the evolution of these two protein families (Eneqvist et al. 2003; Hennebry et al. 2006a). The results of the bioinformatic analyses have provided an important ‘first step’ in our understanding of TLP and have influenced the direction of subsequent research. To date, in excess of 200 individual TLP sequences have been identified using a combination of BLAST and HMM searches (Hennebry et al. 2006a). TLP genes are found in all kingdoms. In contrast, TTR genes are only found in vertebrate genomes. This finding has led to the hypothesis that the TTR gene may have evolved from the duplication of the TLP gene in early vertebrate evolution (Hennebry et al. 2006a).

5.2.2

Motifs Characterising TLP and TTR Sequences

In order to better define the sequence characteristics of TLP and TTR and to distinguish between the two protein families, the sequences were probed for isolated signature motifs (Hennebry et al. 2006a). The high level of similarity between TTR sequences was such that a single motif was found to span the entire protein. Therefore, a set of TTR-specific motifs was unattainable. However, five motifs were identified among TLP sequences (A–E) and were found in the arrangement (E), B, D, C, A along the length of the protein sequence (Fig. 5.1a(i)). Motif A, which corresponds to the region of greatest similarity between TLP sequences, was found at the C-terminal region of the sequences and included a consensus YRGS tetrapeptide which was 100% conserved in all TLP sequences. Motif B is the second strongest motif and was found in the N-terminal region of the protein. Motifs C and D were found in the middle of the TLP sequences. Motif E was only found in TLPs from plant species and from two alphaproteobacteria: Bradyrhizobium japonicum and Magnetospirillum magnetotacticum. Motif E is homologous to the proteins of COG 3195 (Cluster of Orthologous Groups), a group of bacterial proteins where the entire protein is made up of this single domain. Motif E has been subsequently identified as a unique protein, OHCU decarboxylase whose function relative to TLP will be discussed further in chapter ‘Clinical Implications of TTR Amyloidosis’. Three motifs were identified to be in common between TLP and TTR sequences: A’, B’ and C’ (Fig. 5.1a(ii)). Motif A’ is equivalent to Motif A of the TLP-only set: at the C-termini of the proteins. Motif A’ is the most highly conserved among the set of TLP + TTR sequences. Motif B’ in the TLP + TTR set completely overlapped with the Motif B in the TLP only set, and extended for three and five residues further toward the N- and C-termini, respectively. Motif C’ found in the TLP + TTR set was shorter (15 vs. 21 residues) but partially overlapped Motif C in the TLP only set. The absence of a Motif D’ in the TLP + TTR set implied that Motif D is specific for TLP sequences and is the region with the least similarity between TTR and TLP sequences (Fig. 5.1a(ii)).

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S. Hennebry

Fig. 5.1 Motifs identified in TLP sequences and in the set of TLP + TTR sequences. (a) Motifs identified in (i) TLP sequences and (ii) TTR + TLP sequences. (i) In the set of TLP sequences, five motifs were identified (A–E). The motifs are found in the order (E), B, D, C, A, with A being the most highly conserved. Motif E (not shown) is only found in the TLPs from plants and the a-proteobacteria Magnetospirillum magnetotacticum and Bradyrhizobium japonicum. (ii) In the set of TTR + TLP sequences, three motifs were identified. Motif A’ is equivalent to Motif A from the TLP motif set. Motif B’ is similar but extended in the N- and C-terminal regions to Motif B. Motif C’ is shorter than Motif C and its location is shifted towards the N-terminus. Motif D is specific to the TLP set of proteins. Motifs logos were generated using the MEME program (Bailey and Elkan 1994). (c) The motifs identified in the TLPs correlate to regions of structural significance. (i) TheSalmonella dublin sequence is shown with the predicted secondary structure indicated above the sequence. Motifs identified in common between TLPs and TTRs are indicated below the alignment of the S. dublin TLP sequence with that of human TTR. The experimentally determined secondary structure of human TTR is indicated above its sequence. The alignments indicate that the primary and secondary structures of TLPs and TTRs are highly conserved. (ii) The motifs identified in common between TLPs and TTRs were superimposed on the tertiary structure of sea bream TTR (Folli et al. 2003). Motif A’ lines the hydrophobic core. Motif B’ forms the dimer–dimer interface and the entrance to the hydrophobic core. Motif C’ forms the monomer– monomer interface. The program SWISS-PDB viewer was used to generate the figure (from Hennebry et al. 2006a)

5.2.3

Structural Significance of Motifs

In order to determine whether the conserved sequence motifs corresponded to regions of structural significance, a homology model of the Salmonella dublin TLP was constructed (Hennebry et al. 2006a). The S. dublin TLP was selected for this analysis as it was among the first TLP sequences identified (Prapunpoj et al. 2000). In addition, the predicted amino acid sequences from Salmonella sp. were identical, and so the S. dublin TLP would represent TLPs from this genus. The sequence motifs which were determined to be common to both TLP and TTR sequences were aligned with the predicted and experimentally determined secondary structures of TLP and TTR respectively (Fig. 5.1b(i)). Motif A’ corresponded to b-strand Gloop-strand H region of TTR. Motif B’ corresponded to b-strand A-loop-strand B region of TTR. Motif C’ corresponded to b-strand E-turn-a-helix of TTR. The equivalent regions of the S. dublin TLP were predicted to share the same secondary structure features as TTRs (Hennebry et al. 2006a). These results suggested that the


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conserved motifs might correspond to important structural features of the TTR tetramer. The motifs were then superimposed onto the crystal structure coordinates of sea bream TTR (pdb identifier: 1OO2; Folli et al. 2003) (Fig. 5.1b(ii)). The residues comprising Motif A’ (the most highly conserved motif) were found to line the dimer–dimer interface of the protein and were also potentially involved in monomer–monomer interactions. The residues comprising Motif B’ were also found to cluster at the dimer–dimer interface of the protein, this time forming the entrance to the ligand binding channel of TTR. Residues comprising Motif C’ were also found to have a structural role, potentially in monomer–monomer interactions. The finding that the sequence motifs conserved between TLP and TTR corresponded to structurally important regions of the TTR tetramer lent further weight to the prediction that TLP shares similar secondary and tertiary structures to TTR. The identification of the motifs specific to TLP and to the set of TLP + TTR revealed that the regions of greatest similarity between the proteins are in the Nand C-terminal regions and that these correspond to the b-strands (strands A, B, G and H) forming the entrance to and lining the dimer–dimer interface of the TTR tetramer. However, a close examination of the differences between the TLPspecific motifs and TLP + TTR motifs revealed important information regarding the likely ligand(s) of TLPs. The ligands of TTR (thyroid hormones and related compounds) bind deep within the central channel of the protein, formed by the dimer–dimer interface. Important residues involved in this ligand binding include: Lys15, Glu54 and Thr119. An inspection of the dimer–dimer interface in the molecular models generated for the S. dublin TLP suggested a structurally distinct binding channel. The presence of conserved histidine, arginine and tyrosine residues in all TLP sequences at the equivalent regions of Lys15, Glu54 and Thr119 in all TTR sequences suggested that the central channel would be considerably smaller and more positively charged in TLP. These observations suggested that if the dimer–dimer interface is the ligand binding site of TLP, the ligands bound are likely to be smaller than thyroid hormones and carry different distribution of charges.

5.2.4

Variations in the Predicted Subcellular Localisation of TLPs

Whilst all TLP sequences identified have been shown to contain Motifs A–D, further variations were observed in the N-terminal regions of these sequences. Careful inspection of the N-terminal regions of TLP from various organisms revealed that many TLP sequences contained likely subcellular localisation sequences or secretion peptides. On this basis, TLP sequences can be divided into four groups: (1) cytoplasmic TLPs (the majority of TLP sequences); (2) periplasmic TLPs (a small group of gram-negative bacterial TLP sequences mainly from the group Enterobacteriaceae); (3) peroxisomal TLPs (most metazoan TLP sequences and plant TLP sequences); and (4) extracellular TLPs (nematode TLP sequences). As noted, plant and metazoan TLPs are represented by both cytoplasmic and

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peroxisomal sequences. This is due to splice variation of the TLP gene in these organisms which results in the loss of the peroxisomal targeting sequence (Hennebry et al. 2006a). It is postulated that TLPs from plants and metazoan organisms are localised to both the cytoplasm and the peroxisome. In most instances, only one TLP gene was identified per haploid genome. However, several bacteria were found to possess two or three non-identical TLP genes. In these cases, the second or third TLP gene was found to encode a protein with a periplasmic localisation sequence. The functional significance of the dual localisation of TLPs in some bacteria and in plants and metazoans will be discussed later in this chapter.

5.3 5.3.1

Structural Characterisation of the S. dublin TLP Synthesis and Characterisation of Recombinant S. dublin TLP

Given that bioinformatic analysis of the S. dublin TLP suggested shared similar structural characteristics with TTR, this protein was selected for further structural and functional studies. Recombinant S. dublin TLP was synthesised using an E. coli expression system (see Hennebry et al. 2006a). Given the close evolutionary distance between E. coli and Salmonella, the periplasmic localisation signals utilised by both bacteria are identical. Thus, the N-terminal periplasmic localisation signal of the S. dublin TLP was left intact so that secretion of the recombinant protein to the periplasm could be tested in E. coli. Recombinant S. dublin TLP was successfully purified from the periplasm of host E. coli (Hennebry et al. 2006a). N-terminal sequencing of the recombinant protein confirmed that the N-terminal signal peptide had been cleaved upon translocation of the protein into the periplasm (Hennebry et al. 2006a). This indicated that it was likely that in vivo the S. dublin TLP was localised to the periplasm. Recombinant S. dublin TLP was subjected to gel filtration and comparison of its subunit molecular mass (as determined by ionising mass spectrometry) with its apparent native molecular weight suggested that the protein formed homotetramers, similarly to TTR.

5.3.2

The X-Ray Crystal Structure of the S. dublin TLP

The three-dimensional structures of TTR from various organisms have been well characterised (refer to relevant chapter in this book). The first X-ray crystal structure of a TTR to be solved was that of human TTR, in 1978 (Blake et al. 1978). The Protein Database (http://www.pdb.org) contains multiple crystal structure coordinates for human TTR (including multiple amyloidogenic forms


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and with various ligands bound to both wild-type and mutant human TTRs). The crystal structures of TTR from rat (Wojtczak 1997), chicken (Sunde et al. 1996) and sea bream (Folli et al. 2003; Eneqvist et al. 2004) have also been solved. All of these structures demonstrate the remarkable conservation of the prealbumin-like fold (as described by SCOP, http://scop.mrc-lmb.cam.ac.uk) which consists of an eightstranded b-sandwich (strands A–H) with each sheet adopting a Greek-key topology. A two-turn a-helix usually exists between strands E and F in TTR. The two TTR dimers associate via non-polar interactions between the loops joining stands G and H with the loops joining strands A and B, making the TTR tetramer a ‘dimer of dimers’. ˚ (Hennebry The X-ray crystal structure of the S. dublin TLP was solved to 2.5 A et al. 2006b) (Fig. 5.2). Comparison of the structure of S. dublin TLP with that of TTRs from various organisms has enabled a greater understanding of the structural evolution of these paralogous proteins. In the months following the publication of the S. dublin TLP, the X-ray crystal structures of the TLPs from E. coli, B. subtilis and Danio rerio (Lundberg et al. 2006; Jung et al. 2006; Zanotti et al. 2006) were solved. The structures of these proteins will be discussed in the subsequent chapters of this book. The overall structure of the S. dublin TLP strongly resembled the archetypal TTR fold (Blake and Oatley 1977; Blake et al. 1978): a prealbumin-like fold (as defined by the SCOP database: http://scop.mrc-lmb.cam.ac.uk) consisting of an eight-stranded b-sandwich with each sheet adopting a greek-key topology (Hennebry et al. 2006b). A two-turn a-helix was found between strands E and F. The asymmetric unit consisted of two protein molecules (a TLP dimer; Fig. 5.2a) which dimerise via intermolecular hydrogen bonds between H-strands to form an eight-stranded b-sheet. A similar interaction has been found in all TTR crystal structures published to date. Application of tight non-crystallographic symmetry (NCS) restraints during refinement yielded identical molecules in the asymmetric unit. Therefore, the S. dublin TLP, similarly to TTR, was determined to be a tetrameric ‘dimer of dimers’ (see Fig. 5.2b). The two dimers were found to associate via non-polar interactions between the loops joining strands G and H with loops joining strands A and B. This was consistent with the observation from gel filtration analyses, that the TLP formed tetramers in solution (Hennebry et al. 2006a).

5.3.3

Comparison of the Structure of S. dublin TLP with TTRs from Human, Chicken, rat and Seabream

5.3.3.1

Comparison of the Overall Fold of S. dublin TLP with TTR

The structure of S. dublin TLP was overlayed with structures of TTRs from human (1F41), chicken (1TFP), rat (1KGI) and seabream (1SNO) (Hornberg et al. 2000; Sunde et al. 1996; Muziol et al. 2001; Eneqvist et al. 2004). The human, rat and fish ˚ . Chicken TTR and TTR tetramers superimposed to within an RMSD of 1.0 A

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˚ crystal structure of the S. dublin TLP. (a) Dimeric structure within the Fig. 5.2 The 2.5 A asymmetric unit of the crystal, showing the interaction between the b-strands H of each monomer. Secondary structure elements are labelled. The C and N termini are marked with an asterisk (*). (b) The homotetrameric structure generated by crystal symmetry, in an orthogonal orientation to (a). Approximate positions of predicted ligand binding sites at the dimer–dimer interface (shown as a dotted line) are indicated by black circles. Figure generated using PyMol (Delano 2002) (from Hennebry et al. 2006b)


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˚ (Fig. 5.3). S. dublin TLP showed larger variation, with RMSD values up to 4.0 A The structural deviation observed between the S. dublin TLP and the TTR subunits was of the same order of magnitude to that within the set of TTR subunits. The total ˚ ) was contacting surface area between molecules within the tetramer (5,275 A ˚ ˚ ˚ ) and 6–14% less than that of fish (5,625 A), human (5,810 A), chicken (5,828 A ˚ rat (6,133 A) TTR structures. 5.3.3.2

Comparison of the Putative Active Site of S. dublin TLP with the Thyroid Hormone Binding Site of Human TTR

One of the striking features of the quaternary structure of TTR is the central channel of the protein into which the thyroid hormones and other ligands (with the exception of retinol-binding protein) bind. This central channel traverses the entire tetramer. Whilst the overall structure of S. dublin TLP and human TTR were highly homologous, examination of the dimer–dimer interface of the TLP revealed significant differences compared to human TTR. The conserved residues His6, His95 and Tyr108 at the dimer–dimer interface of S. dublin TLP and other TLPs were shown to dramatically alter the physicochemical properties of this region of the protein in comparison to human and other TTRs. Figure 5.4 shows a comparison of the dimer– dimer interface of S. dublin TLP with the equivalent region in human TTR. The Tyr108 residue in S. dublin TLP has a profound effect on the size and shape of the dimer–dimer interface compared with human TTR, dramatically reducing the depth of the central channel. The equivalent residue in human TTR is a conserved threonine (Thr119). Residue Leu99 of S. dublin TLP also adopts a conformation which plugs the base of the pocket, compared to the equivalent residue Leu110 in human TTR. Thus, the residues Leu99 and Tyr108 in the S. dublin TLP contribute to a groove-like dimer–dimer interface. This contrasts with the dimer–dimer interface of the TTR molecule, which forms a deep and funnel-shaped central channel which runs through the middle of the tetramer. Other residues at the dimer–dimer interface of S. dublin TLP which differ considerably to the equivalent interface of TTR, are several positively charged residues (Fig. 5.4), His6, Arg44 and His95. These residues are also 100% conserved among TLP sequences and contribute to a significantly more positively charged dimer–dimer interface of S. dublin TLP compared to the more negatively charged channel of human TTR (Fig. 5.4a(i) and 5.4a(ii)). The comparison of the structures of TTRs and TLPs indicates that following the duplication of the TLP gene in early vertebrate evolution, specific amino acid changes at the dimer–dimer interface of the TLP tetramer resulted in increased negative charge and an ‘opening’ of the central channel of the protein. None of the charged residues identified at the dimer-dimer interfaces of TLPs are conserved in TTR sequences. One exception is the recently identified TTR from the lamprey, Petromyzon marinus (Manzon et al. 2007). This is the only TTR sequence to have a histidine residue at the position equivalent to His95 in S. dublin TLP (which is a conserved histidine in all TLPs). Therefore, it is likely that changes to the dimer– dimer interface of TLP occurred firstly at His6, Arg44 or Tyr108 residues and lastly at His95.

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Fig. 5.3 Structural comparison of the S. dublin TLP with TTR. (a) Stereo diagram showing a superimposition of the S. dublin tetramer (magenta) with human (1F41, Hornberg et al. (2000), cyan), rat (1KGI, Muziol et al. (2001), yellow), chicken (1TFP, Sunde et al. (1996), orange) and fish (1SNO, Eneqvist et al. (2004), green) TTRs. Tetramers were superimposed using the A chain only. (b) Stereo diagram showing a superimposition of monomers of S. dublin TLP (magenta) with human (1F41, cyan), rat (1KGI, yellow), chicken (1TFP, orange) and fish (1SNO, green) TTRs. Bound thyroxine ligand in the human structure is shown as sticks. Regions of plastic deformation are labelled. Residues at the dimer–dimer interface of S. dublin TLP are shown as red sticks (from Hennebry et al. 2006b)


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Fig. 5.4 Comparison of the dimer–dimer interfaces of human TTR and S. dublin TLP. (a) (i) Residues that make up the ligand binding pocket of (i) human TTR are shown. Thyroxine is shown in stick representation, in yellow. The residues Lys15, Glu54 and Thr119 are labelled and form important interactions with thyroxine. Some elements of secondary structure are not shown, for clarity. (ii) The dimer–dimer interface of S. dublin TLP is shown (at the equivalent position to human TTR shown in part (i)). Hydrogen bonds are shown as broken cyan lines. Some elements of secondary structure are not shown, for clarity. Residues known to be important to catalysis in S. dublin TLP are indicated with an asterisk. Residues His6, His95 and Tyr108 of S. dublin TLP are equivalent upon structural alignment to Lys15, Thr106 and Thr119 of TTR, respectively. (b). Surface characteristics and interactions at the ligand binding sites of (i) human TTR and (ii) S. dublin TLP. Positively charged residues are in blue, negatively charged residues are in red. (i) The electrostatic surface potential surface of human TTR (1F41; Hornberg et al. 2000), shown with thyroxine ligand inside the hormone-binding pocket, is negatively charged. (ii) The equivalent region in the S. dublin TLP is positively charged. Figure generated using PyMol (Delano 2002) (from Hennebry et al. 2006b)

5.3.3.3

Comparison of the X-Ray Crystal Structure of S. dublin TLP to TLP Structures from E. coli, B. subtilis and D. rerio

The X-ray crystal structures of the TLP from E. coli (Lundberg et al. 2006), B. subtilis (Jung et al. 2006) and D. rerio (Zanotti et al. 2006) were published shortly after the structure of the S. dublin TLP was published. A comparison of the TLPs from the three species of bacteria with the vertebrate TLP (zebrafish) shows

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little structural divergence. Major differences between the S. dublin TLP and D. rerio TLP were found in the flexible portions of strand B and C that protrude towards the solvent and in the conformation of the loop connecting strands D and E (Zanotti et al. 2006). Greater differences were observed between the structures of TLPs from B. subtilis and D. rerio: the B-C loop was significantly shorter in B. subtilis TLP whilst the loop connecting the short a-helix to strand F was extended. The dimer–dimer interfaces of TLP from prokaryotes and eukaryotes were nearly identical. The location and orientation of the residues present in this region were well maintained, including the highly conserved residues His6, Arg44, His95 and Tyr108 (S. dublin TLP numbering). The only significant difference was found in the C-terminal serine residue (from the YRGS tetrapeptide), which assumed different orientations in the structures from S. dublin, E. coli and B. subtilis compared with D. rario. It is therefore clear that the dimer–dimer interface of TLP has been incredibly well conserved throughout evolution. This suggests that the dimer–dimer interface might be the ligand binding site of TLP.

5.4 5.4.1

The In Vitro Function of the S. dublin TLP Binding of Thyroid Hormones by the S. dublin TLP

Even before the publication of the X-ray crystal structure of a TLP, homology modelling of the likely structures of these proteins led to the hypothesis that it was unlikely that thyroid hormones were the natural ligands of TLPs. Homology modelling of the S. dublin TLP (Sect. 5.2.3) suggested that the shape and charge of the dimer–dimer interface of this protein might preclude the binding of thyroid hormones. To confirm this, experiments characterising the binding of thyroid hormones to the S. dublin TLP were performed using the method published by Chang et al. (1999). The binding of radiolabelled tri-iodothyronine (T3) and tetraiodothyronine (T4) by the S. dublin TLP could not be detected. The resultant scatchard plots suggested low levels of non-specific interactions between the protein and thyroid hormones, indicating that the affinity of the thyroid hormones for TLP was too low to be of physiological significance. This finding supported the hypothesis that it was likely that the dimer–dimer interface of TLP would be too shallow to accommodate bulky compounds bound by TTRs.

5.4.2

Examining the Role of the S. dublin TLP in Purine Metabolism

In order to gain clues as to the likely function of TLP, the genomic context of TLP genes from bacteria was examined. In bacteria, genes encoding proteins which act in a shared metabolic pathway are often found grouped on the chromosome in an


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operon. Genes encoding cytoplasmic TLP sequences were frequently found in a specific genomic context: a purine metabolism operon. Specifically, the cytoplasmic TLP genes were frequently found neighbouring the gene encoding the enzyme uricase. Indeed, a role for TLP in purine metabolism was proposed in 2001. In an effort to develop a greater understanding of purine metabolism in the gram-positive bacterium, Bacillus subtilis, Schultz et al. (2001) generated a series of insertion mutants. One of these mutations was made in the TLP gene which is located immediately downstream of the gene encoding uricase. The bacteria harbouring this mutation were characterised by a reduced rate of proliferation (compared to wild-type) on media containing uric acid as the principal source of nitrogen (Schultz et al. 2001). In gram negative bacteria, the TLP gene was also found in purine metabolism operons. However, the purine metabolism-associated genomic context of the TLP gene was only found for the TLP genes encoding cytoplasmic proteins. There was no consistent genomic context identified for TLP genes encoding periplasmic proteins. For example, the S. dublin TLP gene is immediately downstream of genes (CopR and CopS) encoding a two-component sensor-histidine kinase system for bacterial resistance to copper. Therefore it was unclear whether the S. dublin TLP would share similar functions to TLPs from other bacteria. Purines are significantly smaller compounds that thyroid hormones, whilst sharing similar chemical characteristics. Therefore, the binding of uric acid and allantoin to S. dublin TLP was assessed, using fluorescence quenching. The static quenching of the intrinsic fluorescence of tryptophans results from conformational changes in proteins upon ligand binding (Engelborghs 2001). No such conformational static quenching of the intrinsic fluorescence of the tryptophan residues of S. dublin TLP could be detected following titration with uric acid or allantoin (Hennebry et al. 2006a). These results, however, could not be taken as conclusive evidence of the lack of binding of the compounds by the TLP. Rather, the lack of quenching merely indicated a lack of significant conformational change to the protein in regions containing a tryptophan residue.

5.4.3

Hydrolysis of 5-HIU by the the S. dublin TLP

It had long been reported that that the enzyme uricase oxidises the purine uric acid directly into allantoin (e.g. Howell and Wyngaarden 1960). The enzyme reaction has since been demonstrated to be more complex. Tipton’s group showed in 1997 that uricase oxidises uric acid to a metastable compound, 5-hydroxyisourate (5-HIU) (Kahn et al. 1997). This intermediate decomposes into racemic allantoin nonenzymatically via another unstable intermediate, 2-oxo-4-hydroxy-4-carboxy5-ureidoimadolazine (OHCU) (Fig. 5.5a). The decomposition of 5-HIU was shown to occur over 30 min at pH 7, in vitro (Sarma et al. 1999). In biological systems, only (S)-allantoin is found and the enzyme which degrades this purine, allantoinase, specifically binds the (S) form of the compound (Lee and Roush 1964).

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Fig. 5.5 The hydrolysis of 5-HIU by S. dublin TLP. (a) The uricase degradation pathway showing the role of three enzymes (uricase, TLP and OHCU decarboxylase) in converting uricase to allantoin. (b) Hydrolysis of 5-HIU by wild-type S. dublin TLP and by His6Ala, His95Ala and Tyr108Phe mutant S. dublin TLPs. 0.05 units ml 1 uricase was equilibrated in 50 mM potassium phosphate buffer (pH 7.8) for 3 min. The reaction was commenced with the addition of 50 mM uric acid. The appearance of 5-HIU was monitored spectrophotometrically at 312 nm. The amount of 5-HIU peaked after approximately 3 min and began spontaneous decomposition if left unperturbed. Addition of 0.04 mM wild-type S. dublin TLP resulted in rapid hydrolysis of 5-HIU (10 mmol min 1 mg 1 TLP). Addition of equimolar amounts of His95Ala or Tyr108Phe mutants S. dublin TLPs resulted in reduced hydrolysis (1.2 mmol min 1 mg 1 TLP; activity reduced by 90%). The His6Ala mutation abolished hydrolytic activity (from Hennebry et al. 2006b)

The implication, as hypothesised by Kahn and Tipton (1998), was the involvement of at least two additional enzymes in the degradation of uric acid to allantoin: to increase the rate of decomposition and to facilitate the generation of stereo-specific allantoin. The significance of this hypothesis in the context of a possible function of TLP was not fully appreciated until recently, when it was revealed that the TLP from the gram positive bacterium, B. subtilis, hydrolysed one intermediate in the uric acid degradation pathway, 5-HIU to OHCU (Lee et al. 2005). Importantly, the same researchers established that human TTR did not possess this hydrolytic activity. Ramazzina et al. (2006) have more recently shown that mouse TLP hydrolyses 5-HIU and that the COG3195 protein was responsible for the decarboxylation of


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OHCU to S-allantoin (Fig. 5.5). Thus, the pathway of the conversion of uric acid to S-allantoin via the three enzymes, uricase, TLP (5-HIUase) and OHCU decarboxylase was revealed (Fig. 5.5a). The TLP from B. subtilis was predicted to be cytosolic and its gene was located in a purine metabolism operon. However, it was not clear whether a periplasmic TLP, such as the S. dublin TLP, would also hydrolyse 5-HIU. The recombinant S. dublin TLP was tested for its ability to hydrolyse 5-HIU, using the method published by Lee et al. (2005). One mg of recombinant S. dublin TLP was shown to hydrolyse 10 mol min 1 of 5-HIU (Hennebry et al. 2006b).

5.4.4

Characterisation of the Active Site of S. dublin TLP

In the previous section the ligand binding channel of human TTR was compared with the equivalent region of the crystal structure of S. dublin TLP. Several residues at the dimer–dimer interface of the S. dublin TLP (Fig. 5.4b) were 100% conserved across all TLP sequences but not found in any TTR sequences. Examination of the likely arrangement of these residues indicated a potential catalytic site where 5-HIU might be hydrolysed (Fig. 5.4b). With this in mind, residues which could theoretically perform a role in chemical catalysis were selected for site-directed mutagenesis to see what affect this would have on the hydrolytic activity of TLP. These residues were His6, His95 and Tyr108 (Fig. 5.4b). The mutations His6Ala, His95Ala and Tyr108Phe were introduced into the S. dublin TLP sequence (Hennebry et al. 2006b). The S. dublin TLP His6Ala, His95Ala and Tyr108Phe proteins were synthesised, purified and shown to form homotetramers, in order to rule out the potential global structural importance of the mutated residues. The hydrolytic activity of each mutated S. dublin TLPs was compared with that of wild-type S. dublin TLP. All of these mutations had significant effects on the hydrolytic activity of the protein (Fig. 5.5b). The His6Ala mutation abolished hydrolytic activity which was reduced to a negligible rate of 0.1 mol min 1 mg 1 of protein. The His95Ala and Tyr108Phe S. dublin TLPs had activities of 1.2 mol min 1 mg 1 of protein: a reduction of 90%.The His10Ala mutation completely abolished enzyme activity. The His99Ala and Tyr119Phe mutations reduced activity by more than 90% (Hennebry et al. 2006b).

5.5

Perspectives

We determined that the S. dublin TLP is most likely a periplasmic protein. The periplasm is a layer of peptidoglycan found between the inner and outer membranes of gram-negative bacteria. In these bacteria, the periplasm has a variety of important roles. Several groups of enzymes with specialised functions reside in the

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periplasm. For example, hydrolytic enzymes which can degrade foreign DNA or peptides are important part of the bacteria’s defence system. Some periplasmic enzymes, for example, superoxide dismutase and catalase detoxify oxidising compounds (free radicals) from the external environment. Proteins involved in chemotaxis and nutrient acquisition are localised to the periplasm as are numerous transport proteins which dock to membrane-bound proteins to elicit transport of their substrate into the cytoplasm (for a review, see Oliver 1996). In all bacteria studied to date, the proteins involved in purine metabolism are located in the cytosol (for a review, see Vogels and van der Drift (1976)). Therefore, a role for the S. dublin TLP in purine metabolism is unclear. In addition, Salmonellae lack the upstream enzymes required for generation of 5-HIU: no homologs of xanthine dehydrogenase, uricase or OHCU-decarboxylase have been identified in the genomes of Salmonella species. Nonetheless, import proteins and enzymes for the degradation of downstream compounds, such as allantoin are encoded in the Salmonellae genomes. If 5-HIU is the true ligand of the S. dublin TLP, then the source of 5-HIU must be the external environment, as is the case for allantoin. In vivo, 5-HIU is short-lived and is produced by the non-enzymatic oxidation of uric acid (Santos et al. 1999). 5-HIU is highly reactive and capable of reacting with free radicals, causing lipid oxidation (Santos et al. 1999). It has therefore been proposed that together, TLP and OHCU decarboxylase rapidly detoxify 5-HIU, yielding the relatively inert allantoin (Hennebry et al. 2006a; Ramazzina et al. 2006). In the case of S. dublin, an enteric bacterium, possible sources of oxidised uric acid (i.e. 5-HIU) include the gut of its host: the guts of reptilian and avian Salmonella hosts are particularly high in uric acid (Wright 1995); mammalian hosts secrete uric acid into their guts in response to bacterial infection (Vorbach et al. 2003). The data presented here demonstrate the rapid kinetics by which recombinant S. dublin TLP hydrolyses 5-HIU, a reactive compound known to generate damaging oxidative radicals. Therefore, it is possible that the role of periplasmic TLP in enteric bacteria such as S. dublin is to rapidly detoxify 5-HIU from the external environment. The investigation of the structure and function of the S. dublin TLP represented the first step in furthering our understanding of the non-vertebrate evolution of the TTR molecule. The conservation of catalytic residues at the TLP dimer-dimer interface, which have been demonstrated to be essential for enzymatic activity, indicates that is likely that all TLPs share 5-HIU hydrolytic activity. Furthermore, the distribution of TLPs in all kingdoms, but the representation of TTRs in vertebrates alone, clearly suggests that the transthyretin-like fold originally functioned in purine metabolism. Following the duplication of TLP gene, amino acid substitutions to residues at the dimer-dimer interface dramatically altered the protein’s function whilst its overall structure was unchanged. Thus, the evolution of TLP represents a remarkable case of the divergent evolution from a hydrolytic enzyme (TLP) to a thyroid hormone distributor (TTR).


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References Bailey, T.L., Elkan, C. (1994) Fitting a Mixture Model by Expectation Maximisation to Discover Motifs in Biopolymers. AAAI Press, Menlo Park, CA Blake, C.C.F., Oatley, S.J. (1977) Protein–DNA and protein–hormone interactions in prealbumin: A model of the thyroid hormone nuclear receptor? Nature 268, 115–120 Blake, C.C.F., Geisow, M.J., Oately, S.J. (1978) Structure of prealbumin: Secondary, tertiary and ˚ . J. Mol. Biol. 121, 339–356 quaternary interactions determined by Fourier refinement at 1.8 A Chang, L., Munro, S.L.A., Richardson, S.J., Schreiber, G. (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. Eur. J. Biochem. 259, 534–542 DeLano W.L. (2002) The PyMol User’s Manual. Delano Scientific, San Carlos, CA, USA Eneqvist, T., Lundberg, E., Nilsson, L., Abagyan, R., Sauer-Eriksson, A.E. (2003) The transthyretinrelated protein family. Eur. J. Biochem. 270, 518–532 Eneqvist, T., Lundberg, E., Karlsson, A., Huang, S., Santos, C.R.A., Power, D.M., Sauer-Eriksson, A.E. (2004) High-resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J. Biol. Chem. 279, 26411–26416 Engelborghs, Y. (2001) The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim. Acta A Mol. Biomol. Spectrosc. 57, 2255–2270 Folli, C., Pasquato, N., Ramazzina, I., Battistutta, R., Zanotti, G., Berni, R. (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett. 555, 279–284 Hennebry, S.C., Wright, H.M., Likic´, V.A., Richardson, S.J. (2006a) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64, 1024–1045 Hennebry, S.C., Law, R.H., Richardson, S.J., Buckle, A.M., Whisstock, J.C. (2006b) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J. Mol. Biol. 359, 1389–1399 Hornberg, A., Eneqvist, T., Olofsson, A., Lundgren, A., Sauer-Eriksson, A.E. (2000) A comparative analysis of 23 structures of the amyloidogeneic protein transthyretin. J. Mol. Biol. 032, 649–669 Howell, R.R., Wyngaarden, J.B. (1960) On the mechanism of peroxidation of uric acids by hemoproteins. J. Biol. Chem. 235, 3544–3550 Jung, D.-K., Lee, Y., Park, S.G., Park, B.C., Kim, G.-H., Rhee, S. (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc. Nat. Acad. Sci. U.S.A. 103, 9790–9795 Kahn, K., Serfozo, P., Tipton, P.A. (1997) Identification of the true product of the urate oxidase reaction. J. Am. Chem. Soc. 119, 5435–5442 Kahn, K., Tipton, P.A. (1998) Spectroscopic characterisation of intermediates of the urate oxidase reaction. Biochemistry 37, 11651–11569 Lee, K.W., Roush, A.H. (1964) Allantoinase assays and their application to yeast and soybean allantoinases. Arch. Biochem. Biophys. 108, 460–467 Lee, Y., Lee, D.H., Kho, C.W., Lee, A.Y., Jang, M., Cho, S., Lee, C.H., et al. (2005) Transthyretinrelated proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett. 579, 4769–4774 Lundberg, E., Backstrom, S., Sauer, U.H., Sauer-Eriksson, E. (2006) The transthyretin-related protein: structural investigation of a novel protein family. J. Struct. Biol. 155, 445–457 Manzon, R.G., Neuls, T.M., Manzon, L.A. (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen. Comp. Endocrinol. 151, 55–65 Muziol, T., Cody, V., and Wojtczak, A. (2001) Comparison of binding interaction of dibromoflavonoids with transthyretin. Acta Biochim. Pol. 48, 885–892 Oliver, D.B. (1996) The periplasm. In: Neidhart, F.C., Curtiss, R. III, Ingraham, I.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaecter, M., Umbarger, M.E. (eds.) Escherichia coli and Salmonella: Cellular and molecular biology. ASM Press, Washington. pp 88–103

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Power, D.M., Elias, N.P., Richardson, S.J., Mendes, J., Soares, C.M., Santos, C.R.A. (2000) Evolution of the thyroid hormone-binding protein, transthyretin. Gen. Comp. Endocrinol. 119, 241–255 Prapunpoj, P., Yamauchi, K., Nishiyama, N., Richardson, S.J., Schreiber, G. (2000) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am. J. Physiol. Reg. Integr. Comp. Physiol. 279, R2026–R2041 Ramazzina, I., Folli, C., Secchi, A., Berni, R., Percudani, R. (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat. Chem. Biol. 3, 144–148 Sarma, A.D., Serfozo, P., Kahn, K., Tipton, P. (1999) Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolising enzyme. J. Biol. Chem. 274, 33863–33865 Santos, C.X.C., Anjos, E.I., Augusto, O. (1999) Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation. Arch. Biochem. Biophys. 372, 285–294 Schultz, A.C., Nygaard, P., Saxild, H.H. (2001) Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR Transcription activator. J. Bacteriol. 183, 3293–3302 Sunde, M., Richardson, S.J., Chang, L., Pettersson, T.M., Schreiber, G., Blake, C.C. (1996) The crystal structure of transthyretin from chicken. Eur. J. Biochem. 236, 491–499 Vogels, G.D., van der Drift, C. (1976) Degradation of purines and pyrimidines in microorganisms. Bacteriol. Rev. 40, 403–469 Vorbach, C., Harrison, R., Capecchi, M.R. (2003) Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol. 24, 512–517 ˚ resolution: First report on a Wojtczak, A. (1997) Crystal structure of rat transthyretin at 2.5 A unique tetrameric structure. Acta Biochim. Pol. 44, 505–517 Wright, P.A. (1995) Nitrogen excretion: Three end products, many physiological roles. J. Exp. Biol. 198, 273–281 Zanotti, G., Cendron, L., Ramazzina, I., Folli, C., Percudani, R., Rodolfo, B. (2006) Structure of zebrafish HIUase: Insights into evolution of an enzyme to a hormone transporter. J. Mol. Biol. 363, 1–9

Chapter 6

Vertebrate HIU hydrolase: Identification, Function, Structure, and Evolutionary Relationship with Transthyretin Giuseppe Zanotti, Ileana Ramazzina, Laura Cendron, Claudia Folli, Riccardo Percudani, and Rodolfo Berni

Abstract 5-Hydroxyisourate hydrolase (HIUase) is an enzyme widely distributed in prokaryotic and eukaryotic organisms that catalyzes the hydrolysis of HIU into OHCU in the degradation route from urate to (S)-allantoin. During early vertebrate evolution, a duplication event in the gene encoding HIUase gave rise to the thyroid hormone binding protein transthyretin (TTR). Due to the close evolutionary relationship between HIUase and TTR, these two proteins possess a high degree of amino acid sequence similarity, while performing quite different functions. The 3D structure of zebrafish HIUase compares very well with that of TTR: a highly preserved scaffold harbors distinct functional sites located in the same regions of the two proteins. The residues that are differentially conserved in HIUase as compared to TTR map in catalytic regions occupying the outer portions of the two halves of the central channel that transverses the whole tetrameric proteins. The evolution of HIUase into TTR has been accompanied by remarkable changes of the catalytic sites to give rise to a channel open at both ends, thus allowing free access to hormone molecules. Keywords 5-Hydroxyisourate hydrolase, Transthyretin, Molecular evolution, Uric acid degradation, Thyroid hormones

G. Zanotti (*) Department of Biological Chemistry, University of Padua, and ICB-CNR, Section of Padua, Viale G. Colombo 3, 35121 Padova, Italy e-mail: [email protected]


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Introduction

5-Hydroxyisourate (HIU) is the product of the urate oxidase reaction occurring in the ureide pathway, in which the oxidative degradation of urate to (S)-allantoin is accomplished. Urate is generated in purine metabolism, through the degradation of the nucleic acid components adenine and guanine. Depending on the organism, urate, the anion of uric acid which is predominant under physiological conditions, either represents an end metabolic product or is further degraded oxidatively, involving the enzyme urate oxidase, to produce allantoin in the ureide pathway. Specifically, humans lack a functional urate oxidase, a characteristic they share with apes, Dalmatian dogs, birds, reptiles, yeasts and various bacteria. As a consequence, the concentration of urate in human blood serum is remarkably higher than that present in the blood serum of other primates and mammals that possess urate oxidase. In the presence of hyperuricemia, a pathological situation that may affect humans, the poorly soluble urate salts can precipitate in joints, giving rise to gouty arthritis, and can produce kidney stones and renal failure. The reasons for the absence of a urate degradative pathway in humans, associated with high levels of urate in blood serum, are controversial. It should be pointed out, however, that due to its antioxidant properties urate may exert a beneficial effect by neutralizing reactive oxygen species, so that the advantage of having a large amount of this compound may compensate the potential onset of a painful disease. It has long been known that urate undergoes oxidation catalyzed by urate oxidase in a diverse array of organisms. The longstanding question as to the way by which urate in such organisms produces allantoin was not answered until recently. In particular, based on the observation that allantoin could be generated in vitro upon urate oxidase reaction, it was initially thought that allantoin might represent a product of this enzymatic reaction. More recently, it has been established that HIU is the true product of the urate oxidase reaction and that it undergoes a quite slow nonenzymatic decomposition into racemic allantoin (Kahn 1997). By contrast, (S)-allantoin is produced in living organisms, consistent with the enzymatic conversion of HIU into this allantoin stereoisomer in vivo. The two enzymes involved in the ureide pathway leading to (S)-allantoin from HIU in the urate oxidase reaction have indeed been identified. An enzyme catalyzing the hydrolysis of HIU, and therefore designated HIU hydrolase (HIUase), to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) was first found in soybean root nodules (Sarma et al. 1999; Raychaudhuri and Tipton 2002). Two homologous HIUases, respectively from Bacillus subtilis (Lee et al. 2005) and from mouse (Ramazzina et al. 2006), which lacked homology with the soybean enzyme, have then been heterologously expressed and identified unambiguously by assaying their catalytic activity. OHCU decarboxylase is the next enzyme in the pathway, catalyzing the decarboxylation of OHCU to produce (S)-allantoin (Ramazzina et al. 2006). Very recently, the crystal structures of OHCU decarboxylase from zebrafish (Cendron et al. 2007) and from Arabidopsis thaliana (Kim et al. 2007) have been determined, revealing features consistent with the stereoselective formation of (S)-allantoin as reaction product. The complete degradation

6 Vertebrate HIU Hydrolase Identification, Function, Structure

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Scheme. 6.1 Degradation route from urate to (S)-allantoin

route from urate to (S)-allantoin is illustrated in Scheme. 6.1. It must be considered that HIU can form allantoin nonenzymatically on a time scale of hours, while HIUase and OHCU decarboxylase accomplish this conversion stereoselectively, producing (S)-allantoin, on a time scale of seconds (Ramazzina et al. 2006). Remarkably, the aforementioned HIUases from B. subtilis and mouse have been found to belong to a family of prokaryotic and eukaryotic proteins of unknown function but similar to the vertebrate protein thyroxine binding transthyretin (TTR). The members of this protein family were therefore designated transthyretin-like proteins (TLPs) (Prapunpoj et al. 2000; Hennebry et al. 2006a) or transthyretinrelated proteins (TRPs) (Eneqvist et al. 2003). The presence of members of the TLPs/TRPs family possessing HIUase activity has also been established for the enterobacteria Salmonella dublin (Hennebry et al. 2006b) and Escherichia coli (Lee et al. 2005; Lundberg et al. 2006). We report here on the identification and structural characterization of vertebrate HIUase and on its evolutionary relationship with TTR.

6.2

Identification and Function of Vertebrate HIUase

To find the missing enzymes in the biological conversion of uric acid to allantoin, we sought proteins with a functional link to urate oxidase, the first enzyme of the pathway. Functional links between different proteins can be inferred through genome comparisons, by the identification of a relationship between the corresponding genetic elements. Three criteria were used for this identification. First, we examined genes of unknown function that are found in the neighborhood of the gene coding for urate oxidase. The rationale for this criterion is that genes that have a coordinated expression, and hence a related function, tend to be physically clustered, as observed frequently, but not exclusively, in prokaryotic genomes (Snel et al. 2002; Hurst et al. 2004). Second, we looked for cases in which urate

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oxidase is fused together with other proteins; such instances indicate that the two proteins can interact or be involved in the same metabolic pathway, even when they are encoded by distinct polypeptide chains (Marcotte et al. 1999). Third, we selected genes that show a pattern of presence and absence across a range of genomes that is similar to that observed for urate oxidase; common ‘phylogenetic profiles’ (Pellegrini et al. 1999) of different genes indicate that the different traits are under a common selective pressure and hence there is a functional relationship among them. Two gene families encoding proteins of unknown function have been found to be linked to urate oxidase in accordance with the aforementioned criteria. When the pattern of gene presence or absence across genomes was evaluated in a phylogenetic context (Barker and Pagel 2005), the link between urate oxidase and the other two gene families emerged clearly. Urate oxidase has been gained or lost several times during species evolution; these events have been accompanied by a parallel gain/loss of the other two gene families (Ramazzina et al. 2006). On the basis of the evidence obtained by genome comparison, we devised experiments to investigate the role of the two protein families in urate metabolism. Phylogenetic across-genome correlation indicated that the functional linkage with urate oxidase had been maintained from bacteria to humans (Ramazzina et al. 2006). We therefore chose to probe the activity of mammalian proteins. Genes of murine origin, designated as uraH and uraD, representatives of the two protein families, were expressed in recombinant form, by using E. coli as expression organism, and the corresponding proteins were purified. We then tested the activity of the two proteins on the conversion of HIU produced by the urate oxidase reaction. Because optically active compounds are involved, the formation of the products of the reactions occurring in the presence of the two proteins was conveniently monitored by circular dichroism. In particular, the HIU CD spectrum presents specific maxima (at about 210 and 270 nm) and minima (at about 240 and 310 nm), the OHCU CD spectrum is characterized by a maximum and a minimum at about 230 and 260 nm, respectively, while the CD spectrum of (S)-allantoin shows a maximum at about 215 nm (Ramazzina et al. 2006). The HIU hydrolase and OHCU decarboxylase activities for uraH and uraD, respectively, could thus be definitely established: formation of levorotatory OHCU from levorotatory HIU catalyzed by HIUase and formation of (S)-allantoin from levorotatory OHCU catalyzed by OHCU decarboxylase (Fig. 6.1). The recombinant HIUase orthologue from zebrafish (Danio rerio), possessing a specific activity similar to that of the corresponding murine enzyme, was then obtained in our laboratory and structurally characterized (Zanotti et al. 2006).

6.3

Molecular Evolution of the HIUase/Transthyretin Family

The oxidative degradation of purines is found in bacteria and in the main eukaryotic lineages. In this pathway, there are examples of alternative branches and of single enzymatic steps involving different genes in different organisms, with genes that


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Fig. 6.1 Enzymatic activity of HIUase and OHCU decarboxylase. Time resolved CD spectra of uric acid degradation products in the presence of urate oxidase (a), or in the presence of urate oxidase and HIU hydrolase (b), or in the presence of urate oxidase, HIUase and OHCU decarboxylase (c). Spectra were collected every 2 min. For additional details regarding the kinetics of the enzymatic reactions, see Ramazzina et al. (2006)

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appear to be of more recent acquisition (Ramazzina et al. 2008). Genomic analyses indicate, however, that the enzymatic oxidation of uric acid is always associated with HIU hydrolysis, and that this activity is always encoded by uraH. The uraH gene was thus probably present in the early evolution of the oxidative pathway of purine degradation, which could have started in bacteria after the innovation of oxygenic photosynthesis and the formation of atmospheric oxygen about 2.3 Gyr ago (Mulkidjanian et al. 2006). From bacteria, the pathway could have been horizontally transferred to eukaryotes with mitochondrial symbiosis around 1.8 Gyr ago (Hedges et al. 2001), thus explaining the lack of HIUase and the other enzymes of the pathway in archea. During evolution, most of the uraH-type genes have maintained the original role in purine degradation and a close association with those encoding urate oxidase and OHCU decarboxylase. Similar to urate oxidases and OHCU decarboxylases, HIUases are typically cytosolic proteins in bacteria and peroxisomal proteins in eukarya. However, some innovations have also taken place, as in the case of the periplasmic HIUase of enterobacteria (Hennebry et al. 2006a; Eneqvist et al. 2003) and the expansion and functional diversification of the family observed in nematodes (Jacob et al. 2007; Saverwyns et al. 2008). In vertebrates, the major evolutionary modifications of HIUases have been pseudogenization of the uraH gene, which occurred in man and other vertebrates (Ramazzina et al. 2006), and a gene duplication event that gave rise to TTR (Hennebry et al. 2006a; Zanotti et al. 2006). Consistent with a gene duplication event, in vertebrates the gene encoding HIUase and TTR have the same intron/exon structures (Fig. 6.2a). The first coding exon encodes a signal peptide in TTR proteins and a type 2 peroxisomal targeting signal (PTS) in HIUases. However, the analysis of the Espressed Sequence Tag (EST) of HIUase indicates that, in mouse and other organisms, a shorter protein corresponding to HIUase but lacking the PTS sequence is also produced. Given that HIU is an unstable substrate that should only be produced in peroxisomes, the functional significance of extra-peroxisomal HIUases is unclear. Conservation of the intron position and sequence similarity in the first exon suggest that, after gene duplication, the PTS sequence of HIUase was transformed into the TTR signal peptide by point mutations. The gene duplication event that gave origin to TTR probably occurred after the separation of earliest deuterostoma (500 Myr ago), as suggested by the absence of TTR sequences in the genomes of protochordates. On the other hand, the presence of a TTR sequence in the shark Leucoraja erinacea (GenBank CV221819) indicates that the duplication predates the separation between cartilaginous and bony fishes (400 Myr ago). Consistent with these observations, a phylogenetic reconstruction of the two families shows the branching of TTR slightly before the separation of chordates (Fig. 6.2b). During the early evolution of chordates, profound modifications of the TTR ancestor occurred to adapt the binding cavity to the two thyroid hormones 3,5,30 -triiodo-l-thyronine (T3) and 3,5,30 ,50 -tetraiodo-l-thyronine (thyroxine, T4) (see below).


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Fig. 6.2 Evolutionary comparison of HIUase and TTR. (a) Sequence alignment of the proteins encoded by the uraH genes from mouse (Acc. No: EDL17955), green monkey (Acc. No: ABE02226) and zebra fish (Acc. No: Q06S87) with mouse TTR (Acc. No: EDK96952). The two alternative starting methionines (M1 and M2), the peroxisomal targeting signal (PTS) and the position of introns (with intron phases in parenthesis) are indicated. (b) Phylogenetic relationship of HIUase and TTR proteins. The unrooted phylogenetic tree has been constructed with the maximum-likelihood method implemented in the Proml program of the PHYLIP package (Felsenstein 1981) and displayed with the TreeIllustrator program (Trooskens et al. 2005) with branch length correction for visibility enhancement. Eukaryotic sequences are indicated with the binomial name of species. The major taxonomic groups are indicated

6.4

Crystal Structure of Zebra Fish HIUase

To date, the structure of zebrafish HIUase is the only known structure of a vertebrate HIUase (Zanotti et al., 2006). It has been determined for two crystal forms, monoclinic (P21) and orthorhombic (P212121), both at a relatively good

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Fig. 6.3 (a) Stereo view of a cartoon representation of zebra fish HIUase. The view is along the twofold molecular axis that transverses the central channel of the tetrameric enzyme. (b) Amino acid sequence of zebra fish HIUase aligned with that of human TTR. Secondary structure elements for both proteins are reported (b-strand, arrows; a-helix, spirals). The residues conserved in the two proteins are boxed; those conserved in HIUases and substituted in TTR are denoted by an orange background

˚ , respectively). In the monoclinic crystal form there is resolution (1.98 and 1.70 A one monomer in the asymmetric unit, whilst two monomers are present in the asymmetric unit for the orthorhombic form. The two models are virtually identical, suggesting that the protein conformation is quite rigid and barely influenced by crystal packing. Like TTR, whose molecular model was in fact used to solve the structure by molecular replacement, HIUase is a homo-tetramer, formed by four identical subunits arranged according to a 222 symmetry (Fig. 6.3a: monomers are labeled from A to D). An analysis of a monomer performed with the software Procheck (Laskowski et al. 1993) indicates that it is composed of ten b-strands (position and numbering convention for b-strands are illustrated in Fig. 6.3b), but two of them (strands 1–2 and 8–9) can be considered the two halves of strands bA and bG. Consequently, each HIUase monomer can be described as composed of eight antiparallel b-strands, arranged in a topology similar to the Greek key bbarrel, with a short a-helix located at the end of b-strand E. As in the case of TTR, a dimer is formed through the interactions between strands F and H of a monomer with the corresponding ones of another, resulting in an eight-stranded b-sandwich. In turn, two dimers interact, mainly through hydrophobic contacts, to form the tetramer. Most of the interactions between dimers are established between monomers A and C (and their equivalent B and D), but some contacts are also present between monomers A and D (and B and C).


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Comparison of the Structures of HIUase and Transthyretin

The structure of zebrafish HIUase is very similar to that of human TTR (PDB 1F41, (Hornberg et al. 2000)): a comparison of monomers and dimers of the two proteins ˚ , respectively. Similar values are gives root mean square deviations of 1.0 and 1.2 A found for the comparison between zebrafish HIUase and sea bream (Sparus aurata) TTR (PDB 1OO2, (Folli et al. 2003)). The structures of HIUases from three bacteria (S. dubli, PDB 2GPZ, (Hennebry et al. 2006b); B. subtilis, PDB 2H0E, (Jung et al. 2006); E. coli, PDB 2G2N, (Lundberg et al. 2006)) also compare quite well with that of the zebrafish enzyme. Major differences are found in some loops that are exposed to the solvent and in some cases are flexible in human TTR. The S. Dublin HIUase (PDB 2GPZ) differs from the vertebrate enzyme mainly in the flexible portions of strands B and C that protrude towards the solvent, and in the conformation of the long loop connecting strands D and E. This situation is similar for the E. coli enzyme (PDB 2G2N). More relevant differences are present in the B. subtilis enzyme (PDB 2H0E), in which the loop B–C is shorter, whilst the loop that connects the short a-helix to strand F is longer as compared to zebrafish HIUase. In comparison with TTR, some of the b-strands in zebrafish HIUase present irregularities in the hydrogen bonding pattern. In particular, Pro-105 (residue numbering of zebrafish HIUase, Fig. 6.3b) in the middle of b-strand G hinders the formation of H-bonds with residues of nearby strands: in addition to the lack of interaction between the nitrogen of residue Pro-105 with the carbonyl of Tyr-116 of b-strand H, the carbonyls of Val-104 and Pro-105 do not form H-bonds with nitrogen atoms of His-12 of b-strand A and of Tyr-116 of b-strand H, respectively. This results in irregularities in b-strands A, G and H. However, the major difference between HIUase and TTR is found for the long channel that transverses the entire tetrameric proteins and is coincident with one of their twofold symmetry axes. In TTR, this channel harbors the binding sites for two thyroid hormone molecules, which are accommodated in the two symmetrical halves of the channel (Wojtczak et al. 2001). In HIUase this channel is still present, but is obstructed at both ends by the side chains of Tyr-116, Leu-14, and Leu-107. In particular, the comparison of the molecular surface of the catalytic cavity of HIUase with that of the corresponding region of TTR, i.e. the thyroid hormone binding cavity, shows that the cavity in HIUase is markedly shallower than that of TTR, such that it cannot host the bulky hormone molecule (Fig. 6.4a). The structural comparison also illustrates the evolutionary changes that have made possible the functional transition of the enzyme into a transport protein. In each catalytic cavity of HIUase, two Tyr-116 residues contribute substantially to the formation of the bottom of the cavity. The key molecular event has been the substitution of Tyr-116 with Thr-119 residue of TTR. In TTR, this replacement opens up the two ends of the channel, making it accessible to two hormone molecules. At the same time, all the residues of HIUase potentially relevant for catalysis (see below) are replaced in TTR: His-12/Lys-15, Asp-50/Ser-52, Arg-52/Glu-54, His-103/Thr-106, and Ser-119/Val-122 (Fig. 6.3b). Remarkably, the first three replacements appear to be relevant to allow thyroid

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hormone binding and are therefore associated with the acquisition of the novel functional properties of TTR. The internal portion of the channel, which represents a closed cavity not accessible to the solvent, is filled in the crystal structure of HIUase by five ordered water molecules. The residues lining the internal cavity are quite conserved as compared to TTR.

6.4.2

The HIUase Active Site

Taking into account the positions of conserved residues of putative HIUases from a variety of prokaryotic and eukaryotic organisms, the results of site-directed mutagenesis experiments (Hennebry et al. 2006b; Zanotti et al. 2006; Jung et al. 2006), and the crystal structures of the B. subtilis enzyme in complex with substrate analogues (Jung et al. 2006), the location of two putative catalytic regions in the HIUase tetramer, occupying the two symmetrical entrances of the central channel, can be considered definitely established with high reliability. A model of the crystal structure of zebrafish HIUase with the substrate analogue 8-azaxanthine as it is

a

Fig. 6.4 (continued)


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b

Fig. 6.4 (continued) (a) van der Waals representation of the homo-tetramers of HIUase (left) and TTR (right, PDB ID 1F41). Details of the molecular surfaces of the boxed area of the HIUase active site cavity (left) and of the corresponding area in TTR (right) are shown below van der Waals representations. The TTR-bound thyroxine molecule (stick model, PDB 1IE4, Wojtczak et al. (2001)) is also shown. Different colors correspond to different subunits of the tetrameric proteins. (b) Detail of the active site of zebra fish HIUase, located in between two subunits at one end of the central channel; the residues conserved in the enzyme family are labeled. Due to the molecular symmetry, two active sites are present on opposite sides of the tetramer. Each of them is formed by the same residues belonging to two different subunits

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bound to B. subtilis HIUase (Jung et al. 2006) superimposed reveals that several residues near the HIUase-bound substrate analogue are conserved and identically positioned (not shown), indicating that active site characteristics have been well preserved from prokaryotes to vertebrates during evolution. In particular, His-12, Asp-50, Arg-52, His-103, and Pro-105 are the residues in the area of the active site that are conserved in all HIUases, along with four residues (Tyr-116, Arg-117, Gly-118, and Ser-119) constituting the HIUase signature at its C-terminus. It should be noted that all these residues, due to the twofold molecular axis coincident with the protein central channel, are contributed twice, at each of the two ends of the channel, by two subunits (subunits A and C and subunits B and D) belonging to different dimers (Fig. 6.4b). Site-directed mutagenesis experiments have shown that the replacement of Asp-50 by Asn reduces the enzymatic activity of zebrafish HIUase to about 50%, indicating that this residue is not essential for catalysis (Zanotti et al. 2006). Similar experiments with B. subtilis HIUase (Jung et al. 2006) have shown that the presence of His-12 and of a positively charged residue (Arg or Lys) at position 52 represent absolute requirements for catalytic activity. The residues of the C-terminal signature, except for the last Ser residue, are also essential for the enzymatic activity. While confirming the essential role played by His-12, site-directed mutagenesis experiments with S. Dublin HIUase have shown that mutations replacing His-103 and Tyr-116 affect substantially the activity, without abolishing it completely (Hennebry et al. 2006b). Interestingly, the crystal structure of the E. coli HIUase has revealed Zn2+ and Br ions near the putative enzyme active sites; however, the finding of such ions bound to HIUase might have been caused by their presence in the crystal medium (Lundberg et al. 2006). In this regard, it should be noted that Zn2+ ions do not appear to be required for the catalytic activity of zebrafish HIUase (Zanotti et al. 2006). A catalytic mechanism has been proposed for B. subtilis HIUase (Jung et al. 2006), according to which the major catalytic role is played by His-12 and Arg-52. Since no evident nucleophile provided by a catalytic residue appears to be present near the active site, it has been proposed that His-12 could serve as a general base for the deprotonation of a water molecule, which would carry out a nucleophilic attack on C6 of HIU to generate an oxyanion O6, stabilized by the positively charged Arg-52. In this mechanism, a role for other residues, such as His-103 and Tyr-116, has also been proposed (Jung et al. 2006).

6.5

Conclusions

Despite the similarity with TTR, the function of vertebrate HIUase could be correctly predicted by bioinformatic analysis, through whole genome comparison. HIUase and TTR are proteins with very similar sequence and structure, yet with radically different binding properties and functions. These two families thus illustrate a clear case of functional divergence that occurred in vertebrate proteins. In protein evolution, adaptation of old proteins for new functions is a common


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phenomenon (Todd et al. 2001). This phenomenon is also observed, for example, in the case of another thyroid hormone carrier protein, thyroxine-binding globulin (TGB), which originated from the protease inhibitors serpins (Irving et al. 2000). There are some well-known examples in which a single fold supports both enzymatic and nonenzymatic functions (Todd et al. 2002), as observed in the case of TTR-HIUase. In the majority of these examples, a catalytic progenitor gave rise to a nonenzyme through the substitution of residues that are critical for catalysis and/or substrate binding. However, the substitution of a limited number of residues at the active site pocket is generally sufficient for the acquisition of new functional properties. Almost all of the residues that line the zebrafish HIUase active site have been replaced in human TTR. By contrast, the residues that are located outside the active site are often conserved in the two proteins. In this respect, HIUase and TTR represent a remarkable example of active site modification and functional divergence by accelerated evolution.

References Barker D, Pagel M (2005) Predicting functional gene links from phylogenetic-statistical analyses of whole genomes. PLoS Comput Biol 1:e3 Cendron L, Berni R, Folli C, Ramazzina I, Percudani R, Zanotti G (2007) The structure of 2-oxo-4hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase provides insights into the mechanism of uric acid degradation. J Biol Chem 282:18182–18189 Eneqvist T, Lundberg E, Nilsson L, Abagyan R, Sauer-Eriksson AE (2003) The transthyretinrelated protein family. Eur J Biochem 270:518–532 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555:279–284 Hedges SB, Chen H, Kumar S, Wang DY, Thompson AS, Watanabe H (2001) A genomic timescale for the origin of eukaryotes. BMC Evol Biol 1:4 Hennebry SC, Wright HM, Likic VA, Richardson SJ (2006a) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins Struct Funct Bioinfo 64:1024–1045 Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC (2006b) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359:1389–1399 Hornberg A, Eneqvist T, Olofsson A, Lundgren E, Sauer-Eriksson AE (2000) A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 302:649–669 Hurst LD, Pal C, Lercher MJ (2004) The evolutionary dynamics of eukaryotic gene order. Nat Rev Genet 5:299–310 Irving JA, Pike RN, Lesk AM, Whisstock JC (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 10:1845–1864 Jacob J, Vanholme B, Haegeman A, Gheysen G (2007) Four transthyretin-like genes of the migratory plant-parasitic nematode Radopholus similis: members of an extensive nematodespecific family. Gene 402:9–19 Jung DK, Lee Y, Park SG, Park BC, Kim GH, Rhee S (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc Natl Acad Sci USA 103:9790–9795

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Kahn K, Serfozo P, Tipton PA (1997) Identification of the true product of the urate oxidase reaction. J Am Chem Soc 5435–5442 Kim K, Park J, Rhee S (2007) Structural and functional basis for (S)-allantoin formation in the ureide pathway. J Biol Chem 282:23457–23464 Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck – a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291 Lee Y, Lee DH, Kho CW, Lee AY, Jang M, Cho S, Lee CH, Lee JS, Myung PK, Park BC, Park SG (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774 Lundberg E, Backstrom S, Sauer UH, Sauer-Eriksson AE (2006) The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457 Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D (1999) Detecting protein function and protein–protein interactions from genome sequences. Science 285:751–753 Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, Dufresne A, Partensky F, Burd H, Kaznadzey D, Haselkorn R, Galperin MY (2006) The cyanobacterial genome core and the origin of photosynthesis. Proc Natl Acad Sci USA 103:13126–13131 Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO (1999) Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci USA 96:4285–4288 Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G (2000) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol Regul Integr Comp Physiol 279:R2026–R2041 Ramazzina I, Cendron L, Folli C, Berni R, Monteverdi D, Zanotti G, Percudani R (2008) Logical identification of an allantoinase analog (puuE) recruited from polysaccharide deacetylases. J Biol Chem 283:23295–23304 Ramazzina I, Folli C, Secchi A, Berni R, Percudani R (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2:144–148 Raychaudhuri A, Tipton PA (2002) Cloning and expression of the gene for soybean hydroxyisourate hydrolase. Localization and implications for function and mechanism. Plant Physiol 130:2061–2068 Sarma AD, Serfozo P, Kahn K, Tipton PA (1999) Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme. J Biol Chem 274:33863–33865 Saverwyns H, Visser A, Van Durme J, Power D, Morgado I, Kennedy MW, Knox DP, Schymkowitz J, Rousseau F, Gevaert K, Vercruysse J, Claerebout E, Geldhof P (2008) Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – comparison with other strongylid nematodes and Caenorhabditis elegans. Int J Parasitol. doi 10.1016/j.ijpara.2008.04.004 Snel B, Bork P, Huynen MA (2002) The identification of functional modules from the genomic association of genes. Proc Natl Acad Sci USA 99:5890–5895 Todd AE, Orengo CA, Thornton JM (2002) Sequence and structural differences between enzyme and nonenzyme homologs. Structure 10:1435–1451 Todd AE, Orengo CA, Thornton JM (2001) Evolution of function in protein superfamilies, from a structural perspective. J Mol Biol 307:1113–1143 Trooskens G, De Beule D, Decouttere F, Van Criekinge W (2005) Phylogenetic trees: visualizing, customizing and detecting incongruence. Bioinformatics 21:3801–3802 Wojtczak A, Cody V, Luft JR, Pangborn W (2001) Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr D Biol Crystallogr 57:1061–1070 Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9

Chapter 7

Transthyretin-Related and Transthyretin-like Proteins A. Elisabeth Sauer-Eriksson, Anna Linusson and Erik Lundberg

Abstract Bioinformatics programs are highly accurate in identifying protein families directly from protein sequences, even when the sequence identity is very low. The transthyretin-related proteins (TRPs) are one example of a protein family that has been identified. Whereas transthyretin (TTR) is well-characterized both in vivo and in vitro, only recently has research focused on the TRPs. Their structural similarity to TTR has been verified, and their function as a 5-hydroxyisourate (HIU) hydrolase has been established. In this review we discuss structural aspects of TRP function. We also discuss the still unknown transthyretin-like proteins (TLPs) that are seemingly unique to nematodes. Keywords Transthyretin-related protein, HIU hydrolase, Transthyretin, Amyloid, Fibril formation

7.1

Introduction

The transthyretin-related proteins (TRPs) comprise a relatively recently identified family of proteins (Eneqvist et al. 2003). Homologues of TRP can be found in a wide range of species from bacteria, plants and animals. Extensive biochemical analysis of the TRP, in combination with structural studies, has highlighted the main structural features that govern their tetrameric assembly and function. The proteins function as hydrolases, catalyzing the second reaction which degrades purine nucleotides in the ureide pathway.

A.E. Sauer-Eriksson (*) Department of Chemistry, Umea˚ University, SE-90187 Umea˚, Sweden e-mail: [email protected]


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Whereas the physiological relevance of TRP in prokaryotes is relatively clear, the same is not true for its function in vertebrates. Purines are major components of nucleic acids and nucleotides, and purine degradation pathways can be found in all kingdoms of life (Nygaard 1983). In most mammals, allantoin is the final product of the purine degradation pathway and it is excreted as one of the major components in urine (Fig. 7.1). However, humans and other primates lack the enzyme uricase and excrete uric acid as the final product of purine degradation. Some other species (e.g., fish and amphibians) have enzymes that further degrade allantoin to allantoic acid and finally to glyoxylic acid and urea. In prokaryotes, the purine degradation products are mostly utilized as nitrogen sources, but they may serve other functions depending on the organism. In Escherichia coli the pathway seems incomplete as allantoin is produced but not urea and glyoxylic acid (Xi et al. 2000). E. coli can utilize allantoin as a nitrogen source but only under anaerobic conditions (Cusa et al. 1999). In Bacillus subtilis on the other hand a complete purine degradation pathway is present (Fisher 1999; Schultz et al. 2001). The specific function of TRP in the purine degradation pathway is to facilitate the hydrolysis of 5-hydroxyisourate (5-HIU), the end product of the uricase reaction, and to convert it to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) (Fig. 7.1). This catalytic activity was first identified in the TRPs of B. subtilis and E. coli (Lee et al. 2005; Jung et al. 2006), but it has also been verified in Salmonella dublin (Hennebry et al. 2006a) and Mus musculus TRP (Lee et al. 2006; Ramazzina et al. 2006). The need for a 5-HIU hydrolase for this task was initially not apparent as 5-HIU can nonenzymatically convert into allantoin under in vitro conditions. However, the spontaneous conversion of 5-HIU generates a racemic mix of S- and R-allantoins, and the allantoinase, which constitutes the next enzyme in the purine catabolism pathway, is specific for S-allantoins only. From the observation that racemization of allantoin takes several hours, it became apparent that a novel enzyme might be involved (Kahn and Tipton 1998). One year later the first HIU hydrolase, which facilitated stereo-specific conversion of HIU to S-allantoin, was

Fig. 7.1 The purine catabolic pathway

7 Transthyretin-Related and Transthyretin-like Proteins

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discovered in soybean (Sarma et al. 1999). By producing the less reactive compound allantoin, HIU-hydrolases like TRP could also serve an important function in the periplasm of enterobacteria, to prevent accumulation of toxic free radical species, generated by antioxidant activity of uric acid (Hennebry et al. 2006b). Occasionally, the TRPs are referred to in the literature as transthyretin-like proteins or TLPs. In addition to the TRPs, which comprise the characteristic sequence motif Y-R-G-S at their C-terminal end, there are protein sequences listed as TTR-like in the databases, with a particularly large number of them in Caenorhabditis elegans. We therefore prefer to refer to the ‘‘Y-R-G-S-motif’’ family as TRP or HIU hydrolase with a TTR-like fold (Eneqvist et al. 2003; Lundberg et al. 2006), to distinguish them from the so-far uncharacterized TLP proteins. There are 57 genes in C. elegans which are listed as TTR-1 to TTR-57 in WormBase (a genetic database for C. elegans and related nematodes; www.wormbase.org). Some of these TLP sequences were originally identified as structurally TTR-like by Sonnhammar and Durbin (1997). Of these, the TLP listed as ‘‘TTR-1’’ has been shown to influence aging in C. elegans (Hansen et al. 2005). The 57 TLP sequences present in C. elegans share low sequence identity with each other; however sequences TTR-18 to TTR-31 seem to comprise a subgroup found in other nematodes. Sequence analysis of representatives from TTR, TRP, and TLP suggests that the three protein groups are not functionally related (Fig. 7.2). Whereas residues at the ligand-binding sites are almost completely conserved in the TTR and TRP families, they are not conserved in the TLP group. In contrast, the most conserved regions in TLP can be found at sites corresponding to b-strands B and E and the a-helix in TTR and TRP. The present review will focus on structural aspects of TRP which have advanced our understanding of this protein family. Recently it has also been established that TRP can form amyloid fibrils (Lundberg et al. 2009; Santos et al. 2008). We will also present results from a bioinformatic study on TLP, and a preliminary 3D model of one TLP from the nematode C. elegans. Interestingly, the model suggests that TLP’s structural similarity to TTR and TRP might not be entirely preserved.

7.2 7.2.1

The Structure of TRP Overall Structure

The sequence identity between TRP and TTR is relatively low; E. coli TRP (ecTRP) shares 30% sequence identity with hTTR (Fig. 7.2) (Prapunpoj et al. 2000; Eneqvist et al. 2003). In addition to the C-terminal Y-R-G-S motif, four more motifs have been identified as defining TRPs, and can be used to distinguish them from TTRs (Hennebry et al. 2006b). Structures of TRP have been determined from several species, and include those from E. coli (Lundberg et al. 2006),

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B

TRP-D.rerio (2H1X) TRP-S.dublin (2GPZ) TRP-B.subtilis (2H0E) TRP-E.coli (2G2N)

* * * * * ---------------MNRLQHIRGHIVSADKHINMSATLLSPLSTHVLNIAQGVPGANMT ---------------MKRHILATVIASLVAAPAMALAAGNNILSVHILDQQTGKPAPGVE --------------------------------MSEPESLMGKLTTHILDLTCGKPAANVK --------------MLKRYLVLSVATAAFSLPSLVNAAQQNILSVHILNQQTGKPAADVT

26 20 28 23

TTR-G.gallus (1TFP) TTR-H.sapiens (1F41) TTR-S.aurata (1SN2) TTR-R.norvegicus(1GKE)

------MAFHSTLLVFLAGLVFLSEAAPL--VSHGSVDSKCPLMVKVLDAVRGSPAANVA ------MASHRLLLLCLAGLVFVSEAGP-----TGTGESKCPLMVKVLDAVRGSPAINVA -------MLQPLHCLLLASAVLLCNTAPTPTDKHGGSDTRCPLMVKILDAVKGTPAGSVA ------MASLRLFLLCLAGLIFASEAGP-----GGAGESKCPLMVKVLDAVRGSPAVDVA

32 29 32 22

TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C.briggsae-TTR-18

---MFR--FSLLLLLVPLVR----CVPNPKPLFGIGIGRKQSAGAEGKLTCAGEPLADVK MAPMFLPTVSVFLLLLVVVQQSLLVLASPKPLFGIGIGRTQSAGVEGTLLCEGKPMADVL ---MFR------LLLLLIIS-----LPIVFCAFGGLVGRVQSTGVRGTLMCNGRPAPRVL MEK---------AVLVVCVF------FCLLPVTLSSLGQRQRVIVKGRLLCGNAPASNIR --------MSSVYSCLILLT-----FIVASFCISIEIGNLQSVSVNGTLLCNDKPAKNIK ------------MHQLILVA-----LFVSTASSLPFIGSVQSVRVTGKVTCNGSPAENIK * * . . * : * . * :

35 33 29 26 26 27

B

C

D

E

a

TRP-D.rerio TRP-S.dublin TRP-B.subtilis TRP-E.coli

* * IVLHRLDPVSSAWNILTTGITNDDGRCPG--LITKENFIAGVYKMRFETGKYWDALGE-VVLEQKKDN--GWTQLNTGHTDQDGRIKA--LWPEKAAAPGDYRVIFKTGQYFESKKL-IGLKRLGES-----IMKEVYTNNDGRVDVP-LLAGEELMSGEYVMEFHAGDYFASKNMNA VTLEKKADN--GWLQLNTAKTDKDGRIKA--LWPEQTATTGDYRVVFKTGDYFKKQNL--

82 74 82 77

TTR-G.gallus TTR-H.sapiens TTR-S.aurata TTR-R.norwegicus

VKVFKKAAD-GTWQDFATGKTTEFGEIHE--LTTEEQFVEGVYRVEFDTSSYWKGLGL-VHVFRKAAD-DTWEPFASGKTSESGELHG--LTTEEQFVEGIYKVEIDTKSYWKALGI-LKVSQKTAD-GGWTQIATGVTDATGEIHN--LITEQQFPAGVYRVEFDTKAYWTNQGS-VKVFKRTAD-GSWEPFASGKTAESGELHG--LTTDEKFTEGVYRVELDTKSYWKALGI--

87 84 87 77

TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C-briggsae-TTR-18

VKLYDDDRGVDTDDLMGETRTDSEGRFRLEGYTHEITTIDPKINIYHDCNDGLKPCQR-VKLYDDDRGVDTDDLMAEGKTDSKGRFSLEGYTHEFTTIDPKINIYHDCND-LLPCQR-IKLYDDDRGIDMDDFMGETKSDSQGNFELSGYIHEMSPIDPKFNIYHDCNDGIKPCQR-VKLVDEDDGPDPDDDMDDGYTNDNGEFLLDGQQTEISPIDPVLKIYHDCND-GLPCQR-VKLYEEEAILDV--LLDERFTKDDGTFEMAGSKSEVTTIDPKLNIYHKCN-YDGICVR-VKLYEKEILLDK--LLDERSTDVKGSFSLAGNKKELTAIDPHVNIYHKCN-YKGVCYK-:** :.: * : : :. .* * : * *.:.*** .:***.** * :

93 90 87 83 81 82

F

G

H

TRP-D.rerio TRP-S.dublin TRP-B.subtilis TRP-E.coli

*** * * * * *** --TCFYPYVEIVFTITNTS-QHYHVPLLLSRFSYSTYRGS------DTFFPEIPVEFHISKTN-EHYHVPLLLSQYGYSTYRGS----ADQPFLTIVTVRFQLADPD-AHYHIPLLLSPFGYQVYRGS------ESFFPEIPVEFHINKVN-EHYHVPLLLSQYGYSTYRGS-----

119 111 121 114

TTR-G.gallus TTR-H.sapiens TTR-S.aurata TTR-R.norwegicus

--SPFHEYADVVFTANDSGHRHYTIAALLSPFSYSTTAVVSDPQE --SPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE --TPFHEVAEVVFDAHPEGHRHYTLALLLSPFSYTTTAVVSSVRE --SPFHEYAEVVFTANDSGHRHYTIAALLSPYSYSTTAVVSNPQN

130 127 130 120

TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C-briggsae-TTR-18

--KISIMIPDKYIASGEHPNTYYDAGTVELEGKFSGETRDCLH---KISIMIPDKYISSGKTPERYYNAGEVELEGKFKGEERDCLH---KISIMIPDGYITEAKTPRKWYNAGTIELAGKFAGETRDCIH---KWRFKIPNKYIVDPEDSSTVMNMGTWNLEPIADDEERDCIH---KISILIPTEYITNGEKPARTFNVGELNLASKFSGQSTDCFN---KLKIKIPKSFISEGETAERTFDIGELNLAGSFSGESTDCLN-* : ** :* . : . : * :* .: **::

134 131 128 124 122 123

Fig. 7.2 TRP representatives from six nematodes have been aligned with representatives from TTRs and TRPs with known 3D structures. Sequence numbering for TTR and TRP are identical to the ones in their respective structures, the first residue being marked with a yellow box. The first residue after the predicted signal sequence cleavage (PHOBIUS, Kall et al. (2004)) is marked with a black box. TRPs from D. rerio and B. subtilis are non-cytoplasmic proteins. For human and chicken TTRs, the yellow box is identical to the position of the black box, thus the black box is not shown. Identical and homologous residues within the three subgroups are marked in red and pink, respectively. Similarity was defined as amino acid substitutions within one of the following groups: FYW, IVLMF, RK, QDEN, GA, TS, and HNQ. Identical and similar residues within the TRP family are shown in dark and light green; within the TTR family in dark and light blue; and within the TLP subgroup in dark and light gray. The secondary structure elements are from human TTR (Hornberg et al. 2000). Residues lining the substrate-binding channel in TTR (Wojtczak et al. 2001) are shown in turquoise stars, and in TRP as red stars (Jung et al. 2006). The assignment (*.:) shown below the sequences is directly from ClustalW2 (Larkin et al. 2007) and refers to alignment of the six TLP sequences only

S. dublin (Hennebry et al. 2006a), Danio rerio (Zebra fish) (Zanotti et al. 2006), and B. subtilis (Jung et al. 2006). Despite their low sequence identity the structures are found to be very similar to TTR (Fig. 7.3a, b). Each monomer is composed of an


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Fig. 7.3 (a) The structure of ecTRP drawn from coordinates 2g2n (Lundberg et al. 2006). The four conserved residues that constitute the active site are shown as balls-and-sticks representation. Two 8-azaxanthine molecules are modeled in the active site (Jung et al. 2006). (b) Superposition of the E. coli structure (pdb code 2g2n, green) with the structures of S. dublin (pdb code 2gpz, gray), Zebra fish (pdb code 2hix, red), and B. subtilis (pdb codes 2h0e, blue). The positions of the b-strands A-H are indicated

eight-stranded antiparallel b-sandwich where each sheet has a Greek-key topology. The b-strands in the monomers are named A-H to follow the nomenclature used for TTR. Furthermore there is one short a-helix per monomer. Two monomers

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dimerize through main-chain-main-chain interactions involving the H-strands and the F-strands. Two of the resulting extended 8-stranded b-sheets (DAGHH0 G0 A0 D0 ), one from each dimer, interact face-to-face, which create the ligand-binding site of the TRP tetramers. The tetrameric form of ecTRP is formed mainly by hydrophobic contacts involving residues situated at the AB- and GH-loops. In addition to the structure of ecTRP solved in our lab (pdb code 2g2n (Lundberg et al. 2006)), one more structure of the same protein from E. coli has become available; however, this work has not been published (pdb code 2igl, deposited by Zuo and Malhotra). The two E. coli structures are almost identical as can be ˚ , obtained by deduced from the low root mean square (r.m.s.) deviation of 0.5 A superimposition of the main chain Ca atoms. The structures deviate mostly at their N-terminal ends, and at some loop regions. Two monomers, in the 2igl structure, A and B, have well-defined N-terminal ends in the electron density. In both of them, the His22 residue is positioned at the entrance of the active site, sandwiched against the side-chain of Arg135. The side chain of A-His22 furthermore forms a hydrogen bond with the main-chain oxygen atom of D-Glu39, and the side chain of B-His22 forms a hydrogen bond to the main-chain oxygen atom of C-Gly136. Thus it appears that the AB-His22 side chains are contributing to the tetramerization of the molecule. It should be noted however that the first four amino acids of the construct, His22-Met23, are part of the vector design and not part of the native protein. Consequently, the position of the first two visible amino acids in 2igl, His22, and Met23, is of no biological relevance. The first ordered amino acid in the 2g2n coordinates is Asn4 (Asn27 in 2igl). The 2igl structure was determined from crystals grown in the absence of zinc ions, something our lab has not been successful in doing. It seems plausible that the additional residues His22 and Met23 positioned at the entrance of the active-site cavity could provide the stability needed for crystal-formation without the presence of zinc. Additional structural discrepancies between the 2igl and 2g2n structures are present in loop regions. In particular these occur at the BC-loops, which constitute the ‘‘stirrups’’ of the upper part of the molecule that we refer to as ‘‘the saddle’’ (Fig. 7.3a). These changes are probably due to different crystal packing interactions at this site. Superposition of the E. coli 2g2n structure with the structures of S. dublin (pdb code 2gpz), Zebra fish (pdb code 2hix), and B. subtilis (pdb codes 2h0e) revealed a highly conserved core structure, with structural changes among the TRP structures at the BC-, DE-, and EF-loop regions. These loops are situated on the same side of the b-barrel (Fig. 7.3b). Rms devations of Ca atoms in the monomers ˚. varies between 0.5 and 1.2 A

7.2.2

Composition of the Active site

The localization of the gene supported a functional role for TRP involving purine catabolism linked to urate oxidase (Eneqvist et al. 2003). From modeling of the E. coli TRP structure it was apparent that the putative ligand-binding site was


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almost entirely conserved and positioned at the same site as for TTR (Eneqvist et al. 2003). The first TRP structures provided the detailed descriptions of the ligandbinding site. In parallel, functional studies verified that the protein actually was an enzyme possessing 5-hydroxyisourate (HIU) hydrolase activity. Structures of TRP in complex with substrate analogues, 8-azaxanthine and 5,6,-diaminouracil, were also determined, and revealed the details of the active site composition upon ligand binding (Jung et al. 2006). The complex studies were done on B. subtilis TRP and provided the first structural evidence that these analogues were indeed ligands of TRP. Overall, binding of substrate analogues caused no large changes in the conformation of the tetrameric proteins; r.m.s. deviation values between the apo ˚ (Jung et al. 2006). As for and the analogue-complexed structures were 0.3–0.4 A TTR, modeling and refinement of bound ligands in TRP were hampered by the twofold symmetry-axis positioned right at the ligand-binding site. Due to this twofold axis, one ligand molecule had to be built with an occupancy of 0.5 with the other orientation generated by the crystallographic twofold symmetry. This procedure resulted in a structure in which two ligands were seemingly positioned very close and on some occasions even on top of each other. The electron density map over the bound ligand displayed an average of the two conformations, which made the density harder to interpret. Consequently, the positions of molecule residuals could have quite high positional errors associated with them. In the case of the B. subtilis TRP substrate–analogue complexes, the ligands could be satisfactorily modeled in the electron density, and a plausible mechanism for HIU hydrolysis was proposed which was also supported by site-directed mutagenesis studies (Jung et al. 2006). In this catalytic mechanism, four residues seem central to the composition of the active site (E. coli numbering in coordinates 2g2n are shown in brackets): Arg49 (Arg47) and His105 (His98) are positioned at the entrance of the active site cavity; His14 (His9) makes up its central part; and Tyr118 (Tyr111) is positioned at the innermost site forming the back-wall of the cavity. In the proposed TRP mechanism, unlike other HIU hydrolases, where negatively charged residues, e.g., glutamates, function as a nucleophile, a water molecule has to be deprotonated, presumably by donating a hydrogen to His14, after which it makes a nucleophilic attack on the C6 carbon of the 5-HIU substrate (Fig. 7.4). The resulting oxyanion O6-moiety is negatively charged and neutralized by the proximity of the guanidinium group of Arg49. Subsequent electron rearrangements on the 5-HIU substrate result in ring cleavage at the C6-N1 bond, most likely by abstracting a proton from the guanidinium group of Arg49. The catalytic cycle is then completed with the transfer of the proton from His14 to Arg49 (Jung et al. 2006). Results from site-directed mutagenesis studies conclusively verified the crucial roles of His14 and Arg49. Significantly, a Tyr114Phe mutant lost its activity, suggesting that also this residue is involved in the catalysis, presumably by interacting with atom O8 from the substrate 5-HIU (Jung et al. 2006). The absolute conservation of the residues His14, Arg49, His105, and Tyr114 suggests that the same mechanism is likely to be valid for all members of the TRP family (Fig. 7.2). The ecTRP structure solved in our lab did not crystallize unless zinc ions were included in the crystallization conditions. Overall, five zinc-binding

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Fig. 7.4 The proposed catalytic mechanism for HIU hydrolysis by TRP, based on work from Jung et al. (2006)

sites are present per monomer, of which two are positioned at the active site, tetrahedrally coordinated to residues His9, His96 and His98, Ser114 and three water molecules (Lundberg et al. 2006). The side chains of these residues have locked positions that do not seem compatible with substrate binding, as judged by homology modeling based on the B. subtilis complex structures. However, in the E. coli 2igl structure, where no zinc ions were present in the crystallization conditions, these side chains have rotamers resembling those found in the substrate-analogue complexes of B. subtilis. Since we have not been able to identify any functional role for zinc-binding in the E. coli TRP protein, it is reasonable to assume that the sidechains of the active site residues in E. coli TRP can adopt the rotamer conformation required for catalytic activity as seen from the B. subtilis complex structure.

7.3

The Amyloidogenic Properties of TRP

In TTR amyloidoses, the normally folded, secreted protein cannot assemble into amyloid fibrils unless a preceding partial unfolding event occurs (Colon and Kelly 1992). How important are tertiary similarities as opposed to sequence identity for proteins with a TTR-like fold to form fibrils? In an attempt to answer this question, we and others have investigated the fibril-forming properties of ecTRP in vitro, and compared the results with those from hTTR (Lundberg et al. 2009; Santos et al. 2008). The results showed that ecTRP has the ability to form Congo red-positive


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fibrils in vitro if the temperature is high enough. The fibril heights were found to be 4.0 nm for ecTRP by atomic force microscopy (AFM) (Fig. 7.5) (Lundberg et al. 2009). This is significantly higher than the 2.8 nm height of hTTR also measured by AFM (Lundberg et al. 2009). With transmission electron microscopy (TEM) the fibril diameter size was estimated to 8.0 nm for ecTRP (Santos et al. 2008). Dimensions of molecules estimated by AFM and TEM are however not directly comparable. Interestingly, misfolded and aggregated ecTRP material has been shown to be toxic for neuroblastoma cells, although the soluble protein is not (Santos et al. 2008). Still, it is not clear if the fibril-formation properties of ecTRP have any biological implication (Lundberg et al. 2009; Santos et al. 2008).

7.4

Structural Model of TLP

The sequence identity between TLP., TTR and TRP is very low. For example, pairwise alignment identified no similarity between the C. elegans TLP named TTR-30 and hTTR, and only 22% sequence identity over 79 residues comparing TTR-30

Fig. 7.5 Atomic force microscopy images of ecTRP. The samples were incubated at 65 C for 72 h (courtesy of Anders Olofsson, sample preparation as described in Lundberg et al. (2009)). The white scale bar is 500 nm

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and ecTRP. Comparison of sequence-aligned TLP, TTR, and TRP proteins with the 3D structures of TTR and TRP showed that insertions and deletions are situated exclusively at the N- and C-terminal ends, the surface exposed BC- and DE-loops, and the a-helix (Fig. 7.2). The AB- and GH-loops comprising the dimer–dimer interface at TTR and TRP are well conserved in length although the GH-loop is not well conserved in sequence. Secondary structure prediction with JPRED (Cole et al. 2008) suggested a slightly modified TTR-like fold (Fig. 7.6). Whereas TTR and TRP have only a short three-residue long D-strand, the program predicts an extended D-strand comprising nine residues in TLP. Furthermore, there seems to be no a-helix in the TLP structure. Two comparative models of the C. elegans. protein TTR-30 based on the X-ray crystallographic structures of human TTR (pdb code 1f41) and E. coli TRP (pdb code 2g2n) were made using MOE software (Molecular Operating Environment, Version 2007.09, Chemical Computing Group Inc., www.chemcomp.com) (Fig. 7.7a). The resulting models were very similar to each other, but do not seem to have converged into reasonable structures. We have not attempted however to

A Pred: hTTR:

B

---CCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCEEEEEEECC ---MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAA-37

Pred: ------CCHHHHHHHHHHHHHHHHHHHCCCCCCCCEEEEEECCCCCCCCCCEEEEEECCC ecTRP: ------MLKRYLVLSVATAAFSLPSLVNAAQQNILSVHILNQQTGKPAADVTVTLEKKAD-31 Pred: -----CCHHHHHHHHHHHHHHHHHHHHHHCCCCEEEEEEEEEECCCCCCCCEEEEEECCC TTR-30 -----MSSVYSCLILLTFIVASFCISIEIGNLQSVSVNGTLLCNDKPAKNIKVKLYEEEA-34

C Pred: hTTR:

D

E

F

CCCEEEEEEEECCCCCCCCC--CCCCCCCCCCEEEEEEEEHHHHHCCCCCCCCCEEEEEE DDTWEPFASGKTSESGELHG--LTTEEQFVEGIYKVEIDTKSYWKALGISPFHEHAEVVF-95

Pred: C-CCEEEEEEECCCCCCCCC--CCCCCCCCCCEEEEEEEHHHHHHCCCCCCCCCEEEEEE ecTRP: N-GWLQLNTAKTDKDGRIKA--LWPEQTATTGDYRVVFKTGDYFKKQNLESFFPEIPVEF-88 Pred: CCCC-CCCCCECCCCCEEEEEEEEECCCCEEEEEEEEEECC-CCCCCCEEEEEEECCCCC TTR-30 ILDV-LLDERFTKDDGTFEMAGSKSEVTTIDPKLNIYHKCN-YDGICVRKISILIPTEYI-92

G Pred: hTTR:

H

EEECCCCCCEEEEEEECCCCCCEEEEEECCCC TANDSGPRRYTIAALLSPYSYSTTAVVTNPKE-127

Pred: EEECCC-CCEEEEEEECCCCCCCCCCC----ecTRP: HINKVN-EHYHVPLLLSQYGYSTYRGS------114 Pred: CCCCCCCCEEEEEEEEEECCCCCCCEECCC-TTR-30 TNGEKPARTFNVGELNLASKFSGQSTDCFN---122

Fig. 7.6 Prediction of secondary structure of hTTR, ecTRP, and. TLP (TTR-30) by the program JPRED (Cole et al. 2008). The symbols E, H, and C indicate high probability for b-strands, ahelices, or coiled structures, respectively. The predicted b-strands and a-helices are highlighted in green and yellow boxes, whereas structurally verified b-strands and a-helices are marked in blue and red boxes


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make an extensive refinement of the model, because our purpose was solely to elucidate whether or not TLP structures would have a TTR-like fold. In the TLP models, the hydrophobic core in the monomers is not preserved and includes four buried charged residues. In agreement with the predicted secondary structure, the models also show that the amino acid substitutions at the BC-loop, i.e., ‘‘the stirrups’’ lead to a structural collapse of this b-hairpin and a number of surface-exposed hydrophobic amino acids. Furthermore, conserved amino acids within the TLP family are mostly situated on the surface of the proteins, and in particular at the area we refer to as the ‘‘saddle’’ (Fig. 7.7b). In the saddle, the secondary structure is not predicted to be a-helical, and we note that the a-helix is more distorted in the TLP models. Combined, these features in the TLP models suggest that TLPs have structures not identical to the TTR-like fold. Inconsistencies are present and in particular the saddle-stirrup area most likely deviates from the TTR and TRP proteins. It is plausible that members of the TLP group/family are targeted for ligands other than thyroid-hormones and 5-hydroxyisourate, and therefore their ligand-binding site is not at the dimer-dimer interface. Interestingly, hTTR has been shown to possess a cryptic protease activity that could be inhibited with serine protease inhibitors (Liz et al. 2004). The TTR-mediated proteolysis was not inhibited by thyroid-hormone binding, suggesting that the protease active site is not situated at the hormone-binding site (Liz et al. 2004). The residues lining the hormone/5-hydroxyisourate-binding site in TTR and TRP are not conserved in TLP. However, the four residue stretch D-C-L/F-H at the C-terminus of TLP seems distinctive for its family, or at least for a sub-group of them. These residues are positioned three residues downstream of the Y-R-G-S motif in TRP. In hTTR, this region is unstructured (Hornberg et al. 2000). Another interesting feature of the TLP models, with possible structural implications unrelated to ligand binding, is that two symmetry-related Cys17 residues are modeled right at the dimer-dimer interface. The distance between the symmetry˚ , which indicates that if the protein retains some features of related Sg atoms is 4 A the TTR-like fold, we would expect these two symmetry-related cysteines to form a disulfide-bond. Furthermore, Cys73 and Cys79, positioned in the a-helices of the TRP and TTR structures, might also form a disulphide bond, especially if the a-helix has another conformation in TLP, as indicated by secondary structure prediction.

7.5

Conclusions

Recent structural work on the TRPs and studies of their catalytic process provide a new understanding of how these enzymes work. Structural similarities between TRP and TTR suggest that the TRP gene at some point duplicated in the first primitive vertebrates to produce the TTR gene (Prapunpoj et al. 2000; Eneqvist et al. 2003). The fact that TRP and TTR retain their overall structures points to a stable fold ideally suited to function as a rigid scaffold for differently evolved functions. This is supported by the presence of TLPs, nematode-specific protein

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Fig. 7.7 (a) Model of TLP from C. elegans (TTR-30) based on hTTR (pdb code 1f41, Hornberg et al. (2000)). The sequences were aligned as seen in Fig. 7.2. Water molecules, ions, and metal ions were deleted from the template pdb-files before the modeling procedure. Coordinates of nonidentical side chains were selected from a rotamer library generated from high-resolution PDB data and the energy minimizations of final comparative models were made using Amber99 force field. (b) Visualizing the conservation of the three-dimensional structure. Identical and homolgous residues of six aligned TLP sequences shown in Fig. 7.2 are drawn in black and gray, respectively


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sequences. Bioinformatic analysis and structural modeling suggest that TLPs have, at least in part, a TTR-like fold, but with unrelated function to TRP and TTR. Further investigations of TLPs are needed to determine their functional and biological role, and their structural relationship to TTR and TRP.

Acknowledgements The authors thank Uwe H. Sauer and Tobias Hainzl for valuable discussions, and Terese Bergfors for critical reading of the manuscript.

References Cole C, Barber JD, Barton GJ. 2008. The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36:W197–W201. Colon W, Kelly JW. 1992. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31:8654–8660. Cusa E, Obradors N, Baldoma L, Badia J, Aguilar J. 1999. Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli. J Bacteriol 181:7479–7484. Eneqvist T, Lundberg E, Nilsson L, Abagyan R, Sauer-Eriksson AE. 2003. The transthyretinrelated protein family. Eur J Biochem 270:518–532. Fisher SH. 1999. Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! Mol Microbiol 32:223–232. Hansen M, Hsu AL, Dillin A, Kenyon C. 2005. New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLoS Genet 1:119–128. Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC. 2006a. The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359:1389–1399. Hennebry SC, Wright HM, Likic VA, Richardson SJ. 2006b. Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64:1024–1045. Hornberg A, Eneqvist T, Olofsson A, Lundgren E, Sauer-Eriksson AE. 2000. A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 302:649–669. Jung DK, Lee Y, Park SG, Park BC, Kim GH, Rhee S. 2006. Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc Natl Acad Sci USA 103:9790–9795. Kahn K, Tipton PA. 1998. Spectroscopic characterization of intermediates in the urate oxidase reaction. Biochemistry 37:11651–11659. Kall L, Krogh A, Sonnhammer EL. 2004. A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338:1027–1036. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. Lee Y, Lee DH, Kho CW, Lee AY, Jang M, Cho S, Lee CH, Lee JS, Myung PK, Park BC, Park SG. 2005. Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774. Lee Y, Park BC, Lee do H, Bae KH, Cho S, Lee CH, Lee JS, Myung PK, Park SG. 2006. Mouse transthyretin-related protein is a hydrolase which degrades 5-hydroxyisourate, the end product of the uricase reaction. Mol Cells 22:141–145.

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Liz MA, Faro CJ, Saraiva MJ, Sousa MM. 2004. Transthyretin, a new cryptic protease. J Biol Chem 279:21431–21438. Lundberg E, Backstrom S, Sauer UH, Sauer-Eriksson AE. 2009. The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457. Lundberg E, Olofsson A, Westermark GT, Sauer-Eriksson AE. 2009. Fibril-formation properties of human and piscine transthyretin, and E. coli transthyretin-related protein. FEBS J 276 (7):1999–2011. Nygaard P. 1983. Utilization of preformed purine bases and nucleosides. In: A. Munch-Petersen (ed.), Metabolism of nucleotides, nucleosides and nucleobases in microorganism. Academic Press, London, UK p. 27–93. Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G. 2000. Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol Regul Integr Comp Physiol 279:R2026–R2041. Ramazzina I, Folli C, Secchi A, Berni R, Percudani R. 2006. Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2:144–148. Santos SD, Costa R, Teixeira PF, Gottesman M, Cardoso I, Saraiva MJ. 2008. Amyloidogenic properties of transthyretin-like protein (TLP) from Escherichia coli. FEBS Lett 582:2893– 2898. Sarma AD, Serfozo P, Kahn K, Tipton PA. 1999. Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme. J Biol Chem 274:33863–33865. Schultz AC, Nygaard P, Saxild HH. 2001. Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183:3293–3302. Sonnhammer EL, Durbin R. 1997. Analysis of protein domain families in Caenorhabditis elegans. Genomics 46:200–216. Wojtczak A, Cody V, Luft JR, Pangborn W. 2001. Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr D Biol Crystallogr 57:1061–1070. Xi H, Schneider BL, Reitzer L. 2000. Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage. J Bacteriol 182:5332–5341. Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R. 2006. Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9.

Chapter 8

The Transthyretin–Retinol-Binding Protein Complex Hugo L. Monaco

Abstract Retinol-binding protein (RBP), the specific transporter in plasma of retinol and transthyretin (TTR), transporter of thyroid hormones, form in higher vertebrates under physiological conditions, a macromolecular complex that prevents glomerular filtration of the low molecular mass RBP. The crystal structures of two RBP–TTR complexes have shed light on many aspects of the interaction between the two transporters and explained observations made with other experimental methods. Since higher levels of RBP are associated with insulin resistance in obesity and type 2 diabetes it has been suggested that dissociation of the complex with the consequent renal elimination of RBP might be a way to treat the disease. Keywords Transthyretin, TTR, Retinol-binding-protein, RBP, RBP4, Retinol, Insulin resistance, Stra6

8.1

The Complex

Vertebrates obtain vitamin A from the diet either as carotenoids or as long chain fatty acid esters of retinol which, prior to absorption, are in both cases converted to free retinol, the form of the vitamin taken up by the intestinal mucosa. After cellular uptake the free retinol is reesterified in the mucosal cell by long chain fatty acids and incorporated with other lipids into chylomicrons that are absorbed in the lymphatic system. Chylomicrons transport retinyl esters to the liver, their main storage organ, and there the vitamin is laid away mainly in the stellate cells (Harrison 2005; Senoo et al. 2007). Mobilization of retinol from the liver to the

H.L. Monaco Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy e-mail: [email protected]


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peripheral target tissues takes place after the esters are hydrolysed and the free retinol is bound to plasma retinol-binding protein (plasma RBP or RBP4), the specific protein that solubilises, carries, and protects the vitamin in vertebrate plasma (Kanai et al. 1968). RBP is synthesized mainly in the liver, secreted bound to retinol with a 1:1 stoichiometry and is normally found in plasma forming a macromolecular complex with transthyretin (TTR) (Ingenbleek and Young 1994; Hamilton and Benson 2001, see also the relevant chapters of this book). The complex with TTR can be formed by both apo- and holo-RBP; however, the dissociation constant of the former is significantly higher. This observation supports the proposal of a delivery mechanism of the vitamin in which the stable complex containing retinol is retained in plasma, while the single low-molecular-weight apoRBP is cleared from circulation by glomerular filtration (van Bennekum et al. 2001). The normal RBP4 concentration in human plasma is about 2 mM and that of TTR about 4.5 mM and therefore there is an excess of the latter, and as a consequence virtually all of the holo-RBP molecules present in plasma are complexed to TTR. Each TTR tetramer with a molecular mass of 54,000 binds one RBP molecule with a molecular mass of 21,000 and therefore the molecular mass of the complex present in plasma is about 75,000. If the complex is formed in vitro, this stoichiometry may vary and the crystallographic work on the complex has definitively established that this stoichiometry cannot include more than two RBP molecules per TTR tetramer (Monaco 2000). This result was confirmed by mass spectrometry (Rostom et al. 1998) which was also used to estimate the dissociation constants of the first and the second RBP molecules in the complex. The values found with this method are 1.5 107 M for the first RBP molecule bound and 3.5 105 M for the second, and they are in agreement with the results that were determined with other techniques. The different values of the dissociation constants support an earlier proposal that binding of the first RBP molecule induces cooperative effects that decrease the affinity of TTR for the second RBP molecule. Although it has been shown that RBP has an affinity for other retinoids, the naturally occurring complex contains exclusively all-trans retinol bound to RBP. When the affinity for TTR of RBP bound to different ligands is examined in vitro, it is observed that the formation of the complex is impaired or even prevented when there is a bulky group exposed on the surface of the RBP molecule, confirming the role of retinol in conferring specificity to the interactions between the two macromolecules (Berni and Formelli 1992; Malpelli et al. 1996). The complex has been studied in several different organisms, and cross-reactivity among RBP and TTR from different species, was demonstrated many years ago (Kopelman et al. 1976). The values of the dissociation constants were comparable among the homologous complexes belonging to different species and were also very similar for the chimeric complexes formed by RBP and TTR of different origin. The two proteins that participate in the formation of this complex are very well characterized and their properties have been strongly preserved during evolution. A detailed discussion of the structure of TTR and of its complexes with different ligands is given in the appropriate chapters of this book. Here I will briefly summarize the structural elements needed for our discussion of the structure of the complex with RBP. The three-dimensional structures of human (Blake et al. 1978),

8 The Transthyretin–Retinol-Binding Protein Complex

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rat (Wojtczak et al. 2001), chicken (Sunde et al. 1996) and sea bream (Folli et al. 2003; Eneqvist et al. 2004) TTR are known, as well as the structure of the complexes with several compounds of pharmacological interest (Wojtczak et al. 1993). TTR, the first human plasma protein whose structure was determined by X-ray diffraction, is a tetramer of four identical subunits, each 127 amino acids long, in which each subunit is constructed as a mostly antiparallel b-structure. In the tetramer, the four monomers do not occupy equivalent positions but are organized as a dimer of dimers. Each monomer presents two extensive b-sheets, each composed of four strands that are all antiparallel with one exception (Blake et al. 1974). Two monomers form then a very stable dimer by extending their two b-sheets through hydrogen bonding that involves the four strands (two from each monomer) at the edges of the two subunits. The two dimers of the tetramer are separated by a channel and in contact through symmetrically related loops. The channel, about 10 ˚ in diameter, is the ligand-binding site. The three orthogonal molecular twofold A axes of TTR have been designated x, y, and z (Blake et al. 1974), the latter being coincident with the ligand-binding channel of the molecule. Since TTR is one of the proteins that forms amyloid fibrils in many severe human diseases (Hou et al. 2007), a very large number of mutants that give rise to the abnormal protein deposits have been characterized. In particular, many mutants were characterized by X-ray diffraction and a comparative study of 23 different structures has revealed that for most mutants the conformational changes observed in the crystalline state are quite subtle and indistinguishable from the changes observed in two independently determined non-amyloydogenic molecules (Ho¨rnberg et al. 2000). The TTR fold is not exclusive of this transporter. In recent years the transthyretinrelated protein (TRP) family, a group of proteins predicted by their amino acid sequence similarity to vertebrate TTRs to posses the TTR fold, has been investigated by several groups (Lee et al. 2005 and the appropriate chapters in this book). The biological activity of the enzymes formerly called the TRP related protein family is that of the 5-hydroxyisourate hydrolases and they play an important role in the purine degradation pathway. Several 5-hydroxyisourate hydrolase X-ray structures have confirmed that these enzymes indeed possesses the TTR fold. Their source is bacterial: Escherichia coli (Lundberg et al. 2006), Bacillus subtilis (Jung et al. 2006) and Salmonella dublin (Hennebry et al. 2006) but the enzyme is also present in vertebrates and the structure of zebrafish 5-hydroxyisourate hydrolase has also been solved (Zanotti et al. 2006).

8.2

Plasma Retinol-Binding Protein (RBP4)

Plasma retinol-binding protein was first isolated from human plasma in 1968 (Kanai et al. 1968) and subsequently from the serum of a number of other vertebrates: including birds and fish (Berni et al. 1992). Although its major site of synthesis and secretion is the liver, other tissues are known to express the protein, in particular the adipocyte (Soprano and Blanner 1994). In the liver, the availability of retinol plays a critical role in the secretion of RBP: its secretion is specifically blocked during retinol deficiency and restored upon retinol repletion (Ronne et al. 1983).

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Retinol-binding protein belongs to the super family of proteins known with the name of lipocalins, a group of molecules which do not have a very high similarity in amino acid sequence but are highly similar in tertiary structure and share in many cases the common function of hydrophobic ligand binding in an internal cavity. The name is derived from the Greek words ‘‘lipos,’’ meaning fat, and ‘‘calyx,’’ meaning cup due to the fact that their structure has the shape of a calyx and their ligands are mostly hydrophobic (Pervais and Brew 1987). Human RBP is the first lipocalin whose three-dimensional structure was determined by X-ray diffraction analysis of single crystals (Newcomer et al. 1984). Following this work many other X-ray structures of different RBP4s were solved (Newcomer and Ong 2000; Zanotti and Berni 2004). In particular the three-dimensional structure of human (Cowan et al. 1990; Zanotti et al. 1993a) and bovine (Zanotti et al. 1993b) holo- and apo-RBP, as well as chicken RBP (Zanotti et al. 2001) and the higher resolution structure of porcine RBP (Zanotti et al. 1998) have been extensively described. The main feature of all these structures, highly similar to each other, is an eight-stranded up-and-down beta barrel which delimits the cavity followed topologically by a short a-helical segment close to the C terminus of the molecule. The retinol molecule binds into the calyx with the b-ionone ring buried and with the alcohol moiety pointing to the outside of the surface of the molecule. Examination of the model of bovine RBP4 at different values of pH, determined at the highest resolution attained up to now, has revealed the subtle details of ligand binding to the protein (Calderone et al. 2003). The main conclusions of this study regarding the conformation of the bound retinol at physiological pH are that: (a) the cyclohexenyl ring is in the half-chair conformation, (b) the polyene chain is fully extended and planar and, (c) the methyls and hydroxyls are not in the polyene plane. In this model three water molecules are present in the interior of the mostly hydrophobic ligand binding cavity and a network of other solvent molecules surrounds the hydroxyl moiety of retinol exposed on the surface of the protein. The other important contacts of the ligand and the protein, that have been extensively described for the refined model of human RBP4, are hydrophobic (Cowan et al. 1990). Figure 8.1 is a ribbon representation of the molecule showing the bound retinol as a space-filling model. The secondary structure assignments for human RBP4 are for the beta strands the following: strand A, residues 22–30; B, residues 39–47; C, residues 53–62; D, residues 68–78; E, residues 85–92; F, residues 100–109; G, residues 114–123; and H residues 129–138. The alpha helix spans residues 146– 158. The open end of the barrel is delimited by four loops joining strands A–B (residues 31–38), C–D (residues 63–67), E–F (residues 93–99), and G–H (residues 124–128). The first three are close to the retinol molecule and amino acids present in them participate in the contacts with TTR, the fourth is far from the ligand and is not involved in contacts with TTR in the complex. The loop connecting strands C and D has been found to be disordered in several X-ray structures which indicates a certain degree of flexibility which might be related to retinol release. It has been proposed that release of retinol by RBP4 involves a transition of the protein to the molten globule state triggered by a decrease of the pH near the cell membrane, a mechanism postulated for the ligand release of other lipocalins as well


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Fig. 8.1 Ribbon representation of the plasma RBP (RBP4) molecule. The bound retinol is represented as a CPK model with its OH exposed to the solvent on the surface of the protein. The figure was prepared with the program CCP4 mg (Potterton et al. 2002)

(Ptitsyn el al. 1993; Bychkova et al. 1998). A biased molecular dynamics simulation method has been used to explore the conformation of the partially folded structures of RBP in the molten globule state (Paci et al. 2005). In these simulations significant changes which result in an opening in the ligand binding cavity were observed in beta strands E and F and the loop connecting them, i.e., in residues 85–104. Though it was initially thought that removal of the retinol molecule from the calyx would result in major conformational changes leading to a collapse of the beta barrel structure, X-ray crystallographic studies of human and bovine RBP (Zanotti et al. 1993a, b) have shown that the transition from holo- to apoprotein involves only very subtle modifications. The most important is a conformational change in the loop connecting strands A and B, in particular in the region extending from amino acids 33–37. In this part of the molecule Leu 35 and Phe 36 move from their position in the holoprotein to very well defined positions in the apoprotein. When

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retinol is removed the space left empty by the vitamin is filled, in both human and bovine RBP, by the aromatic ring of Phe 36 and solvent molecules, and the movement of the Phe side chain drags the nearby amino acids into positions which are different from those adopted in the holoprotein. In particular Leu 35, which in the holo protein points towards the binding site moves out and points to the exterior of the RBP molecule. These results, obtained with apo RBP prepared from the holoprotein by removing retinol by organic solvent extraction or hydrophobic interaction chromatography was confirmed by the X-ray structure of recombinant apo RBP (Greene et al. 2001). Apo RBP prepared in vitro can be complexed with several natural and synthetic retinoids, i.e., derivatives of retinol. The interest in these compounds stems from their clinical applications that include treatment of skin disease and cancer (Moise et al. 2007). Co-crystals of RBP with different retinoids have been studied by X-ray diffraction (Zanotti et al. 1993c, 1994). The main conclusion of these studies is that the selected retinoids bind assuming the conformation of bound retinol, the natural ligand and with an overall protein structure that is virtually identical to that of the natural protein–ligand complex. Important differences are found at the entrance of the beta barrel where in some cases bulky groups protrude from the cavity into the solvent and as a consequence either reduce or abolish the interaction of RBP with TTR. The first evidence for the existence of specific cell surface receptors for plasma retinol-binding protein came from the study of the binding of 125I-labeled RBP to bovine pigment epithelium cells (Heller 1975). Both human and bovine RBP were found to bind specifically and completely in about one minute to the bovine epithelium cells. The binding was a linear function of the number of binding sites and was saturable with respect to the labeled RBP. The vitamin entered the cells but the labeled protein did not. Apo RBP was also found to bind to these cells but with a significantly higher dissociation constant. Following this work other putative RBP receptors were identified in different tissues: mucosal epithelial cells from the monkey’s small intestine (Rask and Petersson 1976), placenta (Sivaprasadarao and Findlay 1998), testis (Shingleton et al. 1989), retinal pigment epithelium (Ba˚vik et al. 1991, 1992), choroid plexus (Smeland et al. 1995), hepatocytes (Yamamoto et al. 1997) and macrophages (Hagen et al. 1999). Characterization of the receptor at a molecular level was difficult because of its fragility and the transient nature of its interaction with RBP. Using photo-crosss-linking, high affinity purification and mass spectrometry, the receptor was finally identified as Stra6, a 74 kDa, multitransmembrane protein whose function was previously unknown and whose sequence is not similar to that of any other protein of known structure (Kawaguchi et al. 2007; Wolf 2007; Blaner 2007). The amino acid sequence and hydropathy profile indicate that stra6 is a largely hydrophobic molecule with 11 predicted transmembrane domains. Analysis of the accessibility of a tag inserted in different positions in the topology of the molecule combined with a novel lysine accessibility technique has revealed which are the extracellular and which the intracellular domains of the receptor and identified the regions of the molecule implicated in RBP binding (Kawaguchi et al. 2008a). By studying a large number of random mutants and systematically analyzing their properties a domain


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which plays an essential role in RBP binding was identified. Three residues whose mutation completely abolishes the binding of RBP to stra6 were identified in this domain (Kawaguchi et al. 2008b). Mutations in Stra6 cause a series of very severe disorders in humans: anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation which highlights the crucial role played by this receptor in retinol metabolism (Golzio et al. 2007; Pasutto et al. 2007). Several mutations causing severe birth defects were also found to correlate with vitamin uptake from RBP. In addition, it is worth emphasizing that the uptake of retinol by stra6 has been shown to occur efficiently when cells are in the presence of both holo RBP alone and holo RBP complexed to TTR, i.e., the situation encountered in vivo (Kawaguchi et al. 2007). Adipose tissue is an important endocrine organ that secretes into circulation several signaling proteins that influence energy metabolism. These cell-to-cell signaling molecules are called adipocytokines or adipokines, and they include leptin, adiponectin, resistin, tumour necrosis factor-, and interleukin-6 (Henry and Clarke 2008). A recent addition to this list is plasma RBP which, in addition to the liver, is also synthesized in adipose tissue, and was found to behave as an adipokine, a marker of insulin sensitivity and adiposity in both animal models and humans. Systemic insulin resistance is associated with a decreased expression of the glucose transporter 4 (Glut4) in adipocytes, one of the grave defects of obesity and type 2 diabetes. Glut4 knockout mice show insulin resistance and present increased circulating levels of plasma RBP due to an enhanced expression in the adipose tissue of the gene encoding for the protein (Yang et al. 2005). Furthermore it was also observed that increasing the plasma level of RBP4 correlated with insulin resistance whereas lowering it led to insulin sensitivity enhancement. In humans, plasma RBP concentrations are elevated in insulin resistant patients with obesity impaired glucose tolerance or type 2 diabetes (Graham et al. 2006). These findings have led to the proposal that the measurement of the RBP4 levels in plasma might be a simple method to assess the risk of type 2 diabetes and cardiovascular disease (van Dam and Hu 2007). Although the exact intervention of RBP4 in clinical metabolic disease is not completely defined there appears to be no doubt that in addition to retinol delivery, this protein plays an important role in adipocyte lipid metabolism as well (von Eynatten and Humpert 2008).

8.3

Crystal Structures of the Complex

The first structure of a TTR–RBP complex determined by X-ray diffraction analysis of single crystals was that of a chimeric complex prepared in vitro with two molecules of chicken RBP per tetramer of human TTR (Monaco et al. 1995). These crystals diffracted to a slightly better resolution than those of the homologous complex human TTR-human RBP that were solved a few years later (Naylor and Newcomer 1999). The chimeric complex could only be prepared by imposing a stoichiometry of two molecules of RBP per TTR tetramer (Monaco et al. 1994), and an even higher ratio of 2.5–3 molecules of RBP per tetramer was used to prepare

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the crystals of the homologous human complex. In both cases the species found in the crystals was a hexamer TTR(RBP)2 but the quaternary structure of the two hexamers is different. Figure 8.2a is a ribbon representation of the chimeric hexamer in which the model is viewed looking down the z axis of the TTR tetramer as defined by Blake

a

b

Fig. 8.2 Quaternary structure of the TTR-(RBP)2-TTR hexamers. (a) Structure of the chimeric complex chicken RBP-human TTR (Monaco et al. 1995, PDB code 1RLB). The TTR molecule is oriented so that this is the view down the z axis of the TTR tetramer as defined by Blake et al. (1974). The x and y axes are in the plane of the figure, the first is horizontal and the second vertical. They intercept in the center of the TTR channel, the hormone-binding site, which is represented empty in the figure. (b) Structure of the homologous human TTR-RBP complex (Naylor and Newcomer 1999 PDB code 1QAB). In this case the twofold axis which is preserved in the complex is the z axis, i.e., the axis normal to the plane of the figure running through the TTR channel. The figure was prepared using the programs MOLSCRIPT (Kraulis 1991) and Raster3D (Merritt and Bacon 1997)


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and coworkers (Blake et al. 1974). The x and y axes are in the plane of the figure. The x axis is horizontal and separates the two TTR dimers that together form the tetramer. The vertical y axis is, in the chimeric complex, the twofold axis that relates the two RBP molecules as seen in the figure. Therefore, in this complex the two RBP molecules are found to interact most extensively with the same TTR dimer. Figure 8.2b is an analogous ribbon representation of the homologous human complex. In the crystals of this complex, the twofold axis which is preserved is the z axis instead, i.e., the axis perpendicular to the plane of the figure, and thus the two RBP molecules are found to interact most extensively with the opposite dimers of the TTR tetramer as shown in the figure. The reason why two different twofold axes are preserved in the hexamers in these two crystal forms is not known. More recently, the macromolecular complex of human TTR, human RBP and an antiRBP Fab was crystallized with a stoichiometry assumed for the triple complex in the crystals of 2:2:1 for the Fab, RBP, and TTR respectively, i.e., these new crystals preserve the 2:1 RBP–TTR stoichiometry (Malpelli et al. 1999). Therefore it appears that without the presence in the complex of at least one of the TTR twofold axes crystals cannot be formed. Thus, although the species purified from plasma is the pentamer containing only one RBP molecule per TTR tetramer, the artificial preparation of the hexamer seems to be a requirement to facilitate the packing of the molecules in the crystal lattices. In both the chimeric and the human homologous complexes, the two RBP molecules interact with the two monomers of one TTR dimer and a third monomer of the other dimer and this precludes the possibility of binding of a third and fourth RBP molecule to form a heptameric or octameric complex. In the chimeric hexamer in which the y axis is the preserved twofold axis, the two RBP molecules bind to the TTR tetramer along an axis parallel to the x axis (Fig. 8.2a) and is sufficiently close to it to hinder the potential binding of the two other RBP molecules that would be required to satisfy this symmetry element in an octamer. Something analogous happens in the homologous human hexamer in which the two RBP molecules bind on the opposite sides of the x axis but again too close to it to allow octamer formation (Fig. 8.2b). Thus the two crystal structures are consistent with a stoichiometry of either one or a maximum of two RBP molecules per TTR tetramer. Figure 8.3 is a ribbon representation of a pentamer, the species circulating in plasma, with one RBP molecule bound to a TTR tetramer. Note that the RBP molecule interacts with the two components of a dimer and a third monomer of the other TTR dimer. It is this last interaction that rules out the possibility of binding a second RBP molecule to the second dimer on the same side of the tetramer. The main amino acids involved in the contacts between RBP and TTR are listed in Table 8.1 along with the relevant distances between residues in contact. Note that, in addition to the retinol OH, the RBP residues participating in the contacts are clustered in three areas. The residues listed are Leu 35, which is found in different conformations in the apo and holo protein, Trp 67, in the loop connecting strands C and D and in the area at the end of strand E and in its connection to strand F. The last one is the region where most of the RBP amino acids participating in the contacts are located. The interacting regions of TTR with RBP are found in the following

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Fig. 8.3 Ribbon representation of the pentameric complex RBP–TTR, the species normally circulating in plasma. The retinol molecule is represented as a CPK model and the side-chains of the amino acids participating in the contacts as ball- and stick-models and colored in the case of TTR, according to the monomer to which they belong. The TTR tetramer is oriented as in Fig. 8.2 and the figure was prepared using the same programs

areas of the three monomers: monomer A, the loop joining strands F and G with Asp 99 and Ser 100, monomer B, residues 82–85 connecting the TTR alpha helix to strand F and Tyr 114 (in the loop joining strands G and H) and monomer C, the loop joining the first two strands of beta structure as well as Ile 84. An examination of the distances in Table 8.1 leads to the conclusion that the two X-ray structures are in reasonable agreement with one another. There is, however, one major significant difference, not evident from the Table, between the two models. Chicken RBP lacks the last five amino acids present in mammalian RBPs and those are found to interact with the TTR tetramer in the homologous human complex in one of the two RBP molecules present in the hexamer. Therefore in this complex, the two RBP molecules do not have exactly equivalent interactions with the TTR tetramer and the z twofold axis of symmetry is not strictly obeyed. The physiological significance of this additional interaction is discussed by Naylor and Newcomer but the reasons for this asymmetry in the binding of the two RBP molecules in the case of the human homologous complex are not known. Figure 8.4a, taken from the paper describing the chimeric complex (Monaco et al. 1995), is a cartoon representation of the most important interactions between an RBP molecule and the TTR tetramer. Note that the RBP molecule interacts with three different TTR monomers and there is one amino acid, Ile 84, present in two


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Table 8.1 Significant contacts in the TTR–RBP complex in the two structures determined by X-ray crystallography. The third column gives the distance in the chimeric complex (PDB code 1RLB) and the fifth column gives the distance in the homologous human complex (PDB code 1QAB) RBP chain identity TTR chain Distance TTR chain Distance Amino acid and amino identity chimeric identity homologous number TTR ˚) acid number Chimeric complex homologous complex (A ˚) complex (A complex PDB code PDB code 1RLB 1QAB E - 35 (Leu) [CD2] E - 67 (Trp) [CZ2] E - 67 (Trp) [CD2] E - 89 (Lys) [NZ] E - 89 (Lys) [NZ] E - 91 (Trp) [NE1] E - 95 (Ser) [OG] E - 96 (Phe) [CO] E - 96 (Phe) [N] E - 96 (Phe) [CB] E - 96 (Phe) [CE1] E - 97 (Leu) [CO] E - 99 (Lys) [NZ] E - 99 (Lys) [N] E - (Retinol) [OH]

B C C A A A B B B B C B A B B

3.41 4.20 3.45 2.73 4.54 4.07 3.09 2.69 3.38 3.37 3.44 2.80 2.70 3.87 3.15

D A A C C C D D D D A D C D D

4.24 5.03 4.04 3.57 4.92 3.68 2.71 3.25 3.46 3.30 3.25 3.59 5.47 4.33 3.48

83 (Gly) [CA] 20 (Val) [CG1] 84 (Ile) [CD1] 99 (Asp) [CO] 99 (Asp) [OD2] 100 (Ser) [CO] 114 (Tyr) [OH] 85 (Ser) [N] 114 (Tyr) [OH] 84 (Ile) [CG2] 21 (Arg) [CG] 85 (Ser) [OG] 99 (Asp) [OD2] 85 (Ser) [OG] 83 (Gly) [CO]

different TTR monomers (B & C) participating in the interaction, one in each dimer. The TTR region in the contact area is a hydrophobic zone situated between the two dimers in the tetramer, whereas the RBP area where most of the amino acids participating in the interactions are located is the end of strand E and the loop connecting it to strand F at the entrance of the barrel. These amino acids are represented in Fig. 8.4b, c. Note in the diagrams the prevalence of hydrophobic contacts. The retinol hydroxyl group, which is exposed on the surface of the protein, ˚ from the polypeptide chain of a TTR has its oxygen at a distance of about 3–3.5 A monomer. In uncomplexed TTR, there is a fairly large hydrophobic patch which becomes buried upon binding of an RBP molecule, which is a major driving force for complex formation by the two plasma proteins. Of the total RBP surface ˚ 2), 736 A ˚ 2 (about 8%) are buried when the accessible to the solvent (9,356 A molecule is in contact with TTR in the complex. It was already mentioned that the loop connecting strands C and D has been found partially disordered in some RBP X-ray structures but the electron density in this area is clear and continuous in the crystals of the chimeric complex. Therefore, in order to examine possible movements in this important area, the model of RBP in the complex can be superimposed over that of the uncomplexed holoprotein whose structure is known at high resolution (Zanotti et al. 2001). Using the program

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a

Leu E 35

Trp E 91

OH Lys E 89 Å NH3

O

O

C

C

C

Gly B 83

Asp A 99

Ser A 100

Ser E 95

o -

O

NH

C

Ser B 85

O

o

* OH

NH

He B 84

Å NH3 O Lys E 99

C Leu E 97

H-O

O

Tyr B 114

C

Trp E 67

NH

Phe E 96

N

Å

N

NH

* IIe C 84

N Arg C 21

Val C 20

Fig. 8.4 (a) The main protein–protein interactions found in the chimeric complex. An asterisk identifies Ile 84 which is present in the contact area of two different TTR monomers, B and C, that are part of the two separate dimers of the tetramer. (b) Diagram representing as ball and stick models the RBP amino acid side chains in the region 88–92 in contact with TTR. The TTR residues that make hydrophobic contacts are represented as arches in a different color. (c) The same as (b) but covering the RBP region 94–100. (d) Superposition of the models of chicken RBP bound in the complex (Monaco et al. 1995, PDB code 1RLB) and unbound (Zanotti et al. 2001, PDB code 1IIU). The RBP molecule in the complex is represented red and that isolated green. The TTR molecule is represented blue. The two RBP models were superimposed using the program LSQKAB (Kabsch 1978). Figures (b) and (c) were prepared using the visualization program LIGPLOT (Wallace et al. 1995). Figure (d) with the program CCP4 mg (Potterton et al. 2002)


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Fig. 8.4 (continued)

LSQKAB (Kabsch 1978) the coordinates of the two models were superimposed. The area of contact is represented in Fig. 8.4d where a shift in the position of Asn 65 and Trp 67 is clearly observed. Figures 8.5a, b compare the amino acid sequences of human, other five mammalian and chicken TTR and RBP aligned using the program CLUSTAL W (Thompson et al. 1994). The amino acids participating in the contacts in the complex are

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b

Fig. 8.5 Sequence alignment of six vertebrate (a) RBPs and (b) TTRs of the same species. The sequences were aligned using the program CLUSTAL W (Thompson et al. 1994). The six digit code preceding the name of the species in each sequence is the ExPASy code. The last column on the right hand side gives the percentage identity of each sequence and that of the human protein. A black dot identifies in each case a residue involved in the contacts in the complex

indicated with a back dot and the percentage identity of the sequences with that of human RBP and TTR are given in the last column. Note the very high percentage identity among the sequences and also that the residues participating in the contacts are clustered in three areas in the case of RBP and four in the case of TTR.There are a total of 31 amino acid differences in the primary structure of chicken and human TTR and interestingly six of those are found in the N terminal region of the molecule, which is disordered in the electron density maps of the complex, and three are in the C terminal region of the molecule. The sequences of human and chicken RBP differ in 24 amino acids. When the amino acids participating in the contacts listed in Table 8.1 are inspected in Fig. 8.5 only two amino acids in the TTR sequence are found to be different in human and chicken TTR. They are amino acids number 114, which is a Tyr in human TTR and a Phe in chicken TTR and, quite interestingly, amino acid number 84, which is an Ile in human TTR and a Leu in chicken TTR. A similar comparison of the two RBPs reveals that in this case no amino acids in the list are different in Fig. 8.5, although there is one amino acid present in the contact region of the homologous human complex, residue 64, which is a Leu in human RBP and a Phe in the chicken protein. Thus, the regions of protein-protein contact in the complex are strictly conserved for these species as expected given the physiological importance of this protein complex in animal physiology.


8.4

137

Experimental Evidence that Supports the X-Ray Models

The two X-ray models of the complex have allowed the rationalization of many experimental observations that preceded the X-ray work and with also of others made to test the X-ray structures. The effect of the ligand bound to the two protein carriers is totally different: thyroxine binding to TTR does not have any influence on complex formation (van Jaarsveld et al. 1973), whereas retinol binding plays a crucial role and the affinity of apo-RBP for TTR is drastically reduced. In addition, the binding of retinoids with bulky moieties exposed on the surface of the protein molecule can impair or prevent the formation of the macromolecular complex. This very different behavior of the two ligands with regard to the two carrier proteins has received a very simple explanation from the X-ray models. Thyroxine binds in a channel that does not participate in any way in the contacts with RBP, whereas retinol bound to RBP interacts with TTR through its exposed hydroxyl group on the surface of the RBP molecule. In addition, the models of the complex are consistent with the X-ray structural studies that have shown that in the holo to apo transition in RBP the most significant change involves amino acids 34–37 (Zanotti et al. 1993a, b). This region of the molecule, and in particular Leu 35, is present in the protein-protein contact area in both X-ray models. Another observation that concerns the stability of the complex is that the affinity of RBP for TTR is enhanced at high and reduced at low ionic strength (van Jaarsveld et al. 1973) which is in agreement with the participation of a hydrophobic area in the intermolecular contacts. Site-directed mutagenesis has confirmed the role of Leu 35 and has also shown that in the double mutant in which Leu 63 and Leu 64 are changed to Arg and Ser, the ability to bind to TTR is reduced (Sivaprasadarao and Findlay 1994). Deletion of the loops 92–98 results in complete loss of the ability to interact with TTR and this ability can be transferred to the epididymal retinoic acid-binding protein (that normally does not bind to TTR) by inserting the three RBP loops seen to participate in the intermolecular interactions in the X-ray models (Sundaram et al. 2002). Structural analysis of the chimeric complex using mass spectrometry has led to conclusions that are in complete agreement with the X-ray models (Rostom et al. 1998). Chemical modification of the tryptophan residues of isolated RBP and in the complex has shown that there is at least one Trp which reacts in the isolated protein but is protected upon complex formation, which implies that it is located in the area where the protein-protein contacts are established (Horwitz and Heller 1974). The identity of this residue was not established chemically but the two X-ray models of the complex have the same two Trp residues in the contact area: Trp 67 and 91. Acetylation of holo-RBP with N-acetylimidazole (using conditions under which both Lys and Tyr residues react) disrupts its binding to TTR and deacetylation of the tyrosines with hydroxylamine fails to produce RBP with a normal affinity for TTR (Heller and Horwitz 1975). This result indicates that there is at least a Lys and that there are no Tyr present in the RBP area that participate in the protein-protein contact. As Table 8.1 shows, the two models have Lys 89 and 99 on the surface in contact with TTR.

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The macromolecular complex RBP–TTR does not form in the plasma of fish although both proteins are known to be present in circulation (Folli et al. 2003). If the amino acid sequences of sea bream RBP and TTR are compared with those of other vertebrates in which the complex is known to form, significant differences are found in the regions participating in the contacts. In particular the differences found in sea bream TTR are Ile84Ser, Asp99Pro and Ser100Glu and Lys99Thr in RBP, which as Table 8.1 shows participate in important contacts in the complex. Among the many pathological mutations described in TTR that are associated with different types of amyloidosis (Hou et al. 2007) there is one in which it was found that the affinity of TTR for RBP was drastically reduced (Benson and Dwulet 1983), a reduction that can be convincingly explained by the structural models of the complex. The disease is called Indiana-type hereditary amyloidosis and the mutation consists in the substitution Ile84Ser. When the dissociation constant of the complex of this mutant with normal RBP was determined, it was found that the affinity of the altered TTR for normal RBP was negligible (Berni et al. 1994). Both X-ray models of the complex place this residue, Ile 84, twice at the interface with RBP, and the monomers to which the residues belong are in different TTR dimers, which explains the remarkable influence of this single amino acid on the complex stability. The plasma RBP concentration of several other individuals with single mutations in the TTR gene was examined by Waits et al. (Waits et al. 1995) and the only other case in which the RBP concentration was found to be significantly lowered was the Ile84Asn mutant. Interestingly one of the mutants examined was the Tyr114Cys, i.e., an amino acid involved in the contacts in the complex (see Table 8.1) suggesting that a single mutation might not be enough to lead to dissociation of the complex. Elevated RBP4 levels are correlated with insulin resistance and glucose intolerance and the complex has been identified as a target for the treatment of this widely diffused disease (Mody et al. 2008). Disrupting the stability of the complex might be a way of lowering the plasma RBP levels since it is known that uncomplexed RBP is eliminated by glomerular filtration. In the development of a molecule to implement the strategy to treat insulin-resistant individuals through dissociation of the complex, it may therefore be necessary to disrupt more than one contact in the macromolecular complex.

Acknowledgements

I thank Dr. Massimiliano Perduca for his help in preparing the figures

of this chapter

References Ba˚vik CO, Eriksson U, Allen RA, Peterson PA (1991) Identification and partial characterization of a retinol pigment epithelial membrane receptor for plasma retinol-binding protein. J Biol Chem 266:14978–14985.


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Ba˚vik CO, Busch C, Eriksson U (1992) Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium. J Biol Chem 267:23035– 23042. Benson MD, Dwulet FE (1983) Prealbumin and retinol binding protein serum concentrations in the Indiana type hereditary amyloidosis. Arthritis Rheum 26:1493–1498. Berni R, Formelli F (1992) In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences. FEBS Lett 308:43–45. Berni R, Stoppini M, Zapponi MC (1992) The piscine plasma retinol-binding protein. Purification, partial amino acid sequence and interaction with mammalian transthyretin of rainbow trout (Oncorhynchus mykiss) retinol-binding protein. Eur J Biochem 204:99–106. Berni R, Malpeli G, Folli C, Murrell JR, Liepnieks JJ, Benson MD (1994) The Ile84 !Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinol-binding protein. J Biol Chem 269:23395–23398. Blake CCF, Geisow MJ, Swan IDA, Re´rat C, Re´rat B (1974) Structure of human plasma ˚ resolution. A preliminary report on the polypeptide chain conformation, prealbumin at 2.5 A quaternary structure and thyroxine binding. J Mol Biol 88:1–12. Blake CCF, Geisow MJ, Oatley SJ, Re´rat B, Re´rat C (1978) Structure of prealbumin: secondary, ˚ resolution. J Mol tertiary and quaternary interactions determined by Fourier refinement at 1.8 A Biol 121:339–356. Blaner WS (2007) STRA6, a cell-surface receptor for retinol-binding protein: the plot thickens. Cell Metab 5:164–166. Bychkova VE, Dujsekina AE, Fantuzzi A, Ptitsyn OB, Rossi GL (1998) Release of retinol and denaturation of its plasma carrier, retinol-binding protein. Fold Des 3:285–291. Calderone V, Berni R, Zanotti G (2003) High-resolution structures of retinol-binding protein in complex with retinol: pH-induced protein structural changes in the crystal state. J Mol Biol 329:841–850. Cowan SW, Newcomer ME, Jones TA (1990) Crystallographic refinement of human serum retinol ˚ resolution. Proteins Struct Funct Genet 8:44–61. binding protein at 2 A Eneqvist T, Lundberg E, Karlsson A, Huang S, Santos CR, Power DM, Sauer-Eriksson AE (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J Biol Chem 279:26411–26416. Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555:279–284. Golzio C, Martinovic-Bouriel J, Thomas S, Mougou-Zrelli S, Grattagliano-Bessieres B, Bonniere M, Delahaye S, Munnich A, Encha-Razavi F, Lyonnet S, Vekemans M, Attie-Bitach T, Etchevers HC (2007) Matthew-Wood syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6. Am J Hum Genet 80:1179–1187. Graham TE, Yang Q, Blu¨her M, Hammarstedt A, Ciaraldi TP, Henry RR, Wason CJ, Oberbach A, Jansson PA, Smith U, Kahn BB (2006) Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. N Engl J Med 354:2552–2563. Greene LH, Chrysina ED, Irons LI, Papageorgiou AC, Acharya KR, Brew K (2001) Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily. Protein Sci 10:2301–2316. Hagen E, Myhre AM, Smeland S, Halvorsen B, Norum KR, Blomhoff R (1999) Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants. J Nutr Biochem 10:345–352. Hamilton JA, Benson MD (2001) Transthyretin: a review from a structural perspective. Cell Mol Life Sci 58:1491–1521. Harrison EH (2005) Mechanisms of digestion and absorption of dietary vitamin A. Annu Rev Nutr 25:87–103. Heller J (1975) Interactions of plasma retinol-binding protein with its receptor. Specific binding of bovine and human retinol-binding protein to pigment epithelium cells from bovine eyes. J Biol Chem 250:3613–3619.

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Heller J, Horwitz J (1975) Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. J Biol Chem 250:3019–3023. Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC (2006) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359:1389–1399. Henry BA, Clarke IJ (2008) Adipose tissue hormones and the regulation of food intake. J Neuroendocrinol 20:842–849. Ho¨rnberg A, Eneqvist T, Olofson A, Lundgren E, Sauer-Eriksson AE (2000) A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 302:649–669. Horwitz J, Heller J (1974) Modification of tryptophan residues in retinol-binding protein and prealbumin with 2-hydroxy-5-nitrobenzyl bromide. J Biol Chem 249:7181–7185. Hou X, Aguilar MI, Small DH (2007) Transthyretin and familial amyloidotic polyneuropathy. Recent progress in understanding the molecular mechanism of neurodegeneration. FEBS J 274:1637–1650. Ingenbleek Y, Young V (1994) Transthyretin (prealbumin) in health and disease: nutritional implications. Annu Rev Nutr 14:495–533. Jung DK, Lee Y, Park SG, Park BC, Kim GH, Rhee S (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc Natl Acad Sci USA 103:9790–9795. Kabsch W (1978) A solution for the best rotation to relate two sets of vectors. Acta Cryst A32:922–923. Kanai M, Raz A, Goodman De WS (1968) Retinol-binding protein: the transport protein for vitamin A in human plasma. J Clin Invest 47:2025–2044. Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H (2007) A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315:820–825. Kawaguchi R, Yu J, Wiita P, Ter-Stepanian M, Sun H (2008a) Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor. Biochemistry 47:5387–5395. Kawaguchi R, Yu J, Wiita P, Honda J, Sun H (2008b) An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism. J Biol Chem 283:15160–15168. Kopelman M, Cogan U, Mokady S, Shinitzky M (1976) The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization. Biochim Biophys Acta 439:449–460. Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24:946–950. Lee Y, Lee DH, Kho CW, Lee AY, Jang M, Cho S, Lee CH, Lee JS, Myung PK, Park BC, Park SG (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774. Lundberg E, Ba¨ckstro¨m S, Sauer UH, Sauer-Eriksson AE (2006) The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457. Malpeli G, Folli C, Berni R (1996) Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. Biochim Biophys Acta 1294:48–54. Malpeli G, Zanotti G, Gliubich F, Rizzotto A, Nishida SK, Folli C, Berni R (1999) Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP Fab. Acta Crystallogr D Biol Crystallogr 55:276–278. Merritt EA, Bacon DJ (1997) Raster3D: photorealistic molecular graphics. Methods Enzymol 277:505–524. Mody N, Graham TE, Tsuji Y, Yang Q, Kahn BB (2008) Decreased clearance of serum retinolbinding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice. Am J Physiol Endocrinol Metab 294:E785–E793.


141

Moise AR, Noy N, Palczewski K, Blaner WS (2007) Delivery of retinoid-based therapies to target tissues. Biochemistry 46:4449–4458. Monaco HL, Mancia F, Rizzi M, Coda A (1994) Crystallization of the macromolecular complex transthyretin-retinol-binding protein. J Mol Biol 244:110–113. Monaco HL, Rizzi M, Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041. Monaco HL (2000) The transthyretin–retinol-binding protein complex. Biochim Biophys Acta 1482:65–72. Naylor HM, Newcomer ME (1999) The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP. Biochemistry 38:2647–2653. ˚ qvist J, Sundelin J, Eriksson U, Rask L, Peterson PA (1984) The Newcomer ME, Jones TA, A three-dimensional structure of retinol-binding protein. EMBO J 3:1451–1454. Newcomer ME, Ong DE (2000) Plasma retinol-binding protein: structure and function of the prototypic lipocalin. Biochim Biophys Acta 1482:57–64. Paci E, Greene LH, Jones RM, Smith LJ (2005) Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach. FEBS J 272:4826– 4838. Pasutto F, Sticht H, Hammersen G, Gillessen-Kaesbach G, Fitzpatrick DR, Nu¨rnberg G, Brasch F, Schirmer-Zimmermann H, Tolmie JL, Chitayat D, Houge G, Fernańdez-Martıńez L, Keating S, Mortier G, Hennekam RC, von der Wense A, Slavotinek A, Meinecke P, Bitoun P, Becker C, Nu¨rnberg P, Reis A, Rauch A (2007) Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation. Am J Hum Genet 80:550–560. Pervaiz S, Brew K (1987) Homology and structure-function correlations between alpha 1-acid glycoprotein and serum retinol-binding protein and its relatives. FASEB J 1:209–214. Potterton E, McNicholas S, Krissinel E, Cowtan K, Noble M (2002) The CCP4 molecular-graphics project. Acta Crystallogr D Biol Crystallogr 58:1955–1957. Ptitsyn OB, Zanotti G, Denesyuk AL, Bychkova VE (1993) Mechanism of pH-induced release of retinol from retinol-binding protein. FEBS Lett 317:181–184. Rask L, Peterson PA (1976) In vitro uptake of vitamin A from the retinol-binding plasma protein to mucosal epithelial cells from the monkey’s small intestine. J Biol Chem 251:6360–6366. Ronne H, Ocklind C, Wiman K, Rask L, Obrink B, Peterson PA (1983) Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein. J Cell Biol 96:907–910. Rostom AA, Sunde M, Richardson SJ, Schreiber G, Jarvis S, Baterman R,, Dobson CM, Robinson CV (1998) Dissection of multi-protein complexes using mass spectrometry: subunit interactions in transthyretin and retinol-binding protein complexes. Proteins Struct Funct Genetics (Supp 2):3–11. Senoo H, Kojima N, Sato M (2007) Vitamin A-storing cells (stellate cells). Vitam Horm 75: 131–159. Shingleton JL, Skinner MK, Ong DE (1989) Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells. Biochemistry 28:9641–9647. Sivaprasadarao A, Findlay JBC (1994) Structure-function studies on human retinol-binding protein using site-directed mutagenesis. Biochem J 300:437–442 Sivaprasadarao A, Findlay JBC (1998) The interaction of retinol-binding protein with its plasmamembrane receptor. Biochem J 255:561–569. Smeland S, Bjerknes T, Malaba L, Eskild W, Norum KR, Blomhoff R (1995) Tissue distribution of the receptor for plasma retinol-binding protein. Biochem J 305:419–424. Soprano D, Blanner W (1994) Plasma retinol-binding protein. In: Sporn M, Roberts A, and Goodman D (eds.), The Retinoids: Biology, Chemistry, and Medicine, New York: Raven, p. 257–282.

142

H.L. Monaco

Sundaram A, van Aalten DMF, Findlay JBC (2002) The transfer of transthyretin and receptorbinding properties from the plasma retinol-binding protein to the epididymal retinoic acidbinding protein. Biochem J 362:265–271. Sunde M, Richardson SJ, Chang L, Pettersson TM, Schreiber G, Blake CCF (1996) The crystal structure of transthyretin from chicken. Eur J Biochem 236:491–499. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 van Bennekum AM, Wei S, Gamble MV, Vogel S, Piantedosi R, Gottesman M, Episkopou V, Blaner WS (2001) Biochemical basis for depressed serum retinol levels in transthyretindeficient mice. J Biol Chem 276:1107–1113. van Dam RM, Hu FB (2007) Lipocalins and insulin resistance: etiological role of retinol-binding protein 4 and lipocalin-2?. Clin Chem 53:5–7 van Jaarsveld PP, Edelhoch H, Goodman De WS, Robins J (1973) The interaction of human plasma retinol-binding protein with prealbumin. J Biol Chem 248:4698–4705. von Eynatten M, Humpert PM (2008) Retinol-binding protein-4 in experimental and clinical metabolic disease. Expert Rev Mol Diagn 8:289–299. Waits RP, Yamada T, Uemichi T, Benson MD (1995) Low plasma concentrations of retinolbinding protein in individuals with mutations affecting position 84 of the transthyretin molecule. Clin Chem 41:1288–1291 Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8:127–134. Wojtczak A, Luft JR, Cody V (1993) Structural aspects of inotropic bipyridine binding. Crystal ˚ of the human serum transthyretin-milrinone complex. J Biol structure determination to 1.9 A Chem 268:6202–6206. Wojtczak A, Cody V, Luft JR, Pangborn WJ (2001) Structure of rat transthyretin (rTTR) complex ˚ resolution: first non-biased insight into thyroxine binding reveals with thyroxine at 2.5 A different hormone orientation in two binding sites. Acta Cryst D57:1061–1070. Wolf G (2007) Identification of a membrane receptor for retinol-binding protein functioning in the cellular uptake of retinol. Nutr Rev 65:385–388. Yamamoto Y, Yoshizawa T, Kamio S, Aoki O, Kawamata Y, Masushige S, Kato S (1997) Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes. Exp Cell Res 234:373–378. Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB (2005) Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436:356–362. Zanotti G, Ottonello S, Berni R, Monaco HL (1993a) Crystal structure of the trigonal form of ˚ resolution. J Mol Biol 230:613–624. human plasma retinol-binding protein at 2.5 A Zanotti GR, Berni R, Monaco HL (1993b) Crystal structure of liganded and unliganded forms of bovine plasma retinol-binding protein. J Biol Chem 268:10728–10738. Zanotti G, Malpeli G, Berni R (1993c) The interaction of N-ethyl retinamide with plasma retinol˚ resolubinding protein (RBP) and the crystal structure of the retinoid-RBP complex at 1.9 A tion. J Biol Chem 268:24873–24879. Zanotti G, Marcello M, Malpeli G, Folli C, Sartori G, Berni R (1994) Crystallographic studies on complexes between retinoids and plasma retinol-binding protein. J Biol Chem 269:29613–29620. Zanotti G, Panzalorto M, Marcato A, Malpeli G, Folli C, Berni R (1998) Structure of pig plasma ˚ resolution. Acta Cryst D54:1049–1052. retinol-binding protein at 1.65 A Zanotti G, Calderone V, Beda M, Malpeli G, Folli C, Berni R (2001) Structure of chicken plasma retinol-binding protein. Biochim Biophys Acta 1550:64–69. Zanotti G, Berni R (2004) Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 69:271–295. Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9.

Chapter 9

TTR and RBP: Implications in Fish Physiology Sancia Gaetani and Diana Bellovino

Abstract Transthyretin (TTR), a homotetrameric protein of 54 kDa, is one of the carriers of thyroid hormone and, as a macromolecular complex with retinolbinding protein 4 (RBP4), is involved in the transport of vitamin A in blood, preventing kidney glomerular filtration of the small, 21 kDa RBP4. TTR cDNA has been cloned in many vertebrates (man, rat, chicken, etc.) and more recently also in several teleost fishes. Fish TTR shows a strong homology with the same protein of other vertebrates, with similar monomer–monomer and dimer–dimer interfaces and a conserved tetrameric structure. RBP4 was also isolated from several teleosts and characterized. The expression of the two proteins was investigated during embryonic development and in adult tissues. TTR is expressed in adult fish liver and is therefore present in their serum, even though RBP4 and TTR have never been detected in the blood as a complex. The lack of RBP4–TTR complex formation in fish has been explained, since the residues specifically involved in the interaction between the two proteins in mammals are different from the corresponding residues in fish. Keywords Transthyretin, Retinol-binding protein, Thyroid hormones, Fish retinoids transport

Abbreviations TTR RBP4 ER THs T4-Thyroxine T3

Transthyretin Retinol-binding protein 4 Endoplamic reticulum Thyroid hormones 30 -50 -3-5-Triiodothyronine 30 -3-5-Triiodothyronine

S. Gaetani and D. Bellovino National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178, Roma, Italy e-mail: [email protected]


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THBP TBPA LRAT STRA6 RAR FAP

9.1

Thyroid hormone binding protein Thyroxine-binding prealbumin Lecithin:retinol acyltransferase Stimulated by retinoic acid 6 gene Retinoic Acid Receptor Familial Amyloid Polyneuropathy

Vitamin A Transport

In teleost-fish, as in higher vertebrates, retinoids exert a key role in embryonic development and in several cellular functions throughout life (Chambon 1996; Niederreither and Dolle 2008), as well as in the visual cycle (Blomhoff and Blomhoff 2006). Retinoids introduced via the diet, mainly as carotenoids and retinyl esters, undergo a series of metabolic changes to the final active form, retinoic acid, which interacts with specific transcription factors and modulates the expression of hundreds of genes (Schug et al. 2007). The form involved in the visual cycle is 11-cis-retinal (Thompson and Gal 2003). A schematic description of vitamin A transport and utilization is outlined in Fig. 9.1. As in higher vertebrates, the liver of fish is the organ where vitamin A is stored. In mammals the two proteins involved in the specific binding and efficient transport and targeting of circulating vitamin A (retinol) are expressed in the liver: retinolbinding protein (RBP, now universally denominated RBP4) and transthyretin (TTR). RBP4 is the specific blood retinol carrier, while TTR is involved in thyroid hormone (TH) transport and in higher vertebrates it binds holoRBP4. The formation of the holoRBP4–TTR complex, which takes place in the endoplasmic reticulum (ER) of the hepatocytes before secretion, has been demonstrated and characterized in higher vertebrates (Soprano and Blaner 1994; Bellovino et al. 1996; Melhus et al. 1991; Selvaraj et al. 2008). The secretion of RBP4 is regulated by retinol. In vitamin A deficiency, when retinol is not available in the hepatocyte, RBP4 secretion is inhibited and the protein accumulates in the ER, from where it is promptly secreted upon retinol repletion (Perozzi et al. 1991; Bellovino et al. 1999). Like the other secretory proteins, RBP4 is cotranslationally targeted to the ER where it undergoes signal peptide cleavage and oxidative folding. Secretion of RBP4 is triggered by retinol binding, that induces a conformational change from the ‘‘apo’’ to the ‘‘holo’’ form of the protein and allows its secretion from the ER. Mechanisms underlying such ligand-dependent secretion are still not fully explained. It is believed that ligand binding may relieve retention of RBP4 from the quality control machinery in the ER. However, precise interrelationships between RBP4 folding, ligand binding, TTR assembly and secretion are still not clearly understood (Selvaraj et al. 2008).

9 TTR and RBP: Implications in Fish Physiology

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Fig. 9.1 Outline of vitamin A metabolism. Vitamin A, from vegetable and animal sources, is introduced via the diet. Carotenoids and retinyl esthers are absorbed from enterocytes and are included in lipoprotein particles, in particular chylomicrons. Some retinol is delivered to retina where it is transformed into retinal and takes part to the visual cycle. Circulating chylomicrons are taken up into the liver, where the different components, including the retinyl-esters, are released. According to the vitamin A status of the organism, retinyl esters can be stored in the stellate cells, and/or transformed in retinol in the hepatocytes. Retinol interacts with its specific carrier protein RBP4, thus forming holo-RBP4. Holo-RBP4 forms a complex with TTR and is secreted into blood towards target tissues. Following the interaction of RBP4 with its receptor Stra6, retinol is released from RBP4 into the target cells, and is metabolized to retinoic acid. Retinoic acid is then shuttled to the nucleus, where it regulates gene expression through the activation of specific transcription factors (RARs and RXRs). CRBP-II cellular retinol binding protein II, IRBP interphoto receptor binding protein, RBP retinol binding protein, TTR transthyretin, STRA6 retinoic acid stimulated gene 6, CRBP-I cellular retinol binding protein I, CRABP cellular retinoic acid binding protein, RAR retinoic acid receptor, RXR retinoid X receptor

It is believed that formation of the holoRBP-TTR complex prevents the loss of low molecular weight RBP4 and its bound retinol through the kidney. TTR null mice were generated and were shown to be phenotypically normal and viable. In these animals, plasma level of RBP4, retinol, and THs were significantly decreased (Episkopou et al. 1993).

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Until a few years ago it was known that vitamin A was an essential nutrient for higher vertebrates and for fish, but little was known about its transport and utilization in these animals. The aim of this chapter is to describe the present knowledge of TTR and RBP4 in teleost fish, and their role in controlling the distribution and activity of essential molecules, such as vitamin A and THs, in these organisms.

9.2

Transthyretin in Fish

TTR, formerly called prealbumin for its migration faster than serum albumin during electrophoresis of whole plasma, is a homotetrameric protein forming, in the majority of vertebrates where it has been isolated, a macromolecular complex with holoRBP. It is the carrier of THs: 30 ,50 ,3,5-tetraiodothyronine (thyroxine, T4) and 30 ,3,5-triiodothyronine (T3). In higher vertebrates, TTR is expressed mainly in the liver and in the choroid plexus of the brain. In the hepatocyte, the major storage cell for retinol, both RBP4 (molecular mass 21 kDa) and TTR (molecular mass 54 kDa), are synthesized and secreted as a molecular complex in a ratio of 1:1:1 (holoRBP4-TTR) (Soprano and Blaner 1994; Bellovino et al. 1996). TTR has only been found in vertebrates, and in particular piscine TTR displays the lowest sequence identity with human TTR. In lower species only members of a TTR-related protein family were identified (Eneqvist et al. 2003; Saverwyns et al. 2008), while TTR homologues have been found among vertebrates and share high sequence identity. In adult birds, diprotodont marsupials and eutherian mammals, TTR is expressed both in the liver and choroid plexus of the brain, whereas in reptiles it is expressed only in the choroid plexus. In fish and amphibians the main production site is the liver during development (Schreiber and Richardson 1997; Power et al. 2000; Santos et al. 2002). Interestingly, piscine TTR displays a higher affinity for T3 than for T4, as do the amphibian and avian TTRs (Eneqvist et al. 2004), and in contrast to the marsupial and mammalian TTRs, that have a higher affinity for T4 (Richardson 2007). This characteristic suggests that the binding affinity of TTR changed during evolution. In mammalian TTR, transport of T4 is preferred across the blood-brain barrier, where T4 is converted by 50 -monodeiodination to the biologically more active T3. TTR evolution may correlate with the evolution of deiodinases, which generate T3 from T4 in a tissue specific action. Very recently, a paper was published (Morgado et al. 2008) where the role of the longer N-terminus of fish TTR, thought to influence TH-binding affinity and maybe TTR stability, was investigated. Recombinant wild type sea bream (sbTTRWT) and two mutants lacking the first 6 and 12 N-terminal residues (sbTTRM6 and sbTTRM12, respectively) were produced. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4, indicating that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. The authors suggest that


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sbTTRWT, less prone to dissociate into monomers than human TTR, is more resistant to fibril formation. Four TTR monomers are organized in two very stable dimers that, in turn, interact to form a tetrameric structure with a central hydrophobic channel, in which two hormone-binding sites are present (Monaco et al. 1995; Naylor and Newcomer 1999). The two RBP-binding sites have also been structurally characterized and are situated on the surface of the molecule. Previously determined structures of chicken, rat, and human TTR are very similar, with the exception of the a-helical region, which is different in the avian structure compared with the mammalian protein (Sunde et al. 1996; Wojtczak 1997). In 1985 a paper (Larsson et al. 1985) reported the binding of [125I]T4 to salmon (Salmo salar) serum proteins and the isolation of a protein which, on the basis of its specific properties, was identified as TTR. The thyroxine-binding prealbumin (TBPA), as the authors called it, from all the 15 species (human, monkey, cattle, sheep, goat, water buffalo, horse, swine, dog, cat, rabbit, rat, chicken, frog, and salmon) investigated, except salmon, showed affinity for human RBP. The presence of TBPA in all vertebrates suggests that TTR is a far more important thyroxine carrier than it was thought up to this date. However, attemps to identify TTR in serum of different fish species (Salmo trutta, Onchorhyncus mykiss, Onchorhyncus tshawytscha, Cyprinus carpio) failed (Richardson et al. 1994), and the presence of TTR in fish remained controversial for many years. In 1999, a thyroid hormone-binding-protein (THBP) from serum of masu salmon (Onchorhyncus masou) at the stage of smoltification was isolated, purified, and biochemically characterized (Yamauchi et al. 1999). In salmonids at this developmental stage thyroid hormonal profile is very similar to those found in metamorphosing tadpoles (Dickhoff et al. 1990). The authors detected a single molecular species of THBP by [I125]T3 binding assay and showed that masu salmon THBP is homologous to TTRs of other vertebrates examined. However, its binding affinity for T3 was three times higher than that for T4, and therefore opposite to the higher vertebrate hormone affinity. Santos and Power (Santos and Power 1999) demonstrated unequivocally that TTR is present in teleost fish. It is synthesized mainly in the liver and it is released into the circulation where it binds THs. In this study RNA was extracted from different organs and tissues of juvenile sea breams. A cDNA library was constructed from liver RNA, screened with a TTR cDNA probe generated by RTPCR labeled with [a32P-dCTP] and the full-length TTR cDNA cloned, sequenced and translated in vitro. TTR expression was studied in several sea bream adult tissues and larval samples. TTR was found expressed, by Southern blot analysis of the PCR products, in hatching larvae and in liver, brain, pituitary, gills, kidney, intestine, and testis (not in the ovary). However, by Northern blot hybridization TTR mRNA was detected only in the liver, as a single transcript of 0.7 kb. The authors discuss the possibility that the failure to detect piscine TTR in the past by many investigators could be attributed to the use of T4 as ligand, since their study shows that T3, which is more abundant in fish plasma (Eales and Brown 1995), binds to TTR better than T4. TTR from the amphibian Rana catesbiana (Yamauchi

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Fig. 9.2 Sequence alignment of (a) TTR and (b) RBP4 from different vertebrates. The residues identical in all the sequences presented are shaded in red. The residues identical or chemically similar in at least six sequences are denoted by red characters. The amino acid residues in the human RBP and TTR involved in forming the RBP-TTR complex interactions (Zanotti et al. 2008) are indicated by arrowheads. Sequence alignments were constructed with CLUSTAL W (Thompson et al. 1994) and annotated with Espript (Gouet et al. 1999)

et al. 1993) also showed higher affinity for T3 than for T4. Sea bream TTR is a protein of 130 amino acids with a leader peptide of 20 amino acids. Its molecular mass, estimated by SDS-PAGE migration rate of the in vitro translation product, is about 15 kDa, and it is similar to the monomers characterized in other vertebrates (Santos and Power 1999). It is highly homologous to previously isolated TTRs despite the fact that its amino acid sequence shares only 40–55% identity with the


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sequence of TTR from other vertebrates. It must be emphasized, however, that the amino acids involved in the formation of the central channel in which T3 and T4 bind, are 90% homologous to human TTR and that the amino acids involved in the binding to RBP4 in higher vertebrates are also highly conserved (Fig. 9.2a). The three-dimensional structure was generated based on the known X-ray structures from human, rat, and chicken TTRs and on the sea bream TTR amino acid sequence (Power et al. 2000). According to this model, the overall topology of sea bream TTR has been conserved and the predicted monomer-monomer and dimer-dimer interfaces and tetrameric structures are similar to those determined by X-ray crystallography of human, rat, and chicken TTRs. Funkenstein and coauthors (Funkenstein et al. 1999) used a homologous TTR probe cloned from the liver of the marine fish Sparus aurata, to investigate its ontogeny and tissue distribution. The results showed that TTR mRNA was detected in fish larvae already at the day of hatching, as determined by Northern blot analysis. The expression increased during post-hatching up to the level of the adult liver, that is the major site of TTR expression. However a low level of expression of TTR was detected in all extrahepatic tissues studied (eye, brain, gill, muscle, heart, and kidney). Interestingly, relatively high expression was found in the skin. The aim of the work was to understand the role of sea bream TTR in the transport of THs. It was reported that experimental treatment of several fish species during early stages of development with TH in appropriate doses, induced earlier hatching and accelerated yolk absorption, growth and morphological differentiation (Brown and Kim 1995; Tachihara et al. 1997). Furthermore, TH induces metamorphosis in flounder, Paralichthys olivaceus (Miwa and Inui 1987). The questions the authors wanted to answer were: how the THs are transferred from the maternal circulation to the developing oocyte and how are THs in the developing embryos and larvae transported to their target organs. Exogenous THs administered to larval fish can enter the vascular compartment via skin or gills, but how they are transported into the target organs is not clear. Moreover, the reserves of THs in the yolk will not influence development unless they also enter the circulatory system and then reach the target organs. Most THs circulating in the blood of vertebrates are bound to plasma proteins. Proteins carrying THs in higher vertebrates have been extensively studied, while in fish very little was known at the end of the 1990s. TTR seemed to be the principal carrier of TH in bony fish. In 2003 Apreda et al. (Apreda M., Morgado I., Power D., Gaetani S., Bellovino D., GenBank AJ544193) cloned a full length carp (Cyprinus carpio) TTR cDNA from adult carp liver cDNA library. The protein was translated in vitro and expressed in a bacterial system in order to immunize rabbits and produce an anti carp-TTR polyclonal antibody. Preliminary results showed that carp TTR can be detected in adult carp blood. However, it has been reported that TTR is not present in adult fish, amphibians, reptiles and polyprotodont marsupials blood, since in these vertebrates the TTR gene seems to be turned on in the liver only during specific stages of development, when there is a rise in THs level in blood (Richardson 2007). For fish, this stage is during early development, when the animals are still

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absorbing the yolk. Further research is needed to shed light on this aspect of TTR physiology in fish. A cDNA encoding TTR was then cloned from the Pacific bluefin tuna (Thunnus orientalis) (Kawakami et al. 2006). This cDNA contains a complete open reading frame encoding 151 amino acid residues. The deduced amino acid sequence is 81% and 55% identical to the gilthead seabream and common carp forms respectively, and 33–39% to mammalian, reptilian, and amphibiam forms. A 1.0-kb transcript was found in the liver and ovary, the liver being the main source of the protein. Analysis of T3 and T4 binding demonstrated that both bind to bluefin TTR, but affinity for T3 for the protein is higher than that for T4. The results indicate that bluefin tuna TTR acts as a transporter of THs in the plasma, and therefore contributes to the function of THs in target cells. In 2007, the full-length cDNAs corresponding to TTR were isolated and cloned from two genera of lamprey, Petromyzon marinus and Lampetra appendix (Manzon et al. 2007). These sequences represented the first report of TTR sequences in vertebrates basal to the teleost fishes (superorder Euteleostei). The N-terminal region of the lamprey TTR subunits is nine amino acids longer than those in mammals and four to six amino acids longer than those in other vertebrates. The authors showed that the longer N-terminal region is due to the position of the intron 1/exon 2 splice site. The lamprey TTR gene is expressed mainly in the liver during all phases of the lifecycle. Moreover, it is expressed at low levels in a variety of other tissues and, as in other vertebrates, is upregulated during lamprey metamorphosis. The deduced amino acid sequence of lamprey TTR cDNAs are between 36% and 47% identical with those from other vertebrates. However, unlike other vertebrate TTRs, which show virtually 100% conservation of amino acids lining the central channel and therefore involved in TH binding, the deduced amino acid sequence of lamprey TTR cDNAs indicates that there are five nonconservative substitutions in these amino acids. Of these five substitutions, four are predicted to be neutral with respect to protein function; only the Ser to Phe substitution at position 115 is predicted to be a disfavored substitution. The observation that the TTR gene is developmentally regulated in lampreys, consistent with observations in other vertebrates, is a paradox. In fact in lampreys, one of the most ancient living vertebrates, the elevation of TTR gene expression during metamorphosis occurs when THs levels are at their nadir, whereas in other vertebrates elevation of TTR levels correlates with, and is thought to facilitate, a developmentally important surge in TH levels.

9.3

Piscine Retinol-Binding Protein 4

As mentioned above, besides its role as a TH carrier, in higher vertebrates TTR is also involved in vitamin A transport through its interaction with RBP4. The main form of circulating vitamin A is all-trans retinol, a small hydrophobic molecule that is stored mainly in the liver as retinyl-esters droplets. RBP4, the specific retinol carrier, is essential to mobilize retinol from storage in the liver and


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to target this molecule to specific peripheric tissues (Soprano and Blaner 1994). In fact, it has been demonstrated that in RBP/ knock-out mice fed a normal diet, vitamin A transport can be carried out by other circulating plasma proteins like albumin, but under vitamin A deficiency retinol stored in the liver cannot be efficiently mobilized and retinal function in the visual cycle is impaired (Quadro et al. 1999). Moreover, the recent identification of the specific RBP4 membrane receptor STRA6 (Stimulated by retinoic acid 6 gene) (Kawaguchi et al. 2007), expressed by selected organs (as eye and placenta), demonstrates that RBP4 plays a key role in targeting retinol delivery. In teleost fish, RBP4 is synthesized mainly in the liver and, to a lower extent, in kidney and ovary. It plays essentially the same role as in higher vertebrates, since it binds and transports retinol from the liver to target tissues. In particular, in these organisms RBP4 has been proven to exert a key role in providing vitamin A during zebrafish development (Isken et al. 2008) and to ovaries in order to support retinol accumulation in eggs, and therefore embryonic development (Lubzens et al. 2003; Levi et al. 2008). RBP4 function is strictly related to the expression of the membrane receptor STRA6, that in turn is responsible for clearing holo-RBP4 from the circulation. Isken and coauthors (2008) have demonstrated that the lack of STRA6 receptor in zebrafish embryonic tissues causes an excess of RBP4-bound vitamin A and the subsequent local retinol/retinoic acid accumulation. Retinoic acid accumulation provokes impaired retinoic acid receptor (RAR) signaling and gene regulation, and therefore causes developmental abnormalities and retinoid deficiency to the eye. In particular, it has been suggested that the correct vitamin A concentration at target tissues is maintained by the coordinated action of RBP4, STRA6, and lecithin: retinol acyltransferase (LRAT) proteins. STRA6 acts as a bidirectional retinol transporter, which mediates between RBP4 retinol supply from circulation and intracellular accumulation of retinyl esters performed by LRAT. The trout ovary has been defined by Levi et al. (Levi et al. 2008) as ‘‘an important site for retinoid and carotenoid metabolism.’’ In fact, they have demonstrated that in this organ several crucial genes involved in carotenoid and retinoid metabolism, including rbp4, stra6, and lrat, are expressed and contribute to ensure ovarian follicle viability and egg quality. To date RBP4 has been identified and characterized, at amino acid and/or nucleotide level, in several fish species: yellowtail Seriola quinqueradiata (Shidoji and Muto 1977), rainbow trout (Berni et al.1992; Zapponi et al. 1992), sea bream (Funkenstein et al. 1999; Brown and Kim 1995; Tachihara et al. 1997; Miwa and Inui 1987; Kawakami et al. 2006; Manzon et al. 2007; Quadro et al. 1999; Kawaguchi et al. 2007; Isken et al. 2008; Lubzens et al. 2003; Levi et al. 2008; Shidoji and Muto 1977; Berni et al. 1992; Zapponi et al. 1992; Sammar et al. 2001), carp (Bellovino et al. 2001) and zebrafish (Tingaud-Sequeira et al. 2006). All these proteins share a high degree of homology with higher vertebrates RBP4s, in particular in the regions involved in essential functions such as retinol binding (Fig. 9.2b). RBP4 is member of the lipocalin superfamily, which includes several small soluble proteins involved in the transport of hydrophobic molecules.

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All members of this group share a overall ‘‘calix’’ structure, characterized by an hydrophobic ‘‘pocket’’ in which the ligand is embedded (Flower 1996). Three-dimensional structure prediction (Zapponi et al. 1992) and primary sequence analysis (Bellovino et al. 2002; Lubzens et al. 2003; Folli et al. 2003) suggest that the residues involved in the retinol-binding pocket and the cysteines that coordinate three disulfide bridges (residues 22, 88, 138, 147, 178, and 192 according to human RBP4 sequence in Fig. 9.2b) are highly conserved among all vertebrates studied. Disulfide bridges contribute to the organization of the tertiary structure of RBP4, that in turn is related to its specific function as a lipocalin. Residues in the binding pocket create an environment able to accomodate the hydrophobic portion of retinol, while the polyene chain and the terminal hydroxyl group of retinol are exposed to the surface of the protein, near the entrance of the hydrophobic pocket, in a region involved in the interaction with TTR. As a consequence, the ‘‘apo’’ form of RBP4 has a lower affinity for TTR compared to the ‘‘holo’’ form (Zanotti et al. 1993; Malpeli et al. 1996). This feature in higher vertebrates contributes to the stability of the circulating holoRBP4-TTR complex, while upon release of retinol to target cells through the interaction with STRA6, apoRBP4 is presumably filtered by the kidneys. Circulating fish-RBP4 transports all-trans retinol (Berni et al. 1992), even though it has been shown that 3,4-dehydroretinol (vitamin A2), abundant in fish tissues (Ganguly 1989) and in particular in the liver, in vitro is able to displace alltrans retinol from the ligand binding site on RBP4 (Shidoji and Muto 1977; Berni et al. 1992).

9.4

RBP/TTR Interaction in Fish

In past years, the crystal structure and the putative regions involved in heterologous (human TTR–chicken RBP4, (Monaco et al. 1995)) or homologous (human TTR– human RBP4 (Naylor and Newcomer 1999)) RBP–TTR interactions were analyzed (Sivaprasadarao and Findlay 1994; Berni et al. 1994; Monaco 2000). Unlike the situation in mammals, fish RBP4 does not circulate as a complex with TTR. The RBP4–TTR complex has never been isolated from fish plasma and the in vitro affinity between the two proteins, though specific, is very low (Shidoji and Muto 1977; Berni et al. 1992). In 1992 Berni and colleagues (Berni et al. 1992) purified two forms of RBP4 by gel filtration from plasma of rainbow trout (Onchorhyncus mykiss). They were very similar to mammalial RBPs, but not physiologically involved in the formation of any protein–protein complex in plasma, although capable of interacting with mammalian TTR with low binding affinity. Since the two forms of trout RBP also possessed the region that in mammalian RBP4 has a functional role in binding TTR, the authors suggested that during the phylogenetic development of the nonmammalian vertebrates, TTR was modified to acquire a binding site for RBP4.


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In 2003, the same group performed other experiments of in vitro binding between sea bream or carp TTR and human holo-RBP4 (Folli et al. 2003). The interaction between the two proteins was evaluated by analyzing the changes of the fluorescence anisotropy of RBP4-bound retinol associated with the formation of the RBP4–TTR complex, the molecular mass of which (76 kDa) is higher than that of uncomplexed RBP4 (21 kDa). When sea bream or carp TTR was added to human holo-RBP4, no significant increase in fluorescence anisotropy of the RBP4-bound retinol was revealed under conditions suitable for the formation of the human TTR– RBP4 complex. This result was also obtained when sea bream or carp TTR was added to piscine (carp) holoRBP4. This issue has been resolved by recent research by Zanotti and colleagues (Zanotti et al. 2008), in which the crystal structure of the human RBP4–TTR complex, bound to an anti-RBP4 Fab, was determined. In parallel, the TTR amino acids involved in the interaction between the two proteins have been identified by site-directed mutagenesis. In particular, some of these mutations are characteristic of amyloidogenic variants of TTR. TTR, in fact, is a potential amyloidogenic protein, since some genetic variants are able to form fibrils that accumulate in tissues (e.g., brain) and cause the related degenerative pathology (FAP) (Saraiva 2001). In particular, the Ile84Ser amyloidogenic form causes the loss of RBP4–TTR interaction, and in fact individuals carrying this mutation present an altered retinol-RBP4 plasma transport. Interestingly, it has been demonstrated that some TTR residues (Leu82, Ile84, Asp99, and Ser100), that in amyloidogenic human TTR weaken or abolish the interaction with RBP4, are present in piscine TTR. This supports and explains the lack of interaction between RBP4 and TTR in fish. For instance, human TTR Ile84 is replaced by Ser84 in seabream, while human TTR Ser100 is replaced by Glu100 in seabream, zebra fish, and trout TTR. In the same study it was shown that the RBP4 regions involved in the complex formation have limited differences between terrestrial and aquatic vertebrates, explaining why piscine RBP4 is more prone to form in vitro heterologous complex, albeit weak, with TTRs from higher vertebrates (Saraiva 2001; Folli et al. 2003). Fish RBP4s, as well as Xenopus and chicken, lack a short C-terminal aminoacid sequence that in mammals has been proposed to stabilize the RBP4–TTR interaction (Naylor and Newcomer 1999). According to Zanotti et al. (2008), this portion of the protein is not crucial for the stability of the human RBP4–TTR complex; on the other hand, chicken RBP4, lacking this part of the molecule, is able to form a complex with human TTR (Monaco et al. 1995). It is therefore generally agreed that the interaction between RBP4 and TTR, that is thought to prevent glomerular filtration of RBP4, has evolved in terrestrial vertebrates by mutations affecting a limited number of TTR and RBP4 residues. Carp RBP4 presents biochemical features that are unique among the fish RBPs characterized to date (Bellovino et al. 2001): the signal sequence is not removed (as usually happens for secretory proteins after translocation across the ER membrane) and two N-glycosylations are present at the N-terminal portion of the protein (AsnX-Thr consensus sites at residues 25 and 46). Indeed, the presence of N-glycosylation has also been detected in other two members of Cyprinidae family: zebrafish

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(Danio rerio) and goldfish (Carassius auratus) (Devirgiliis et al. 2005). While for zebrafish RBP4 the single N-glycosylation site can be predicted from the amino acidic sequence, in goldfish the modification has been revealed only through the removal of the glycan moiety by N-Glycosidase F digestion, followed by electrophoretic separation by molecular weight, since neither the amino acid sequence nor the corresponding cDNA sequence for this protein are available. The possible function of this secondary modification on carp RBP4 is not clear. An hypothesis is that the glycan moiety, rich in sialic acid, increases the apparent molecular weight and the net negative charge of the protein, slowing down its kidney filtration rate (Elger et al. 1988) and ultimately compensateing for the lack of interaction with TTR.

9.5

Conclusions

In conclusion, since many years it has been established that vitamin A is essential throughout the life of teleosts, but very little is known about its transport, metabolism, and distribution. In this chapter the results of the isolation and characterization of RBP4 and TTR in fish have been surveyed, but still very little information is available on their mechanism of action and on the implications in fish physiology. Hopefully research in this important field will soon progress rapidly and successfully.

References Bellovino D, Lanyau Y, Garaguso I, Amicone L, Cavallari C, Tripodi M, and Gaetani S (1999) MMH cells: An in vitro model for the study of retinol-binding protein secretion regulated by retinol. J Cell Physiol 181, 24–32. Bellovino D, Morimoto T, Apreda M, Devirgiliis C, Mengheri E, and Gaetani S (2002) Isolation, expression and characterization of carp retinol-binding protein. Gene 295, 231–240. Bellovino D, Morimoto T, Mengheri E, Perozzi G, Garaguso I, Nobili F, and Gaetani S (2001) Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide. J Biol Chem 276, 13949–13956. Bellovino D, Morimoto T, Tosetti F, and Gaetani S (1996) Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells. Exp Cell Res 222, 77–83. Berni R, Malpeli G, Folli C, Murrell JR, Liepnieks JJ, and Benson MD (1994) The Ile-84 ! Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinolbinding protein. J Biol Chem 269, 23395–23398. Berni R, Stoppini M, and Zapponi MC (1992) The piscine plasma retinol-binding protein. Purification, partial amino acid sequence and interaction with mammalian transthyretin of rainbow trout (Oncorhynchus mykiss) retinol-binding protein. Eur J Biochem 204, 99–106. Blomhoff R and Blomhoff HK (2006) Overview of retinoid metabolism and function. J Neurobiol 66, 606–630. Brown C and Kim B (1995) Combined application of cortisol and triiodothyronine in marine finfish culture. Aquaculture 135, 79–86.


155

Chambon P (1996) A decade of molecular biology of retinoic acid receptors. FASEB J 10, 940–954. Devirgiliis C, Gaetani S, Apreda M, and Bellovino D (2005) Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane. Biochem Biophys Res Commun 332, 504–511. Dickhoff WW, and Brown CL, Sullivan CV, and Bern H (1990) Fish and amphibian models for developmental endocrinology. J Exp Zool Supplement 4, 90–97. Eales JG and Brown SB (1995) Regulation and measurement of thyroidal status in teleost fish. Neth J Zool 45, 175–180. Elger B, Ruhs H, and Hentschel H (1988) Glomerular permselectivity to serum proteins in rainbow trout (Salmo gairdneri). Am J Physiol 255, R418–R423. Eneqvist T, Lundberg E, Karlsson A, Huang S, Santos CR, Power DM, and Sauer-Eriksson AE (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J Biol Chem 279, 26411–26416. Eneqvist T, Lundberg E, Nilsson L, Abagyan R, and Sauer-Eriksson AE (2003) The transthyretinrelated protein family. Eur J Biochem 270, 518–532. Episkopou V, Maeda S, Nishiguchi S, Shimada K, Gaitanaris GA, Gottesman ME, and Robertson EJ (1993) Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone. Proc Natl Acad Sci USA 90, 2375–2379. Flower DR (1996) The lipocalin protein family: structure and function. Biochem J 318(Pt 1), 1–14. Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, and Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555, 279–284. Funkenstein B, Perrot V, and Brown CL (1999) Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution. Mol Cell Endocrinol 157, 67–73. Ganguly J (1989) Biochemistry of Vitamin A. Boca Raton FL: CRC Press. Gouet P, Courcelle E, Stuart D, and Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308. Isken A, Golczak M, Oberhauser V, Hunzelmann S, Driever W, Imanishi Y, Palczewski K, and von Lintig J (2008) RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome. Cell Metab 7, 258–268. Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, and Sun H (2007) A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315, 820–825. Kawakami Y, Seoka M, Miyashita S, Kumai H, and Ohta H (2006) Characterization of transthyretin in the Pacific bluefin tuna, Thunnus orientalis. Zoolog Sci 23, 443–448. Larsson M, Pettersson T, and Carlstrom A (1985) Thyroid hormone binding in serum of 15 vertebrate species: isolation of thyroxine-binding globulin and prealbumin analogs. Gen Comp Endocrinol 58, 360–375. Levi L, Levavi-Sivan B, and Lubzens E (2008) Expression of genes associated with retinoid metabolism in the trout ovarian follicle. Biol Reprod 79, 570–577. Lubzens E, Lissauer L, Levavi-Sivan B, Avarre JC, and Sammar M (2003) Carotenoid and retinoid transport to fish oocytes and eggs: what is the role of retinol binding protein?. Mol Aspects Med 24, 441–457. Malpeli G, Folli C, and Berni R (1996) Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. Biochim Biophys Acta 1294, 48–54. Manzon RG, Neuls TM, and Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151, 55–65. Melhus H, Nilsson T, Peterson PA, and Rask L (1991) Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol. Exp Cell Res 197, 119–124. Miwa S and Inui Y (1987) Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus). Gen Comp Endocrinol 67, 356–363. Monaco HL (2000) The transthyretin-retinol-binding protein complex. Biochim Biophys Acta 1482, 65–72.

156


Monaco HL, Rizzi M, and Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268, 1039–1041. Morgado I, Melo EP, Lundberg E, Estrela NL, Sauer-Eriksson AE, and Power DM (2008) Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal. Mol Cell Endocrinol 295, 48–58 Naylor HM and Newcomer ME (1999) The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP. Biochemistry 38, 2647–2653. Niederreither K and Dolle P (2008) Retinoic acid in development: towards an integrated view. Nat Rev Genet 9, 541–553. Perozzi G, Mengheri E, Colantuoni V, and Gaetani S (1991) Vitamin A intake and in vivo expression of the genes involved in retinol transport. Eur J Biochem 196, 211–217. Power DM, Elias NP, Richardson SJ, Mendes J, Soares CM, and Santos CR (2000) Evolution of the thyroid hormone-binding protein, transthyretin. Gen Comp Endocrinol 119, 241–255. Quadro L, Blaner WS, Salchow DJ, Vogel S, Piantedosi R, Gouras P, Freeman S, Cosma MP, Colantuoni V, and Gottesman ME (1999) Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein. EMBO J 18, 4633–4644. Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258, 137–193. Richardson SJ, Bradley AJ, Duan W, Wettenhall RE, Harms PJ, Babon JJ, Southwell BR, Nicol S, Donnellan SC, and Schreiber G (1994) Evolution of marsupial and other vertebrate thyroxinebinding plasma proteins. Am J Physiol 266, R1359–R1370. Sammar M, Babin PJ, Durliat M, Meiri I, Zchori I, Elizur A, and Lubzens E (2001) Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues. Gen Comp Endocrinol 123, 51–61. Santos CR, Anjos L, and Power DM (2002) Transthyretin in fish: state of the art. Clin Chem Lab Med 40, 1244–1249. Santos CR and Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140, 2430–2433. Saraiva MJ (2001) Transthyretin amyloidosis: a tale of weak interactions. FEBS Lett 498, 201–203. Saverwyns H, Visser A, Van Durme J, Power D, Morgado I, Kennedy MW, Knox DP, Schymkowitz J, Rousseau F, Gevaert K, Vercruysse J, Claerebout E, and Geldhof P (2008) Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – Comparison with other strongylid nematodes and Caenorhabditis elegans. Int J Parasitol 38, 1545–1556 Schreiber G and Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B Biochem Mol Biol 116, 137–160. Schug TT, Berry DC, Shaw NS, Travis SN, and Noy N (2007) Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell 129, 723–733. Selvaraj SR, Bhatia V, and Tatu U (2008) Oxidative folding and assembly with transthyretin are sequential events in the biogenesis of retinol binding protein in the endoplasmic reticulum. Mol Biol Cell 19, 5579–5592. Shidoji Y and Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J Lipid Res 18, 679–691. Sivaprasadarao A and Findlay JB (1994) Structure-function studies on human retinol-binding protein using site-directed mutagenesis. Biochem J 300(Pt 2), 437–442. Soprano D and Blaner W (1994) Plasma Retinol-Binding Protein. In The Retinoids: Biology, Chemistry and Medicine [Sporn MB, Goodman DS, editor]. New York: Raven Press. Sunde M, Richardson SJ, Chang L, Pettersson TM, Schreiber G, and Blake CC (1996) The crystal structure of transthyretin from chicken. Eur J Biochem 236, 491–499. Tachihara K, Lel-Zidbeh M, Ishimatsu A, and Tagawa M (1997) Improved seed production of gold-striped amberjack Seriola lalandii under hatchery conditions by injection of triiodothyronine (T3) to broodstock fish. J World Aquacult Soc 28, 34–44.


157

Thompson DA and Gal A (2003) Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases. Prog Retin Eye Res 22, 683–703. Thompson J, Higgins D, and Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 22, 4673–4680. Tingaud-Sequeira A, Forgue J, Andre M, and Babin PJ (2006) Epidermal transient down-regulation of retinol-binding protein 4 and mirror expression of apolipoprotein Eb and estrogen receptor 2a during zebrafish fin and scale development. Dev Dyn 235, 3071–3079. Wojtczak A (1997) Crystal structure of rat transthyretin at 2.5 A resolution: first report on a unique tetrameric structure. Acta Biochim Pol 44, 505–517. Yamauchi K, Kasahara T, Hayashi H, and Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132, 2254–2261. Yamauchi K, Nakajima J, Hayashi H, and Hara A (1999) Purification and characterization of thyroid-hormone-binding protein from masu salmon serum. A homolog of higher-vertebrate transthyretin. Eur J Biochem 265, 944–949. Zanotti G, Berni R, and Monaco HL (1993) Crystal structure of liganded and unliganded forms of bovine plasma retinol-binding protein. J Biol Chem 268, 10728–10738. Zanotti G, Folli C, Cendron L, Alfieri B, Nishida SK, Gliubich F, Pasquato N, Negro A, and Berni R (2008) Structural and mutational analyses of protein-protein interactions between transthyretin and retinol-binding protein. FEBS J 275, 5841–5854 Zapponi MC, Zanotti G, Stoppini M, and Berni R (1992) The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein. Eur J Biochem 210, 937–943.

Chapter 10

TTR and Endocrine Disruptors Kiyoshi Yamauchi and Akinori Ishihara

Abstract In addition to binding thyroid hormones (THs), its endogenous ligands, transthyretin (TTR) can bind a large number of chemicals, including flavonoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and environmental pollutants. By binding to TTR, these chemicals have the potential to change TH homeostasis in plasma and interfere with the thyroid system, particularly in rodents and lower vertebrates. A structure–activity relationship of those chemicals that compete with TH for binding to TTR was found for several groups of chemicals. Owing to the binding specificity of these chemicals for TTR, a competitive binding assay for TH binding to TTR has been used to detect potent TH-disrupting chemicals in samples obtained from various environments. Recent advances in pharmacological and physiological research indicate that some chemicals that bind to TTR may have a chemotherapeutically beneficial effect on TTR-mediated amyloid fibril formation, whereas other chemicals that bind to TTR may have an adverse effect on retinoid homeostasis. Possible roles for the binding of chemicals to TTR are discussed. Keywords Competitive binding, Structure–activity relationship, TH-disrupting activity

10.1

Introduction

In humans, more than 99% of thyroid hormones (THs), including the precursor form l-thyroxine (T4) and the active form 3,30 ,5-triiodo-l-thyronine (T3), in plasma are bound to proteins. These proteins include thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin. TBG has a high affinity for THs and, as such, 70–80% of THs are bound to TBG, despite its low concentration in plasma K. Yamauchi (*) Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan e-mail: [email protected]


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K. Yamauchi and A. Ishihara

(0.27 mM). In contrast, only 10–20% of THs are bound to TTR and albumin, which, compared with TBG, are found in higher concentrations in plasma (4.6 and 640 mM, respectively) (Robbins 1996). As the concentrations of T4 and T3 in plasma are 110 and 2.1 nM, respectively (Chopra 1996), 30% of TBG, 0.2–0.3% of TTR, and 0.002–0.003% of albumin are responsible for TH binding in vivo. This situation is not true in rodents, some marsupials, and lower vertebrates because they lack TBG in plasma. Studies of TH binding to TTR also revealed that eutherian TTRs have higher affinity for T4 than for T3, whereas lower vertebrate TTRs have higher affinity for T3 than for T4 (Schreiber and Richardson 1997). Furthermore, TTR interacts with retinol-binding protein (RBP) in plasma from mammals and birds but not in plasma from amphibians and fish. These comparative aspects suggest that the physiological functions of TTR and the potential for environmental chemicals to interfere with TH binding to TTR in the plasma of rodents and/or the lower vertebrates could be quantitatively and qualitatively different from those of eutherians. Humans and wildlife are exposed to many environmental chemicals that are found in food, air, and water. The plasma protein(s) to which these chemicals bind in plasma is one of the important factors that will affect the tissue distribution, metabolism, excretion, and cellular actions of these chemicals. TTR has significant binding affinity for a large number of environmental chemicals including native and synthetic flavonoids as well as medical/pharmaceutical, agricultural, industrial, and nonintentional chemicals such as dioxins and furans (Brucker-Davis 1998). If environmental chemicals, or their metabolites, displace THs from TTR in vivo, they could alter TH homeostasis in plasma by increasing the free concentration of THs. For this reason, TTR has been posited as a molecular target for thyroid disruption by these chemicals (Brouwer et al. 1998; DeVito et al. 1999; Schmutzler et al. 2007). Data accumulated over the past two decades have indicated that chemicals that have binding affinity for TTR can alter the TH levels in plasma and/or modulate cellular actions of THs in rodents or cell culture systems. However, whether chemicals that bind to TTR affect plasma TH levels, particularly in large eutherians given that TTR is responsible for binding only a small proportion of total THs in plasma, is unclear. In addition to the thyroid disruption by these chemicals, medical/ pharmacological and physiological interests in TTR research have increased. In this chapter, we will discuss how environmental chemicals can interfere with the thyroid system by binding to TTR and the possible physiological significance of this interference, as well as the application of a TTR competitive binding assay to detect TH-disrupting chemicals in environment samples.

10.2

Chemicals That Interact with the TH-Binding Sites of TTR

Flavonoids are known to affect the thyroid system, including the function of the thyroid gland, the secretion of THs from the thyroid gland, and the availability of THs for uptake into target cells, partly because of the structural similarity between

10 TTR and Endocrine Disruptors

161 I

thyroid hormones

I

O

HO

I

NH2 COOH

(I) 3’ 2’ 8

9 O 2

7

flavonoids

6

10

5

1’

8 4’

7

5’

6

6’

5

3

4

10

2

4

3

O

1’

6

2’

5

3’

4

4’

6’ 5’

O

OH

O

F F

O

H N

F

(flufenamic acid*,1.75)

OH H N

CH3

other medical drugs

8 9

O 3

1’

OH O O

6’

2

O

OH

CH3

(mefenamic acid*, 0.27) O

5’

2’

aurones (3’,5’-dibromo-2’,4,4’,6tetrahydroxyaurone) OH O

isoflavone (genistein*)

flavone (F21388, 2.5)

NSAIDs

7

9 O

4’

3’

(L-thyroxine*, 1) (3,3’,5-triiodo-L-thyronine)

(diflunisal*, 0.22)

F

F

OH

N trans-stilbene (diethylstilbestrol*, 37) (resveratrol*)

F

F environmental pollutants

2’

2

3

3

3’

6’

6

5

(Cl)n

5’

2

(Cl)n

4

(Br)n

6 5

3’

6

4’

6’ 5’

2’

3’

(Br)n

polybrominated diphenyl esters and their hydroxylated derivatives

6

CH3

6’

5’

(Cl)n or (Br)n

(Cl)n or (Br)n

2’

O

CH3

OH 5

PCBs and their hydroxylated derivatives (4-OH-3,3’,4’,5-tetrachlorobiphenyl*, 2.56)

3

2

HO

4’

4

(N-(m-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid*)

F

halogenated derivatives of bisphenol A (3,3’,5,5’-tetrabromobisphenol A,10.6) O OH 6 O 5 2 OH

5

3 4

phenols (triiodophenol*, 3.84) (pentabromophenol, 7.12)

4

(Cl)n

2 3

phenoxy acids (2,4-dichlorophenoxy acid)

Fig. 10.1 Thyroid hormones and chemicals with binding affinity for transthyretin (TTR). Core structures or typical examples (in parentheses) of groups of chemicals are indicated. Asterisk (*), Inhibitory activity against amyloid fibril formation (see section on TTR Amyloid). Relative affinities are given as the ratio of IC50 (T4)/IC50 (chemical) except for the value for diethylstilbestrol, which is the ratio of IC50 (T3)/IC50 (chemical). These ratios are from Ko¨hrle et al. (1988), Green et al. (2005), Ciszak et al. (1992), Munro et al. (1989), Ishihara et al. (2003), Brouwer et al. (1990), Van den Berg et al. (1991), Meerts et al. (2000), McKinney et al. (1985), Miroy et al. (1996), Peterson et al. (1998); Klabunde et al. (2000), Purkey et al. (2001), McCammon et al. (2002), Miller et al. (2004), Purkey et al. (2004), and Morais-de-Sa´ et al. (2004)

flavonoids and THs (Schro¨der-van der Elst et al. 2003; van der Heide et al. 2003; Hamann et al. 2006). Flavonoids are multihydroxylated polyphenols and have a basic structure consisting of three rings (Fig. 10.1). Among them, the flavone apigenin (van der Heide et al. 2003) and the isoflavone genistein (Green et al. 2005) have a binding affinity for TTR. These natural, plant-derived flavonoids are

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ingested from food daily, at a rate of up to 2 g per day. Even if the affinities of these flavonoids for TTR were lower than that of TH, these flavonoids are likely to compete with TH for binding to TTR because of the high concentrations of flavonoids in plasma that result from their high intake. The flavone derivatives 4,40 , 6-trihydroxyaurone (Auf’mkolk et al. 1986) and 30 ,50 -dibromo-20 ,4,40 ,6-tetrahydroxyaurone (Ciszak et al. 1992) also have a binding affinity for TTR. The potential of flavonoids to interfere with the thyroid system in rats was studied in detail using a synthetic flavonoid 3-methyl-40 ,6-dihydroxy-30 ,50 -dibromoflavone (F21388). The half maximum inhibitory concentration (IC50) of F21388 for [125I]T4 binding to TTR was 2.5 times lower than that of T4 (Ko¨hrle et al. 1988). In vivo, F21388 displaces TH from TTR (Ko¨hrle et al. 1989; Lueprasitsakul et al. 1990; Mendel et al. 1992) and alters TH levels in plasma (Ko¨hrle et al. 1989; Lueprasitsakul et al. 1990; Schro¨der-van der Elst et al. 1997) and TH metabolism (Schro¨der-van der Elst et al. 1991). These findings have been comprehensively reviewed elsewhere (Ko¨hrle et al. 1988; Hamann et al. 2006). Nonsteroidal anti-inflammatory drugs (NSAIDs) also interfere with TH binding to TTR. Munro et al. (1989) investigated the competitive inhibition potency of 26 drugs, most of which were NSAIDs, with [125I]T4 for binding to TTR in vitro. Of those drugs that competed with TH for binding to TTR, the order of the potency was flufenamic acid>mefenamic acid>diflunisal>meclofenamic acid>diclofenac> fenclofenac>>aspirin. In a later study, the binding properties of NSAIDs were directly confirmed by an antibody capture method in combination with highperformance liquid chromatography (HPLC) analysis (Purkey et al. 2001) and mass spectrometry (McCammon et al. 2002). The affinities of NSAIDs for TTR were several orders of magnitude greater than those for TBG (Munro et al. 1989). Salsalate and its metabolite, salicylate, inhibit TH binding to serum transport proteins and decrease TH levels in plasma in vivo. However, these compounds do not bind specifically to TTR, and can displace TH from TBG and albumin too (Wang et al. 1999). Diethylstilbestrol, a synthetic nonsteroidal estrogen, was the most potent competitor, among 17 candidates, of [125I]T3 binding to chicken TTR and had an IC50 37 times less than that for T3 (Ishihara et al. 2003). Resveratrol, a phytoalexin produced naturally by several plants, and N-(m-trifluoromethylphenyl)phenoxazine 4,6-dicarboxylic acid also have binding affinity for TTR (Klabunde et al. 2000; McCammon et al. 2002). As diethylstilbestrol can cause estrogenic effects, the binding of the chemical to TTR could limit its access to target cells, thereby modulating their cellular actions. Polychlorinated biphenyls (PCBs) are industrial chemicals consisting of paired phenyl rings that have various degrees of chlorination. There are 209 possible PCB congeners, depending on the position and number of chlorine atoms. Although their production was banned in the mid-1970s, PCBs are still widespread and persist in the environment, and are concentrated in the food chain. Analysis of the competitive binding of PCBs and [125I]T4 to TTR demonstrates that laterally (3,30 ,5,50 -) substituted PCBs have a higher affinity for TTR than nonlateral (2,20 ,6,60 -) substituted PCBs (Richenbacker et al. 1986). In addition, the PCB congeners that have


163

a di-meta substitution in one or both rings, such as 3,30 ,5,50 -tetrachlorobiphenyl (TCB), have the highest binding activity among the PCBs (Chauhan et al. 2000). Hydroxylated PCBs are more potent competitors of [125I]T4 binding to TTR than their parent PCBs (Richenbacker et al. 1986; Lans et al. 1993). Of the hydroxylated PCBs, those that have the hydroxyl group substituted on the meta or para positions, and one or more adjacent chlorine substituents, have the highest competitive binding potency, with Kd’s ranging from 8 to 40 nM (Lans et al. 1993). The hydroxylated derivatives of polychlorinated dibenzo-p-dioxins and dibenzofurans, which have a chlorine substitution adjacent to the hydroxyl group, also have a relatively high binding potency for TTR (Lans et al. 1993). These binding properties were confirmed directly by the antibody capture HPLC method (Purkey et al. 2004). Although there is no direct evidence that supports the hypothesis that the binding of hydroxylated PCBs to TTR lowers total TH levels in plasma by displacing THs bound to TTR, there is a significant inverse relationship between PCB and TH levels in plasma from water birds, polar bears, and humans (Brouwer et al. 1998). Common seals fed on PCB-contaminated fish have significantly lower concentrations of total T4 and T3 and free T4 than those fed on control fish (Brouwer et al. 1989). Of the 65 agricultural and industrial chemicals from 12 chemical groups tested, 21 chemicals from 6 chemical groups, including chlorophenols, phenoxy acids, nitrophenols, chlorobenzenes, dichloro-diphenyl-trichloroethanes (dicofol), and others (bromoxynil), were the most potent competitors for [125I]T4 binding to human TTR in vitro (Van den Berg et al. 1991). In contrast, an in vivo study showed that 2,4dichlorophenoxybutyric acid, dinoseb, bromoxynil, and pentachlorophenol of the 21 chemicals were associated with a decrease in plasma levels of total T4 in rats 6 h after the chemicals were administered (Van den Berg et al. 1991). The competitive binding potency of these chemicals for TTR from various vertebrates differs because of variations in the binding specificity of TTR for TH and in the composition of plasma TH-binding proteins (Ishihara et al. 2003). Structure–activity relationships demonstrate the importance of the hydroxyl group and one or more adjacent halogen substituents for binding to TTR in the chlorinated and brominated derivatives of bisphenol A and nonylphenol (Yamauchi and Ishihara 2006); brominated flame retardants and their related compounds, such as polybrominated diphenyl ethers, pentabromophenol, and tetrabromobisphenol A (Meerts et al. 2000); and chlorinated phenols (Van den Berg 1990). This structural characteristic of these chemicals resembles the structure of THs. However, this structural characteristic does not appear to be essential for chemicals to bind to TTR given that most flavonoids, NSAIDs, and stilbenes have no halogen atoms.

10.3

Chemicals That Affect the Stability of the TTR Tetramer and the Formation of the TTR–RBP Complex

A recent intriguing finding is that ligand binding stabilizes the tetrameric structure of human TTR and prevents TTR-mediated amyloid fibril formation, which is closely linked to the human amyloid diseases, senile systemic amyloidosis, and

164


familial amyloid polyneuropathy (Raghu et al. 2004). Most of the compounds that have been found to bind to TTR inhibit TTR-mediated amyloid fibril formation (asterisks in Fig. 10.1), including T4 (Miroy et al. 1996; White and Kelly 2001); flavonoids such as genistein and daidzein (Green et al. 2005); NSAIDs such as flufenanic acid, diclofenac, and diflunisal (Klabunde et al. 2000; Purkey et al. 2001; McCammon et al. 2002; Miller et al. 2004); resveratrol (Klabunde et al. 2000; Purkey et al. 2001; McCammon et al. 2002; Morais-de-Sa´ et al. 2004); diethylstilbestrol (Morais-de-Sa´ et al. 2004); phenoxazine derivatives (Klabunde et al. 2000; McCammon et al. 2002); hydroxylated PCBs (McCammon et al. 2002; Purkey et al. 2004); triiodophenol (Miroy et al. 1996); and 2,4-dinitrophenol (Morais-de-Sa´ et al. 2006). The majority of the chemicals tested by McCammon et al. (2002) exhibit negative cooperativity in binding to TTR, whereas the others exhibit positive cooperativity (e.g., hydroxylated PCB) or noncooperative (e.g., derivatives of Nphenyl phenoxazine) binding modes. A combination of new technical approaches for structure-based drug design, screening, and inhibitory assays facilitated the discovery of a number of novel inhibitors of TTR-mediated amyloid fibril formation, such as derivatives of NSAIDs (Oza et al. 2002; Adamski-Werner et al. 2004; Almeida et al. 2004), bisaryloxime ethers (Johnson et al. 2005), dibenzofurans including dibenzofuran-4,6-dicarboxylic acid (Klabunde et al. 2000; Petrassi et al. 2005), carborane pharmacophores (Julius et al. 2007), and 2-arylbenzoxazoles (Johnson et al. 2008). The structural information of the chemical–TTR complexes will provide a rationale for chemotherapeutic approaches to prevent TTR-associated amyloid diseases. These chemotherapeutic approaches have recently been reviewed in detail (Raghu et al. 2004; see section on TTR Amyloid). When TTR binds RBP, TTR can also transport, indirectly, retinoids and their derivatives, which bind to RBP (Berni et al. 1993). RPB circulates as a 1:1 complex with TTR in blood at a concentration of 1 mM in humans. The TTR–RBP complex prevents filtration of the relatively small RBP molecule through kidney glomeruli (Zanotti and Berni 2004). Retinoid binding to RBP strengthens the TTR–RBP interaction (Fex et al. 1979). When apoRBP is incubated with TTR in vitro, the binding affinity of RBP for TTR was less than that when all-trans retinol or alltrans retinoic acid was added to the reaction mixture (Kd = 1.2 mM vs. 0.2 and 0.8 mM, respectively) (Malpeli et al. 1996). However, the binding affinity of RBP for TTR was decreased with the addition of all-trans retinyl methyl ether (Kd = 6 mM) (Malpeli et al. 1996). In contrast, the synthetic retinoids fenretinide and N-ethylretinamide abolished the binding affinity of RBP for TTR without the apparent loss of binding affinity of RBP for these ligands (Berni and Formelli 1992; Zanotti et al. 1993; Malpeli et al. 1996), indicating that the substitution of the hydroxyl group with an amide group on retinol affects the TTR–RBP interaction. Interestingly, piscine TTR and RBP do not form a complex in plasma (Shidoji and Muto 1977). In support of this finding, the amino acid sequences in piscine TTR and RBP differ from those in human TTR and RBP at positions critical for the interaction between RBP and TTR in higher vertebrates (Zanotti and Berni 2004). Another example demonstrating that a small ligand weakens the TTR-RBP interaction is a metabolite of 3,30 ,4,40 -TCB, but not TCB (Brouwer and Van den


165

Berg 1986; Brouwer et al. 1986; Brouwer et al. 1989). Within two days of exposing rats to TCB, plasma levels of RBP, retinol, and T4 in rats had decreased (Brouwer and Van den Berg 1986; Brouwer et al. 1986). A subsequent study identified 4-OH-3,30 ,40 ,5-TCB as the metabolite of TCB that bound to TTR (Brouwer et al. 1990). Given that a number of PCB congeners are enzymatically hydroxylated by members of the cytochrome P-450 family (Bergman et al. 1994), and that several hydroxylated PCBs bind TTR in vitro with high affinity (Rickenbacker et al. 1986; Lans et al. 1993; Cheek et al. 1999; Purkey et al. 2004), other hydroxylated PCBs may have similar effects to those observed for 4-OH-3,30 ,40 ,5-TCB. However, no reports have appeared to date indicating such an action. In contrast, a recent study demonstrated that the in vitro binding of 4,40 diOH-3,30 ,5,50 -TCB to TTR did not inhibit the association of TTR with holoRBP (MaCammon et al. 2002). The binding of holoRBP to TTR, as well as ligand binding to TTR, inhibits TTRmediated amyloid fibril formation (White and Kelly 2001). RBP is secreted from hepatocytes as the holoRBP–TTR complex (Bellovino et al. 1996). Therefore, plasma levels of the natural ligands for TTR and RBP, and of the TTR-RBP complex, would be critical factors for the inhibition of TTR-mediated amyloid fibril formation (White and Kelly 2001). For chemotherapeutical approaches to prevent TTR-mediated amyloid diseases successfully, it would be essential that the binding of chemicals to TTR neither disrupt the thyroid or retinoid system nor exert any adverse effects on humans.

10.4

Application of Competitive TTR Binding Assay to Detect Potential TH-Disrupting Chemicals

The competitive binding assay for TH binding to TTR has been applied to detect TH-disrupting chemicals in various environmental samples, such as airborne particulate matter extracts (Heussen et al. 1993), surface waters (Yamauchi et al. 2003; Murata and Yamauchi 2007), and sediments (Houtman et al. 2004). Extracts from samples of air collected outdoors and of cigarette smoke, which contained 6.6 and 0.23 ng pentachlorophenol per m3 of sample air, respectively, inhibited [125I]T4 binding to TTR in a dose-dependent manner but not [125I]T4 binding to TBG. Plasma levels of total T3 and T4, and free T4, decreased significantly within 6 h after rats were intraperitoneally injected with the extracts from a sample of air collected outdoors (Heussen et al. 1993). Utilizing a similar competitive assay, TH-displacing activity was also detected in effluent from, and downstream of, paper manufacturing plants in Japan (Yamauchi et al. 2003). Contaminants in water samples collected from various locations inhibited [125I]T3 binding to TTR in the following order: effluent>industrial drain>river month>harbor entrance (Fig. 10.2). This result indicated that the causative contaminants are released from the plants and are diluted downstream.

166


Fig. 10.2 Detection of potential thyroid hormone-disrupting chemicals in surface water downstream from paper manufacturing plants by TTR competition assay. Open columns, water samples used directly in this assay; shaded columns, contaminant samples extracted from the water samples with dichloromethane. Original data were from Yamauchi et al. (2003)

Although the assay is performed using water samples (open columns in Fig. 10.2) or samples of contaminants that have been extracted from the water samples using solid phase extraction (shaded columns in Fig. 10.2), both samples (at 0.8 times their original concentrations) have similar TH-displacing activity, suggesting that extraction and concentration of contaminants are not necessary at least for surface water samples (Yamauchi et al. 2003; Murata and Yamauchi 2007). There is no direct evidence indicating that the effluent investigated in the aforementioned studies disturbs the thyroid system of the native aquatic inhabitants. This is in contrast to the finding in Finland that juvenile female rainbow trout exposed to diluted pulp mill effluents had increased T4 levels and decreased T3 levels in their plasma (Mattsson et al. 2001). More recently, this competitive binding assay for TH binding to TTR was used to detect contaminants that had TH-displacing activity in sediments from the Rhine Meuse estuary in the Netherlands (Houtman et al. 2004). The Rhine Meuse estuary acts as a sedimentary basin for the river, and contaminants discharged upstream of the estuary tend to accumulate here. There is evidence that indicates that the endocrine system in aquatic wildlife is disturbed following exposure to contaminated sediments in the Rhine Meuse estuary (Vos et al. 2000). Of the samples of sediment collected from 15 locations in the Rhine Meuse estuary, half had significant TH-displacing


167

activity that was dose dependent (6–16 pmol T4-equivalents per gram dry weight). In addition to T4 competition binding activity, the authors investigated dioxin responsiveness and estrogen responsiveness, using chemical-activated luciferase gene expression assays, and genotoxicity and nonspecific toxicity in the samples of sediment. However, there were no location-dependent correlations between the T4 competition binding activity and the activities recorded using the other assays. Thus, the sediments potentially contain different endocrine-disrupting activities.

10.5

Perspectives

Chemicals that bind to TTR have the potential to affect TH homeostasis in plasma if they displace TH from TTR. In addition to those chemicals that directly interfere with the thyroid system, a hydroxylated PCB also affects retinoid homeostasis in plasma indirectly by decreasing the TTR–RBP association. Several lines of evidence indicate other physiological roles for TTR and RBP, which are likely to be affected by these chemicals: (1) Chemicals with binding affinity for TTR are also potent inhibitors of TTRmediated amyloid fibril formation (Fig. 10.1). This finding may aid the research of chemotherapeutic approaches that prevent the TTR-associated amyloid diseases, familial amyloid polyneuropathy, and senile systemic amyloidosis. More than 99% of TTR in plasma from eutherians, including that from humans, is not bound by THs. Does this mean that more than 99% of TTR in plasma is apoTTR? It is not known whether endogenous ligands other than THs that bind to TTR, such as all-trans retinoic acid (Kd = 107 M; Smith et al. 1994), inhibit TTR-mediated amyloid fibril formation. (2) Novel roles of TTR within specific cells or cerebrospinal fluid have been suggested in two reports. First, TTR has been found to interact with metalothionein 2 in human liver (Kd = 245 nM), and the complex of the two proteins is found in rat choroid plexus and kidney (Gonçalves et al. 2008). Second, in pancreatic b-cells, TTR promotes glucose-induced increases in cytoplasmic free Ca2+ concentration and protects against b-cell apoptosis (Refai et al. 2005). (3) A positive relationship between the level of RBP in plasma and insulin resistance/glucose intolerance in mice and humans who are obese and have type 2 diabetes has been discovered. The synthesis of RBP in, and secretion of RBP from, adipose tissue, but not the liver, resulted in increased levels of RBP in plasma (Yang et al. 2005). This result indicates that RBP may act as an adipocyte-derived signal. Whether these other physiological roles of TTR and RBP are affected by natural and synthetic ligands for TTR or RBP remains to be clarified.

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References Adamski-Werner SL, Palaninathan SK, Sacchettini JC, Kelly JW (2004) Diflunisal analogues stabilize the native state of transthyretin. Potent inhibition of amyloidogenesis. J Med Chem 47:355–374 Almeida MR, Macedo B, Cardoso I, Alves I, Valencia G, Arsequell G, Planas A, Saraiva MJ (2004) Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative. Biochem J 381:351–356 Auf’mkolk M, Koehrle J, Hesch RD, Cody V (1986) Inhibition of rat liver iodothyronine deiodinase. Interaction of aurones with the iodothyronine ligand-binding site. J Biol Chem 261:11623–11630 Bellovino D, Morimoto T, Tosetti F, Gaetani S (1996) Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells. Exp Cell Res 222:77–83 Bergman A, Klasson-Wehler E, Kuroki H (1994) Selective retention of hydroxylated PCB metabolites in blood. Environ Health Perspect 102:464–469 Berni R, Formelli F (1992) In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences. FEBS Lett 308:43–45 Berni R, Zanotti G, Sartori G, Monaco HL (1993) Plasma retinol-binding protein and its interaction with transthyretin. In: Livrea MA, Packer L (eds) Retinoids, Progress in Research and Clinical Applications, Marcel Dekker, New York, pp 91–102 Brouwer A, van den Berg KJ (1986) Binding of a metabolite of 3,4,30 ,40 -tetrachlorobiphenyl to transthyretin reduces serum vitamin A transport by inhibiting the formation of the protein complex carrying both retinol and thyroxin. Toxicol Appl Pharmacol 85:301–312 Brouwer A, Reijnders PJH, Koeman JH (1989) Polychlorinated biphenyl (PCB)-contaminated fish induces vitamin A and thyroid hormone deficiency in the common seal (Phoca vitulina). Aquat Toxicol 15:99–105 Brouwer A, Klasson-Wehler E, Bokdam M, Morse DC, Traag WA (1990) Competitive inhibition of thyroxin binding to transthyretin by monohy-droxy metabolites of 3,4,30 ,40 -tetrachlorobiphenyl. Chemosphere 20:1257–1262 ˚ , Visser TJ Brouwer A, Morse DC, Lans MC, Schuur AG, Murk AJ, Klasson-Wehler E, Bergman A (1998) Interactions of persistent environmental organohalogens with thyroid hormone system: mechanisms and possible consequences for animal and human health. Toxicol Ind Health 14:59–84 Brouwer A, van den Berg KJ, Blaner WS, Goodman DS (1986) Transthyretin (prealbumin) binding of PCBS, a model for the mechanism of interference with vitamin A and thyroid hormone metabolism. Chemosphere 15:1699–1706 Brucker-Davis F (1998) Effects of environmental synthetic chemicals on thyroid function. Thyroid 8:827–856 Chauhan KR, Kodavanti PR, McKinney JD (2000) Assessing the role of ortho-substitution on polychlorinated biphenyl binding to transthyretin, a thyroxine transport protein. Toxicol Appl Pharmacol 162:10–21 Cheek AO, Kow K, Chen J, McLachlan JA (1999) Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107:273–278 Chopra IJ (1996) Nature, source, and relative biologic significance of circulating thyroid hormones. In: Braverman LE, Utiger RD (eds) Weiner and Ingbar’s The Thyroid, 7th edn. Lippincott-Raven, Philadelphia, pp 111–124 ˚ resolution of human Ciszak E, Cody V, Luft JR (1992) Crystal structure determination at 2.3-A transthyretin-30 ,50 -dibromo-20 ,4,40 ,6-tetrahydroxyaurone complex. Proc Natl Acad Sci USA 89:6644–6648


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DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R (1999) Screening methods for thyroid hormone disruptors. Environ Health Perspect 107:407–414 Fex G, Albertsson PA, Hansson B (1979) Interaction between prealbumin and retinol-binding protein studied by affinity chromatography, gel filtration and two-phase partition. Eur J Biochem 99:353–360 Gonçalves I, Quintela T, Baltazar G, Almeida MR, Saraiva MJ, Santos CR (2008) Transthyretin interacts with metallothionein 2. Biochemistry 47:2244–2251 Green NS, Foss TR, Kelly JW (2005) Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc Natl Acad Sci USA 102:14545–14550 Hamann I, Seidlova-Wuttke D, Wuttke W, Ko¨hrle J (2006) Effects of isoflavonoids and other plant-derived compounds on the hypothalamus–pituitary–thyroid hormone axis. Maturitas 55S:S14–S25 Heussen GA, Schefferlie GJ, Talsma MJ, van Til H, Dohmen MJ, Brouwer A, Alink GM (1993) Effects on thyroid hormone metabolism and depletion of lung vitamin A in rats by airborne particulate matter. J Toxicol Environ Health 38:419–434 Houtman CJ, Cenijn PH, Hamers T, Lamoree MH, Legler J, Murk AJ, Brouwer A (2004) Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption. Environ Toxicol Chem 23:32–40 Ishihara A, Sawatsubashi S, Yamauchi K (2003) Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors. Mol Cell Endocrinol 199:105–117 Johnson SM, Connelly S, Wilson IA, Kelly JW (2008) Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J Med Chem 51:260–270 Johnson SM, Petrassi HM, Palaninathan SK, Mohamedmohaideen NN, Purkey HE, Nichols C, Chiang KP, Walkup T, Sacchettini JC, Sharpless KB, Kelly JW (2005) Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation. J Med Chem 48:1576–1587 Julius RL, Farha OK, Chiang J, Perry LJ, Hawthorne MF (2007) Synthesis and evaluation of transthyretin amyloidosis inhibitors containing carborane pharmacophores. Proc Natl Acad Sci USA 104:4808–4813 Klabunde T, Petrassi HM, Oza VB, Raman P, Kelly JW, Sacchettini JC (2000) Rational design of potent human transthyretin amyloid disease inhibitors. Nat Struct Biol 7:312–321 Ko¨hrle J, Fang SL, Yang Y, Irmscher K, Hesch RD, Pino S, Alex S, Braverman LE (1989) Rapid effects of the flavonoid EMD 21388 on serum thyroid hormone binding and thyrotropin regulation in the rat. Endocrinology 125:532–537 Ko¨hrle J, Spanka M, Irmscher K, Hesch RD (1988) Flavonoid effects on transport, metabolism and action of thyroid hormones. In: Cody V, Middleton E, Harborne JB, Beretz A (eds), Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medical Properties, Alan R Liss, New York, pp 323–340 Lans MC, Klasson-Wehler E, Willemsen M, Meussen E, Safe S, Brouwer A (1993) Structuredependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem Biol Interact 88:7–21 Lueprasitsakul W, Alex S, Fang SL, Pino S, Irmscher K, Ko¨hrle J, Braverman LE (1990) Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat. Endocrinology 126:2890–2895 Malpeli G, Folli C, Berni R (1996) Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. Biochim Biophys Acta 1294:48–54 Mattsson K, Lehtinen KJ, Tana J, Ha¨rdig J, Kukkonen J, Nakari T, Engstro¨m C (2001) Effects of pulp mill effluents and restricted diet on growth and physiology of rainbow trout (Oncorhynchus mykiss). Ecotoxicol Environ Saf 49:144–154

170


McCammon MG, Scott DJ, Keetch CA, Greene LH, Purkey HE, Petrassi HM, Kelly JW, Robinson CV (2002) Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry. Structure 10:851–863 McKinney JD, Chae K, Oatley SJ, Blake CC (1985) Molecular interactions of toxic chlorinated dibenzo-p-dioxins and dibenzofurans with thyroxine binding prealbumin. J Med Chem 28:375–381 Meerts IATM, van Zanden JJ, Luijks EAC, van Leeuwen-Bol I, Marsh G, Jakobsson E, Bergman ˚ , Brouwer A (2000) Potent competitive interactions of some brominated flame retardants and A related compounds with human transthyretin in vitro. Toxicol Sci 56:95–104 Mendel CM, Cavalieri RR, Ko¨hrle J (1992) Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin. Endocrinology 130:1525–1532 Miller SR, Sekijima Y, Kelly JW (2004) Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants. Lab Invest 84:545–552 Miroy GJ, Lai Z, Lashuel HA, Peterson SA, Strang C, Kelly JW (1996) Inhibiting transthyretin amyloid fibril formation via protein stabilization. Proc Natl Acad Sci USA 93:15051–15056 Morais-de-Sa´ E, Neto-Silva RM, Pereira PJ, Saraiva MJ, Damas AM (2006) The binding of 2,4dinitrophenol to wild-type and amyloidogenic transthyretin. Acta Crystallogr D Biol Crystallogr 62:512–519 Morais-de-Sa´ E, Pereira PJ, Saraiva MJ, Damas AM (2004) The crystal structure of transthyretin in complex with diethylstilbestrol: a promising template for the design of amyloid inhibitors. J Biol Chem 279:53483–53490 Munro SL, Lim CF, Hall JG, Barlow JW, Craik DJ, Topliss DJ, Stockigt JR (1989) Drug competition for thyroxine binding to transthyretin (prealbumin): comparison with effects on thyroxine-binding globulin. J Clin Endocrinol Metab 68:1141–1147 Murata T, Yamauchi K (2007) 3,30 ,5-Triiodo-l-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays. Toxicol Appl Pharmacol 226:309–317 Oza VB, Smith C, Raman P, Koepf EK, Lashuel HA, Petrassi HM, Chiang KP, Powers ET, Sachettinni J, Kelly JW (2002) Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors. J Med Chem 45:321–332 Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95:12956–12960 Petrassi HM, Johnson SM, Purkey HE, Chiang KP, Walkup T, Jiang X, Powers ET, Kelly JW (2005) Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J Am Chem Soc 127:6662–6671 Purkey HE, Dorrell MI, Kelly JW (2001) Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. Proc Natl Acad Sci USA 98:5566–5571 Purkey HE, Palaninathan SK, Kent KC, Smith C, Safe SH, Sacchettini JC, Kelly JW (2004) Hydroxylated polychlorinated biphenyls selectively bind transthyretin in blood and inhibit amyloidogenesis: rationalizing rodent PCB toxicity. Chem Biol 11:1719–1728 Raghu P, Reddy GB, Sivakumar B (2004) Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization. Arch Biochem Biophys 400:43–47 Refai E, Dekki N, Yang SN, Imreh G, Cabrera O, Yu L, Yang G, Norgren S, Ro¨ssner SM, Inverardi L, Ricordi C, Olivecrona G, Andersson M, Jo¨rnvall H, Berggren PO, Juntti-Berggren L (2005) Transthyretin constitutes a functional component in pancreatic b-cell stimulussecretion coupling. Proc Natl Acad Sci USA 102:17020–17025 Rickenbacher U, McKinney JD, Oatley SJ, Blake CC (1986) Structurally specific binding of halogenated biphenyls to thyroxine transport protein. J Med Chem 29:641–648


171

Robbins J (1996) Thyroid hormone transport proteins and the physiology of hormone binding. In: Braverman LE, Utiger RD (eds) Weiner and Ingbar’s The Thyroid, 7th edn. Lippincott-Raven, Philadelphia, pp 96–110 Schmutzler C, Gotthardt I, Hofmann PJ, Radovic B, Kovacs G, Stemmler L, Nobis I, Bacinski A, Mentrup B, Ambrugger P, Gru¨ters A, Malendowicz LK, Christoffel J, Jarry H, Seidlova`Wuttke D, Wuttke W, Ko¨hrle J (2007) Endocrine disruptors and the thyroid gland-a combined in vitro and in vivo analysis of potential new biomarkers. Environ Health Perspect 115 (Suppl 1):77–83 Schro¨der-van der Elst JP, Smit JW, Romijn HA, van der Heide D (2003) Dietary flavonoids and iodine metabolism. Biofactors 19:171–176 Schro¨der-van der Elst JP, van der Heide D, Ko¨hrle J (1991) In vivo effects of flavonoid EMD 21388 on thyroid hormone secretion and metabolism in rats. Am J Physiol 261:E227–E232 Schro¨der-van der Elst JP, van der Heide D, Rokos H, Ko¨hrle J, Morreale de Escobar G (1997) Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat. Endocrinology 138:79–84 Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B Biochem Mol Biol 116:137–160 Shidoji Y, Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J Lipid Res 18:679–691 Smith TJ, Davis FB, Deziel MR, Davis PJ, Ramsden DB, Schoenl M (1994) Retinoic acid inhibition of thyroxine binding to human transthyretin. Biochim Biophys Acta 1199:76–80 Van den Berg KJ (1990) Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin. Chem Biol Interact 76:63–75 Van den Berg KJ, van Raaij JA, Bragt PC, Notten WR (1991) Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Arch Toxicol 65:15–19 van der Heide D, Kastelijn J, Schro¨der-van der Elst JP (2003) Flavonoids and thyroid disease. Biofactors 19:113–119 Vos JG, Dybing E, Greim HA, Ladefoged O, Lambre´ C, Tarazona JV, Brandt I, Vethaak AD (2000) Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol 30:71–133 Wang R, Nelson JC, Wilcox RB (1999) Salsalate and salicylate binding to and their displacement of thyroxine from thyroxine-binding globulin, transthyretin, and albumin. Thyroid 9:359–364 White JT, Kelly JW (2001) Support for the multigenic hypothesis of amyloidosis: the binding stoichiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloidogenicity in vitro. Proc Natl Acad Sci USA 98:13019–13024 Yamauchi K, Ishihara A (2006) Thyroid system-disrupting chemicals: interference with thyroid hormone binding to plasma proteins and the cellular thyroid hormone signaling pathway. Rev Environ Health 21:229–251 Yamauchi K, Ishihara A, Fukazawa H, Terao Y (2003) Competitive interactions of chlorinated phenol compounds with 3,30 ,5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water. Toxicol Appl Pharmacol 187: 110–117 Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB (2005) Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436:356–362 Zanotti G, Malpeli G, Berni R (1993) The interaction of N-ethyl retinamide with plasma retinol˚ resolubinding protein (RBP) and the crystal structure of the retinoid-RBP complex at 1.9-A tion. J Biol Chem 268:24873–24879 Zanotti G, Berni R (2004) Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 69:271–295

Chapter 11

Genetics: Clinical Implications of TTR Amyloidosis Merrill D Benson

Abstract Transthyretin (TTR) is a fascinating protein. Its transport properties for thyroxin and Vitamin A, its classic b-barrel structure, stable homo-tetrameric structure in solution and its phylogenetic evolution have all been of great interest to scientific study. Even the lack of pathology in its absence, in TTR gene knockout mice, has been of great interest. Its entire notoriety in the area of human pathology, however, is that it readily forms amyloid fibrils, usually due to single amino acid mutations in its primary structure but in some cases even when there is no gene or amino acid structural alteration. TTR amyloidosis is strictly a human disease and more than 100 TTR mutations having been associated with the disease. A few of the mutations are found in large kindreds worldwide while many mutations have been found in only single individuals or single families. In depth analysis of this autosomal dominant disease with an appreciation of the variations in phenotypes suggests clues which may help rationalize basic physical and chemical studies of TTR with biochemical processes which must be involved in the generation of clinical disease. This chapter discusses the clinical aspects of the disease and tries to use these observations to contemplate the intricacies of pathogenesis. Keywords Amyloid, Amyloidosis, Cardiomyopthy, Neuropathy

11.1

Introduction

Transthyretin (TTR) amyloidosis is the most common type of hereditary amyloidosis. It may also occur as a sporadic disease of the elderly as senile systemic amyloidosis (Benson 2003; Cornwell et al. 1981; Pitka¨nen et al. 1984). The hereditary M.D. Benson (*) Indiana University School of Medicine, 635 Barnhill Drive, MS-128, Indianapolis, IN 462025126 USA e-mail: [email protected]


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form is associated with mutations in TTR of which over a 100 have been identified (Connors et al. 2003). In senile systemic amyloidosis (SSA), also known as senile cardiac amyloidosis (SCA), amyloid deposits are derived from normal TTR and, thus far, no genetic predisposition has been identified (Westermark et al. 1990). This occurrence suggests that the highly b-sheet structured TTR protein is innately prone to form b-pleated sheet fibrils and alterations in its primary structure are likely to encourage this fate. However, the story is not that simple. Variations in the age of the disease onset, penetrance, organ involvement by amyloid deposits, and male versus female disease presentation indicate that there are factors, yet unknown, that must be involved in the disease process that is called TTR amyloidosis. Analysis of the data on TTR amyloidosis that have been accumulated since Andrade’s description of ‘‘A peculiar form of peripheral neuropathy. . .,’’ later called Familial Amyloidotic Polyneuropathy (FAP), requires weighing the importance of each observation of the disease and integration of these observations into an overall picture which may aid in understanding the pathogenesis of the disease (Andrade 1952). The key word is ‘‘observation’’ for we have little to rely on in the realm of experimentation. TTR amyloidosis is a disease of humans. No other species has been found to have a similar disease, and attempts to create an animal model of the disease have limited success in revealing the pathogenic mechanisms that are involved in the transition of a soluble plasma protein to the tissue deposits of inert b-pleated fibrils which we call amyloid. Acknowledging that we must start with predominantly descriptive research we will try to piece together the puzzle of clinical TTR amyloidosis with the hope that it will eventually be validated by experimental reasoning and research. To start with, we will consider the clinical presentation of TTR amyloidosis and the ‘‘variations on a theme’’ that may add to the overall picture. This requires a composite view of the disease plus a consideration of the clinical disease in the larger kindreds with specific TTR mutations. Then it is important to ask what might be learned by the analysis of clinical variations which are seen in families or single individuals where rare, uncommon mutations have been found. If we are able to identify from our clinical observations those factors which are most pertinent to the evolution of the clinical disease, we can attempt to reconcile them with the sparse experimental data we have on TTR expression and metabolism to formulate hypotheses on pathogenesis.

11.2

Clinical Picture of TTR Amyloidosis

The original description of FAP by Andrade emphasized the peripheral neuropathy which – while a major feature of most of the syndromes associated with TTR mutations – is now known to be only one part of the picture (Andrade 1952). Andrade also noted extensive renal amyloid deposition in some cases, but we now know that this is relatively uncommon, especially when compared with the incidence of amyloid cardiomyopathy (Dubrey et al. 1997).

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The typical clinical presentation in Portuguese patients is with loss of small fiber function in the lower extremities. Loss of temperature perception followed by loss of touch and pain modality is the rule with progression in a distal to proximal pattern (Fig. 11.1) (Benson and Kincaid 2007). Parasthesiae and painful dysasthesias may occur mainly in the lower extremities. Motor impairment occurs with foot drop and muscle atrophy. The need for walking aids and, finally, the use of a wheelchair is not uncommon. At the same time bowel dysfunction with constipation but more frequently persistent diarrhea and early satiety results in varying degrees of weight loss and malnutrition. It may be difficult to judge if the degree of physical impairment is due to neuropathy or to generalized inanition. Sexual impotence tends to occur in males and occasionally may be the first manifestation of the clinical disease. Features noted by Andrade included trophic skin changes and nonhealing ulcers of the feet, hand muscle atrophy which was probably the result of median nerve compression (carpal tunnel syndrome) and, rarely, irregular pupil conformation (Fig. 11.2; Lessell et al. 1975). In Portugal, the age of onset of the clinical disease was originally noted as the late twenties to early thirties and this has remained consistent over the past 50 years. Some individuals in Portugal have been found to have late onset of the clinical disease (after age 60) but this is a small percentage of the whole (Sequeiros and Saraiva 1987). Life expectancy was originally reported as less than ten years from disease onset and modern medicine has not changed this figure dramatically. Early descriptions of TTR amyloidosis in Japanese patients were similar to those of the Portuguese disease with an emphasis on neuropathy and muscle wasting (Araki et al. 1968). Disease onset was similar (20–30 years) and with progression of peripheral neuropathy, diarrhea, and generalized debilitation. More recently we have realized the importance of amyloid cardiomyopathy with congestive heart

Fig. 11.1 Distribution of typical nerve function loss in TTR amyloidosis

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Fig. 11.2 Scalloped pupil deformity due to amyloid involvement of ciliary nerves in a patient with TTR Val30Met

failure and arrhythmias in overall survival. It is generally considered that the larger kindreds with FAP in Japan represent introduction of the TTR Val30Met mutation by Portuguese colonization. This is supported by DNA studies showing a common TTR allele haplotype for Portuguese and most Japanese patients (Yoshioka et al. 1980). TTR amyloidosis was also recognized as a hereditary disease at an early date in Northern Sweden. As early as 1968 Andersen reported amyloidosis in association with vitreous opacities and in 1970 emphasized the hereditary nature of this peripheral neuropathy (Andersson 1970, 1968). While we now know that the Swedish patients have the same TTR mutation (Val30Met) as the Portuguese and the Japanese, the clinical disease is quite different. Age of disease onset is in the late fifties and survival after diagnosis is often 10–20 years (Holmgren et al. 1988). Cardiomyopathy and vitreous opacities are common and peripheral neuropathy, while a significant part of the syndrome, tends to be less rapid in progression and, therefore, not as debilitating (Sandgren 1995). Diarrhea, however, can be problematic with resultant weight loss and physical disability (Shur et al. 1994).

11.3

Larger Kindreds

Over 100 TTR mutations have been identified which are associated with amyloidosis. Most mutations have been found in only one family and some in only a single individual. While many of the mutations have not been linked to an inheritance

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pattern, only two have been shown to represent new mutation events (Murakami et al.1992; Yazaki et al. 2002b). Many of these uncommon mutations have been found in individuals who presented with vitreous opacities or cardiomyopathy which have brought the patients to medical attention (Yazaki et al. 2002a). On the other hand, often a family has historically been afflicted with peripheral neuropathy or heart disease and several generations have passed before the causative amyloid has been recognized (Benson and Cohen 1977). This has been the case with some of the TTR mutations that are well represented by extensive kindreds. Included in this group are: Val30Met, Leu58His, Thr60Ala, Ser77Tyr, Ile84Ser, and Val122Ile. To appreciate the phenotypes of clinical disease special consideration of the more common TTR mutations is in order to see variations between kindreds and within kindreds and families (Table 11.1).

11.4

Clinical Aspects of Common TTR Mutations

Val30Met amyloidosis characteristically presents as sensorimotor polyneuropathy with varying degrees of cardiomyopathy. Bowel dysfunction is common with constipation but more often persistent diarrhea is a major manifestation of the disease. Vitreous opacities may or may not occur and this varies within families. Renal amyloid may be present but is less common than cardiac amyloid. The wide variation in mean age of the onset of the disease that is seen for Portuguese and Japanese patients (25–40 years) compared with Swedish patients (55–60 years) tells us that there must be factors, most likely related to metabolism and aging, that modify the disease. The high incidence of vitreous amyloid in older Swedish patients is another observation that suggests age related modifiers (Sandgren 1995).

Table 11.1 Clinical features of the most frequent TTR amyloidoses Mutation Typical age Neuropathy Cardiac Intestinal Eye of onset/ 0–4+ 0–4+ 0–4+ vitreous years yes = +/ no = Val30Met

Leu58His Thr60Ala Ser77Tyr Ile84Ser Val122Ile

30–35 (Portugal, Japan) 55–60 (Sweden) 50–60 60–65 50–60 45–55 60–65

Leptomeninges yes = +/ no =

Renal yes = +/ no =

3–4

2–3

2–4

Rare

2–3 1–2 3–4 2–3 1–2

3–4 4 3–4 4 4

– 0–4 2–3 2–3 1–2

– – – + –

– – – – –

– – – –

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In addition, DNA studies have identified at least three haplotypes that segregate with the Val30Met mutation (Yoshioka et al. 1980). Portuguese, Swedish, and most Japanese patients share TTR haplotype I. Many English patients have TTR haplotype III and show later onset of disease, less extensive neuropathy and more prominent cardiomyopathy (Kincaid et al. 1989). Haplotype II was described only in the original report of this genetic analysis. Leu58His amyloidosis was described in 1969 but the TTR mutation was not identified until 1989 (Mahloudji et al. 1969; Nichols et al. 1991). The disease presented in Americans of German heritage and the mutation has subsequently been found in families in the Rhine Valley of Germany (Goebel et al. 1997). The clinical phenotype is notable for the early onset of the carpal tunnel syndrome which is followed much later by polyneuropathy and cardiomyopathy. Bowel dysfunction is less common and vitreous opacities have not been reported. Thr60Ala was discovered in members of a large kindred in the Appalachian region of the United States (Wallace et al. 1986; Benson et al. 1987). The clinical picture is characterized by cardiomyopathy presenting around age 60 but older onset and decreased penetrance has obscured the genetic nature of the disease in some families. Polyneuropathy tends to be slowly progressive over 10–20 years but bowel dysfunction with diarrhea can be very debilitating. Vitreous amyloid has not been found with this mutation. Ser77Tyr amyloidosis was discovered in a Midwest kindred but has subsequently been found in other American and many European families (Wallace et al. 1988; Libbey et al. 1984; Satier et al. 1990; Zhao et al. 1994). Both cardiac and renal amyloid may occur in addition to the characteristic polyneuropathy starting around age 50 years. Vitreous opacities are not a part of this syndrome. Ile84Ser amyloidosis was first noted in an Indiana family because of vitreous amyloidosis (Falls et al. 1955). Essentially all affected individuals have eye involvement and this often occurs (age 40–50) before systemic manifestations (Rukavina et al. 1956). Carpal tunnel syndrome occurs early as seen with Leu58His. Death is usually from cardiomyopathy. A feature of this disease is the essentially 100% penetrance of the trait. The Indiana kindred is of Swiss origin and affected individuals in Switzerland have been identified but not been the subject of scientific publication (Dwulet and Benson 1986). A family in Hungary carrying the Ile84Ser mutation, with the same clinical phenotype has the same TTR haplotype but has not been linked by genealogy (Zo´lyomi et al. 1998). Val122Ile amyloidosis is mainly a disease of African Americans. The disease allele most likely originated in the West Coast of Africa (Sierra Leone, Gold Coast, etc.) but was first identified in African Americans with onset of cardiomyopathy after age of 60 (Gorevic et al. 1989; Nichols et al. 1991). Approximately 3–4% of African Americans carry this trait but the late age of onset for the clinical disease and the predominance of cardiomyopathy rather than neuropathy has obscured the true incidence of the disease (Jacobson et al. 1997; Yamashita et al. 2005). The degree of penetrance is still to be ascertained.

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179

Clinical Syndromes of FAP

11.5.1 Neuropathy Two forms of neuropathy are common in TTR amyloidosis: 1) Carpal tunnel syndrome – a compression neuropathy at the wrist, and; 2) Polyneuropathy affecting the longest fibers first (Benson and Kincaid 2007). The carpal tunnel syndrome may occur as the first sign of TTR amyloidosis or it may occur later, after bowel, cardiac, or peripheral nerve symptoms are present. Clinically it causes numbness and tingling in the distribution of the median nerve which is compressed in the carpal tunnel. It often disrupts sleep and patients report waking to shake their hands to relieve symptoms. Later it may cause sensory loss, atrophy of the thenar muscles with loss of hand grip and dexterity. Nerve conduction studies of the median nerve typically show delayed latency across the wrist. Nerve conduction may improve after surgical decompression of the median nerve but usually remains in the abnormal range. The peripheral neuropathy of TTR amyloidosis is axonal. Nerve conduction velocities are relatively maintained but response amplitude is decreased. The polyneuropathy is progressive with loss of response in longer sensory nerves (e.g., sural) occurring early. Autonomic neuropathy is often a significant aspect of TTR amyloidosis. Impotence is common in males and bladder retention may occur. Rectal sphincter dysfunction can be very debilitating when coupled with persistent diarrhea. Orthostatic hypotension is common and, when present along with restrictive cardiomyopathy, may severely limit physical activity. Autonomic neuropathy may be implicated in delayed gastric emptying but much of the intestinal dysfunction may be related to amyloid disruption of intrinsic bowel motility.

11.5.2 Cardiomyopathy A majority of the TTR mutations cause amyloidosis with cardiac involvement. This is also the case with SSA (SCA) in which cardiac amyloid deposition is the most common and usually the only clinically significant feature. The cardiomyopathy produces restrictive hemodynamics due to poor compliance of the ventricular walls. Left ventricular filling is impeded and stroke volume is lowered. The result is decreased cardiac output, rise in pressures of the pulmonary bed and subsequent features of congestive heart failure with generalized weakness, fatigue and fluid retention. Decreased cardiac output may commonly lead to hypotension and orthostatic symptoms which limit physical activity. In terminal stages, cardiac function may fall below that needed for tissue perfusion, especially the kidneys, and death may be the result of renal failure. Left atrial enlargement, the result of left ventricular diastolic dysfunction, often leads to atrial

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fibrillation which impairs the left ventricular filling with resultant further reduction in cardiac output. Amyloid deposition also may impair the cardiac conduction leading to AV block, atrial arrhythmias or sinus exit block. Ventricular tachycardia leading to ventricular fibrillation may be a terminal event.

11.5.3 Gastroenteropathy Bowel motility is often a major problem for patients with TTR amyloidosis. Clinically, the gastrointestinal (GI) problems associated with TTR amyloidosis occur in two anatomic areas: the upper GI track and the lower GI track. Upper GI dysfunction is mainly a result of gastroparesis. The stomach does not empty in the appropriate fashion after ingestion of a meal and the patient reports early satiety. Nausea and vomiting are not significant problems. The patient just feels ‘‘full’’ after taking a small portion of nourishment. The problem is one of decreased gastric motility which may result from a combination of factors. Autonomic neuropathy affecting the vagus nerve may lead to gastric motility deficit. In addition, infiltration of the submucosa and muscular layers of the stomach may lead to loss of interstitial cells of Cajal with a decline in intrinsic gastric motility. Lower bowel dysfunction may be a major manifestation of TTR amyloidosis and most frequently is expressed as chronic and unremitting diarrhea. A decrease in transit time of the liquefied intestinal contents seems to be provoked by any attempt at oral intake. The rapid gastrointestinal transit time, coupled with the psychological impact of eating followed by diarrhea, probably are the major factors in poor nutritional status and progressive body weight loss. Neurologically the lower intestinal dysfunction is probably related to the loss of neural inhibitory signals in the lower GI track. Normally, motility of the colon is inhibited by the autonomic nervous system and this allows increased time for reabsorbtion of water resulting in the formation of stools with a normal soft consistency. This process allows for storage in the rectosigmoid part of the colon and with normal function of the intrinsic bowel mechanisms along with higher Central Nervous System control allows for normal defecation.

11.5.4 Leptomeningeal Amyloidosis Leptomeningeal amyloidosis is a rare manifestation of TTR amyloidosis. It has been associated with 12 different mutations of TTR, and with these mutations it may be the most clinically significant and life threatening aspect of the disease. TTR mutations which have been associated with this phenotype range from the Val30Met mutation where leptomeningeal involvement is rarely of clinical significance to other mutations including Val30Gly and Thr114Cys, where most individuals

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in the kindreds have this manifestation (Goren et al. 1980; Petersen et al. 1998; Ueno et al. 1990). Clinically, leptomeningeal amyloidosis is characterized by repeated intra-cerebral hemorrhages which may manifest as seizures or obtundation. Occasionally episodes of obtundation without radiologic evidence of cerebral hemorrhage or infarction may occur. Some affected individuals develop hydrocephalus with increased cerebral spinal fluid (CSF) pressure and relatively high CSF protein levels. Leptomeningeal amyloidosis may or may not be associated with vitreous opacities but when this occurs, such as in the Val30Gly kindred, it is typically termed oculoleptomeningeal amyloidosis. This is a frequent finding with the Thr114Cys patients. Vitreous opacities have been described for approximately 25% of the TTR mutations and may or may not be associated with leptomeningeal manifestations. Pathologically dense amyloid deposition is found in cerebral vessels including those perforating the cortex. The leptomeninges are thickened by deposition of amyloid and this can be appreciated by MRI (Fig. 11.3) (Dowell et al. 2007). Leptomeningeal involvement extends down the spinal column and can give symptoms related to spinal cord or spinal nerve compression. Death is very often due to massive cerebral hemorrhage. Many of the TTR mutations associated with leptomeningeal amyloidosis have been described in only single families, but as with the larger kindreds with this phenotype, affected individuals tend to be relatively young (i.e., fourth or fifth decade of life). Survival is governed by the degree of leptomeningeal involvement. Most patients have little, if any, systemic deposition of amyloid. It is generally considered that leptomeningeal amyloid is the result of TTR produced by the choroid plexus and is not of hepatic origin as is systemically deposited amyloid. In favor of this hypothesis is the fact that in individuals heterozygous for a TTR mutation, the leptomeningeal amyloid is, for all practical purposes, the product of only the variant protein. This is unlike systemic amyloid isolated from the heart or the kidney where typically 65–70% of the amyloid is from a variant TTR and the remainder is from normal TTR. Similar pathogenic mechanisms may be operative in the eye where typically 85–90% of the vitreous amyloid fibrils are derived from a variant TTR (Liepnieks et al. 2006).

Fig. 11.3 Brain MRI of a patient with TTR Val30Gly amyloidosis. (a) Sagital T1 post gadolinium view. Arrows indicate thickened leptomeninges. (b) Sagital view of cervical spine. Arrow indicates leptomeningeal thickening extending down the spinal column

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11.6


Pathogenesis of TTR Amyloidosis From a Clinical Aspect

The pathogenesis of TTR amyloidosis has been the subject of considerable experimental study. Since the predisposition to TTR amyloidosis is associated with inheritance of mutations in the TTR protein, it is logical that alterations in protein structure should be a determining factor in the development of amyloid fibril formation. Tertiary structures of a number of amyloid associated TTR variant proteins have been determined by X-ray crystallography using either native variant plasma TTR or variant TTR produced by recombinant means (Hamilton and Benson 2001). These studies have shown definite alterations in tertiary structure; however, no unifying structural variation has been identified that would aid in predicting amyloid fibril formation. Thyroxin binding by a number of amyloid associated TTR variants has been studied showing considerable variation in hormone affinity (Rosen et al. 1993). This finding has no obvious correlation with the amyloid forming potential of TTR variants that were studied. Subsequent studies have shown that thyroxin binding to TTR thermodynamically stabilizes the tetramer in vitro (Peterson et al. 1998). This is the basis for the hypothesis that tetramer stability is an important factor in the formation of amyloid fibrils. Several small organic molecules have been identified which have high affinity for the TTR thyroxin binding pocket and, thereby, cause increased thermodynamic stability of the TTR tetramer (Baures et al. 1999; Hammarstro¨m et al. 2003). Considerable data have been generated from studying the primary structure of TTR peptides isolated from amyloid deposits. A most consistent finding has been the fragmentation of TTR with cleavage at or around amino residue 49; although some amount of full length TTR protein is always present in the tissue extracts (Liepnieks and Benson 2007). Studies in Westermark’s lab have shown experimentally that the carboxyl terminal portion of TTR is more prone to form fibril structures in vitro (Gustavsson et al. 1994). Several studies have shown that subjects heterozygous for an amyloid associated TTR mutant have lower plasma levels of TTR than normal (Benson and Dwulet 1983; Skinner et al. 1985; Westermark et al. 1985). This would not appear to be due to altered nutrition since it has been observed in young healthy individuals long before any evidence of amyloid has occurred (Waits et al. 1995). Low plasma levels of TTR may be a result of increased metabolic turnover associated with variant TTR proteins and this could be a significant factor in pathogenesis. Studies by Vasquez et al., more than 50 years ago, showed that the plasma half-life of TTR (prealbumin) was only one or two days, and subsequent work has shown increased plasma clearance of Val30Met TTR in both affected and nonaffected subjects (Benson et al. 1996; Vahlquist et al. 1973). Transgenic animal models of TTR amyloidosis have been studied but have not, in general, shed much light on the pathogenic mechanisms of the disease. From a clinical perspective a number of factors speak to the pathogenesis of TTR amyloidosis. These are all observational; none is the result of experimental design. The disease displays autosomal dominant inheritance. Most patients are heterozygous

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for one of the 100 plus TTR mutations. A few patients are homozygous for Val30Met or Val122Ile and have been reported but do not have earlier onset or more aggressive disease than their heterozygous kin (Holmgren et al. 1988; Nichols et al. 1991). While patients homozygous for Leu58His have been reported to have more severe disease, the general lack of clinical difference between homozygous and heterozygous subjects is against significant importance either of TTR variant concentration or tetramer stability from hormone binding. Not all TTR variant gene carriers develop amyloidosis, indicating that other factors must be involved in expressing the clinical disease. TTR amyloidosis is an adult onset disease and, while a few individuals have been reported affected in their teenage years (Leu55Pro, Trp41Leu), most do not develop symptoms of the disease until the fourth or fifth decade of life. It is obvious that the clinical disease is significantly affected by age. Organ impairment from amyloid deposition is, in general, faster in early onset disease patients as seen in the Portuguese and Japanese Val30Met kindreds. Progression is slower in the Swedish and English Val30Met patients who have later onset of the clinical disease. Senile systemic TTR (senile cardiac) amyloidosis occurs in patients over the age of 60 and is usually not of clinical significance until after the age of 70 years. The disease occurs in the absence of any TTR mutation and no other inherited factor has been identified except gender. Essentially all patients with SSA (SCA) are male. This suggests that the male sex is a significant disease modifier and this is consistent with the approximately 60:40 male to female ratio of patients that are diagnosed with TTR amyloidosis. This, as yet unidentified factor, may be a general metabolic variable since the 60:40 ratio of affected individuals is also observed with AL (immunoglobulin light chain) amyloidosis in most published series. Advanced age onset of amyloid deposition from normal protein is also seen in Alzheimer disease (AD). In both TTR and AD amyloidoses the presence of a mutation in the amyloid precursor protein appears to cause an earlier onset of amyloid formation. In the Alzheimer disease, in the absence of a mutation, the disease is affected by inheritance of the apolipoprotein E haplotype. In TTR amyloidosis no similar modifier has been identified (Saunders et al. 1993). Organ system involvement by TTR amyloidosis is quite variable but does suggest underlying modifiers and, therefore, possible clues for pathogenesis. The nervous system (both peripheral and autonomic), the heart, kidneys, bowel, eyes, skin and leptomeningeal tissue may all be sites of amyloid deposition. The apparent syndromic picture associated with certain TTR mutations might suggest dominant control of phenotype by certain specific mutations. However, many of the mutations are in limited kindreds where shared genetic background may not allow diversity. The Val30Met mutation, which is the most widespread worldwide, shows the greatest clinical diversity and, while usually characterized principally by peripheral neuropathy, many patients have cardiomyopathy, vitreous opacities, nephropathy, and occasionally leptomeningeal amyloid deposition. The Thr60Ala mutation, however, has been widely dispersed but, so far, has not been found to be associated with renal, vitreous or leptomeningeal amyloid. This would suggest a greater influence on pathogenesis by this specific TTR mutation.

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So even when combining experimental data on TTR structure and function with clinical observations of TTR amyloidosis we are far from understanding the pathogenesis of the disease. We can, however, consider specific studies that might pertain to pathogenesis and/or search for effective therapies. More information on TTR metabolism including cellular uptake and catabolic processing can be undertaken in animal studies. Specific information on amyloid formation will have to result from human experimentation until an animal model that faithfully represents the human disease is available.

11.7

Therapy

Nonspecific therapy aimed at alternating the ravages of organ system destruction has been, and remains, the most important aspect of treatment for the patient with TTR amyloidosis. Treatments for the neuropathic and psychological consequences of TTR amyloidosis are addressed in other chapters of this book. Analgesic, antiepileptic and psychotropic drugs are a mainstay of treatment for TTR neuropathic symptoms. Surgical decompression for the carpal tunnel syndrome is very effective when done early in the course of disease. Laminectomy has been shown to be effective in a few cases of spinal claudication due to epidural amyloid deposition (Harats et al. 1989). A few notes on treatment of cardiomyopathy are in order since this is a major manifestation in many patients with TTR amyloidosis. Amyloid deposition in the heart results in restrictive hemodynamics. Cardiac walls are thickened and noncompliant (Figs. 11.4a, b). The amyloid causes impairment of ventricular filling during diastole and, therefore, decreased cardiac output. This effect is amplified by any increase in heart rate since the time for diastolic filling is shortened. Thus an increase in heart rate from sudden exercise will decrease ventricular filling and may cause syncope due to decreased cerebral blood flow. Sustained impairment of

Fig. 11.4 Echocardiographic and gross anatomical findings in TTR cardiomyopathy. (a) Echocardiographic image in a patient with TTR Thr60Ala showing thickened left ventricular walls and a dilated left atrium. (b) Transverse section of a heart showing thickened ventricular walls and a small left ventricular cavity

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ventricular filling will lead to congestive heart failure with weakness, fatigue and fluid retention. This often occurs during prolonged tachyarrhymias such as atrial fibrillation, a result of atrial enlargement from restrictive hemodynamics. Therapeutically, it is important to maintain normal sinus heart rhythm for as long as possible. Arrhythmias are bad for cardiac function and may be prevented with use of modest doses of antiarrhythmics such as Beta adrenergic blocking agents. Calcium channel blocking agents should be avoided (Pollak and Falk 1993). They have major negative inotropic properties and may exacerbate congestive heart failure. Diuretics are a major therapeutic modality for treatment of congestive heart failure due to amyloid cardiomyopathy. It is important, however, to adjust their use so that systolic blood pressure is maintained well enough for adequate renal perfusion. Some patients develop cardiac conduction disorders such as sinus exit, atrial, or atrioventricular blocks and then require cardiac pacemaking. Cardiac defibrillators (ICD’s) are of questionable value in amyloid cardiomyopathy. Cardiac transplantation with or without concomitant liver transplant has proven effective for a number of patients with TTR amyloidosis. In patients who have had a heart transplant in conjunction with orthotopic liver transplant, none has developed amyloid in the heart graft even though some have died from amyloid progression in other organs (lung, nerves and bowel). Limited clinical experience indicates that amyloid deposition in cardiac grafts without concomitant liver transplant may be delayed for several years or may not become manifest in the lifetime of the recipient. The only treatment for TTR amyloidosis that has been proven to be effective is orthotopic liver transplantation (OLT) (Holmgren et al. 1991). As noted elsewhere in this book, many patients have benefited from liver transplantation. Even so, some patients have had progression of disease after OLT and this has been shown to be due to continued amyloid formation from normal TTR (Dubrey et al. 1997; Liepnieks and Benson 2007). This emphasizes the importance of developing new therapies. To date, trials are ongoing to determine the effect of small organic molecules to stabilize the TTR tetramer and delay fibril formation. Studies designed to restrict production of TTR fibril precursor synthesis with antisense oligonucleotide or siRNA technology are in progress but are not yet ready for human trials (Benson et al. 2006). Clinical studies resulting from advances in medical diagnostic technology are showing that TTR amyloidosis is not such a rare disease after all. New therapies are sorely needed.

11.8

Conclusion

So here we are. We have amassed a tremendous amount of knowledge about TTR and its single human disease, amyloidosis. This type of amyloidosis has so much phenotypic variation that it could be argued that we are really dealing with many diseases. Each, however, is characterized by the formation and deposition of b-structured protein fibrils with resultant disruption of organ function. As with most types of

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amyloidosis, the major question remains: how does a physiological protein transit from its normal soluble state to one of insoluble proteolytically resistant fibrils? Hopefully, the answer to this question will lead to the development of means to disrupt this pathological process.

References Andersson R (1970) Hereditary amyloidosis with polyneuropathy. Acta Med Scand 188:85–94. Andersson R, Kassman T (1968) Vitreous opacities in primary familial amyloidosis. Acta Ophthal (Copenh) 46:441–447. Andrade C (1952) A peculiar form of peripheral neuropathy. Familial atypical generalized amyloidosis with special involvement of the peripheral nerves. Brain 75:408–427. Araki S, Mawatari S, Ohta M, Nakajima A, Kuroiwa Y (1968) Polyneuritic amyloidosis in Japanese family. Arch Neurol 18:593–602. Baures PW, Oza VB, Peterson SA, Kelly JW (1999) Synthesis and evaluation of inhibitors of transthyretin amyloid formation based on the non-steroidal anti-inflammatory drug, Flufenamic Acid. Bioorganic Med Chem 7:1339–1347. Benson MD (2003) The hereditary amyloidoses. In: Elsevier UK (ed) Best Practice and Research Clinical Rheumatology, chap 2, vol 17, pp 909 927. Benson MD, Kluve-Beckerman B, Zeldenrust SR, Siesky AM, Bodenmiller DM, Showalter AD, Sloop KW (2006) Targeted suppression of an amyloidogenic transthyretin with antisense oligonucleotides. Muscle Nerve 33:609–618. Benson MD, Kluve-Beckerman BB, Liepnieks JJ, Murrell JR, Hanes D, Uemichi T (1996) Metabolism of amyloid proteins. In. Bock GR, Goode JA (eds) The Nature and Origin of Amyloid Fibrils, Ciba Foundation Symposium 199. Wiley, New York, pp. 104–118. Benson MD, Cohen AS (1977) Generalized amyloid in a family of Swedish origin. A study of 426 family members in 7 generations of a new kinship with neuropathy, nephropathy and central nervous system involvement. Ann Intern Med 86:419–424. Benson MD, Dwulet FE (1983) Prealbumin and retinol binding protein serum concentrations in the Indiana type hereditary amyloidosis. Arthritis Rheum 26:1493–1498. Benson MD, Kincaid JC (2007) The molecular biology and clinical features of amyloid neuropathy. Muscle Nerve 36:411–423. Benson MD, Wallace MR, Tejada E, Bauman H, Page B (1987) Hereditary amyloidosis: description of a new American kindred with late onset cardiomyopathy. Arthritis Rheum 30:195–200. Block WD, Jackson CE, Falls HF, Carey JH, Curtis AC, Rukavina JG (1956) Primary systemic amyloidosis: A review and an experimental, genetic, and clinical study of 29 cases with particular emphasis on the familial form. Medicine 35:239–334. Connors LH, Lim A, Prokaeva T, Roskens VA, Costello CE (2003) Tabulation of human transthyretin (TTR) variants. Amyloid 10:160–184. Cornwell GG, Westermark P, Natvig JB, Murdoch W (1981) Senile cardiac amyloid: evidence that fibrils contain a protein immunologically related to prealbumin. Immunology 44:447–452. Dowell JD, Fleck JD, Vakili ST, Benson MD (2007) Familial oculoleptomeningeal amyloidosis with primary angiitis of the CNS. Neurology 68:77–78. Dubrey SW, Davidoff R, Skinner M, Bergethon P, Lewis D, Falk R (1997) Progression of ventricular wall thickening after liver transplantation for familial amyloidosis. Transplantation 64:74–80. Dwulet FE, Benson MD (1986) Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss). J Clin Invest 78:880–886. Falls HF, Jackson JH, Carey JG, Rukavina JG, Block WD (1955) Ocular manifestations of hereditary primary systemic amyloidosis. Arch Ophthalmol 54:660–664.

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Goebel HH, Seddigh S, Hopf HC, Uemichi T, Benson MD, McKusick VA (1997) A European family with histidine 58 transthyretin mutation in familial amyloid polyneuropathy. Neuromuscular Disorders 7:229–230. Gorevic PD, Prelli FC, Wright J, Pras M, Frangione B (1989) Systemic senile amyloidosis. Identification of a newprealbumin (transthyretin) variant in cardiac tissue: immunologic and biochemical similarity to one form of familial amyloidotic polyneuropathy. Clin Invest 83: 836–843. Goren H, Steinberg MC, Farboody GH (1980) Familial oculoleptomeningeal amyloidosis. Brain 103:473–495. ˚ , Engstro¨m U, Westermark P (1994) Mechanisms of transthyretin amyloidogenesis. Gustavsson A Antigenic mapping of transthyretin purified from plasma and amyloid fibrils and within in situ tissue localizations. Am J Pathol 144:1301–1311. Hamilton JA, Benson MD (2001) Transthyretin: A review from a structural perspective. Cell Mol Life Sci 58:1491–1521. Hammarstro¨m P, Wiseman RL, Powers ET, Kelly JW (2003) Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299:713–716. Harats N, Worth R, Benson MD (1989) Spinal claudication in systemic amyloidosis. J Rheum 16:1003–1006. Holmgren G, Haettner E, Nordenson I, Sandgren O, Steen L, Lundgren E (1988) Homozygosity for the transthyretin-Met30-gene in two Swedish sibs with familial amyloidotic polyneuropathy. Clin Genet 34:333–338. Holmgren G, Holmberg E, Lindstrom A, Lindstrom E, Nordenson I, Sandgren O, Steen L, Svensson B, Lndgren E, von Gabain A (1988) Diagnosis of familial amyloidotic polyneuropathy in Sweden by RFLP analysis. Clin Genet 33:176–180. Holmgren G, Steen L, Ekstedt J, Groth CG, Ericzon BG, Eriksson S, Andersen O, Karlberg I, Norden G, Nakazato M, Hawkins P, Richardson S, Pepys M (1991) Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic polyneuropathy (FAPmet30). Clin Genet 40:242–246. Jacobson DR, Pastore RD, Yaghoubian R, Kane I, Gallo G, Buck FS, Buxbaum JN (1997) Variantsequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans. N Engl J Med 336:466–473. Kincaid JC, Wallace RW, Benson MD (1989) Late onset familial amyloid polyneuropathy in an American family of English origin. Neurology 39:861–863. Lessell S, Wolf PA, Benson MD, Cohen AS (1975) Scalloped pupils in familial amyloidosis. N Engl J Med 293:914–915. Libbey CA, Rubinow A, Shirahama T, Deal C, Cohen AS (1984) Familial amyloid polyneuropathy. Demonstration of prealbumin in a kinship of German/English ancestry with onset in the seventh decade. Am J Med 76:18–24. Liepnieks JJ, Benson MD (2007) Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation. Amyloid 14:277–282. Liepnieks JJ, Wilson DL, Benson MD (2006) Biochemical characterization of vitreous and cardiac amyloid in Ile84Ser transthyretin amyloidosis. Amyloid 13:170–177. Mahloudji M, Teasdall RD, Adamkiewicz JJ, Hartmann WH, Lambird PA, McKusick VA (1969) The genetic amyloidoses. With particular reference to hereditary neuropathic amyloidosis, type II (Indiana or Rukavina type). Medicine 48:1–37. Murakami T, Maeda S, Yi S, Ikegawa S, Kawashima E, Onodera S, Shimada K, Araki S (1992) A novel transthyretin mutation associated with familial amyloidotic polyneuropoathy. Biochem Biophys Res Commun 182:520–526. Nichols WC, Liepnieks JJ, McKusick VA, Benson MD (1989) Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allelespecific enzymatic amplification. Genomics 5:535–540. Nichols WC, Liepnieks JJ, Snyder EL, Benson MD (1991). Senile cardiac amyloidosis associated with homozygosity for a transthyretin variant (Ile-122). J Lab Clin Med 117:175–180.

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Petersen RB, Goren H, Cohen M, Richardson SL, Tresser N, Lynn A, Gali M, Estes M, Gambetti P. (1997) Transthyretin amyloidosis: a new mutation associated with dementia. Ann Neurol 41: 307–313. Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95:12957–12960. Pitka¨nen P, Westermark P, Cornwell GG (1984) Senile systemic amyloidosis. Am J Pathol 117: 391–399. Pollak A, Falk RH (1993) Left ventricular systeolic dysfunction precipitated by verapamil in cardiac amyloidosis. Chest 104:618–620. Rosen HN, Moses AC, Murrell JR, Liepnieks JJ, Holmgren G, Sandgren O, Steen L, Benson MD (1993) Thyroxine interactions with transthyretin: a comparison of 10 different naturallyoccurring transthyretin variants. J Clin Endo Metab 77:370–374 Rukavina JG, Block WD, Jackson CE, Falls HF, Carey JH, Curtis AC (1956) Primary systemic amyloidosis: a review and an experimental, genetic, and clinical study of 29 cases with particular emphasis on the familial form. Medicine 35:239–334. Saunders AM, Schmader K, Breitner JC, Benson MD, Brown WT, Goldfard L, Goldgaber D, Manwaring MG, Szymanski MH, McCrown N, Dole KC, Schmechel DE, Strittmatter WJ, Pericak-Vance MA, Roses AD (1993) Apolipoprotein E 4 allele distributions in late-onset Alzheimer’s disease and in other amyloid-forming diseases. Lancet 342:710–711. Sandgren O (1995) Ocular amyloidosis, with special reference to the hereditary forms with vitreous involvement. Surv Ophthalmol 40:173–196. Satier F, Nichols WC, Benson MD (1990) Diagnosis of familial amyloidotic polyneuropathy in France. Clin Genet 38:469–473. Sequeiros J, Saraiva MJ (1987) Onset in the seventh decade and lack of symptoms in heterozygotes for the TTRMet30 mutation in hereditary amyloid neuropathy type I (Portuguese, Andrade). Am J Med Genet 27:345–357. Shur O, Danielsson A, Holmgren G, Steen L (1994) Malnutrition and gastrointestinal dysfunction as prognostic factors for survival in familial amyloidotic polyneuropathy. J Int Med 479-485. Skinner M, Connors LH, Rubinow A, Libbey C, Sipe JD, Cohen AS (1985) Lowered prealbumin levels in patients with familial amyloid polyneuropathy (FAP) and their non-affected but at risk relatives. Am J Med Sci 289:17–21. Ueno S, Uemichi T, Yorifuji S, Tarui S (1990) A novel variant of transthyretin (Tyr114 to Cys) deduced from the nucleotide sequences of gene fragments from familial amyloidotic polyneuropathy in Japanese sibling cases. Biochem Biophys Res Commun 169:143–147. Vahlquist A, Peterson PA, Wibell L (1973) Metabolism of the vitamin A transporting protein complex. 1. Turnover studies in normal persons and in patients with chronic renal failure. Eur J Clin Invest 3:352–362. Waits RP, Yamada T, Uemichi T, Benson MD (1995) Low plasma concentrations of retinol-binding protein in individuals with mutations affecting position 84 of the transthyretin molecule. Clin Chem 41:1288–1291. Wallace MR, Dwulet FE, Conneally PM, Benson MD (1986) Biochemical and molecular genetic characteristic of a new variant prealbumin associated with hereditary amyloidosis. J Clin Invest 78:6–12. Wallace MR, Dwulet FE, Williams EC, Conneally PM, Benson MD (1988) Identification of a new hereditary amyloidosis prealbumin variant, tyr77, and detection of the gene by DNA analysis. J Clin Invest 81:189–193. Westermark P, Pitkanen P, Benson L, Vahlquist A, Olofsson BO, Cornwell GG III (1985) Serum prealbumin and retinol-binding protein in the prealbumin-related senile and familial forms of systemic amyloidosis. Lab Invest 52:314–318. Westermark P, Sletten K, Johansson B, Cornwell GG (1990) Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA 87:2843–2845.

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Yamashita T, Hamidi Asl K, Yazaki M, Benson MD (2005) A prospective evaluation of the transthyretin Ile122 allele frequency in an African-American population. Amyloid J Protein Folding Disord 12:127–130. Yazaki M, Connors LH, Eagle Jr, RC, Leff SR, Skinner M, Benson MD (2002) Transthyretin amyloidosis associated with a novel variant (Trp41Leu) presenting with vitreous opacities. Amyloid J Protein Folding Disord 9:263–267. Yazaki M, Yamashita T, Kincaid J, Scott J, Auger R, Dyck P, Benson MD (2002) Rapidly progressive amyloid polyneuropathy associated with a novel variant transthyretin Ser 25. Muscle Nerve 25:244–250. Yoshioka K, Furuya H, Sasaki H, Saraiva MJM, Costa PP, Sakaki Y (1980) Haplotype analysis of familial amyloidotic polyneuropathy. Hum Genet 82:9–13. Zhao N, Aoyama N, Benson MD, Skinner M, Satier F, Sakaki Y (1994) Haplotype analysis of His58, Ala60 and Tyr77 types of familial amyloidotic polyneuropathy. Amyloid Int J Exp Clin Invest 1:75–79. Zo´lyomi Z, Benson MD, Hala´sz K, Uemichi T, Fekete G (1998) Transthyretin mutation (Serine 84) associated with familial amyloid polyneuropathy in a Hungarian family. Amyloid Int J Exp Clin Invest 5:30–34.

Chapter 12

Molecular Pathogenesis Associated with Familial Amyloidotic Polyneuropathy Maria Joaõ Saraiva

Abstract Human plasma transthyretin (TTR) can undergo conformational changes and form amyloid fibrils, in both acquired and hereditary forms of systemic amyloidosis. More than 100 TTR mutations have been associated with autosomal dominant amyloidosis, usually presenting with peripheral and autonomic neuropathy (Familial amyloidotic polyneuropathy (FAP)) and/or cardiomyopathy (Familial amyloidotic cardiomyopathy (FAC)). The mechanisms underlying cell death in TTR-related amyloidoses need to be addressed for the development of future therapies in FAP. These issues are the subject of this review. Keywords Amyloid, Aggregation, Stress response

12.1

Introduction

Hereditary transthyretin (TTR) amyloidosis is a genetically transmitted disease that results from a mutation in the gene encoding the plasma TTR protein. TTR is a transport protein for thyroid hormones and vitamin A and is predominantly synthesized in the liver. Most known TTR mutations increase the potential for the protein to destabilize and aggregate as amyloid fibrils extracellularly in different organs and tissues, with predominance in the peripheral nervous system (PNS). Brain and liver are spared from deposition. The most common TTR mutation associated with peripheral neuropathy in familial amyloidotic polyneuropathy (FAP) is TTR Val30Met described in the Portuguese population (Saraiva et al. 1984; for mutations M.J. Saraiva Molecular Neurobiology Group, IBMC – Instituto de Biologia Molecular e Celular and ICBAS – Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, 4150 Porto Portugal e-mail: [email protected]


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listing see http://www.ibmc.up.pt/mjsaraiva/ttrmut.html). The first symptoms relate to a sensory neuropathy beginning at the extremities of the lower limbs, with paresthesias, dysaesthesias and loss of thermal and pain sensations. The disease advances proximally and the upper extremities are affected later, when the loss of sensation has reached the level of the knee. Usually 2 or 3 years after the first sensory manifestations, motor disturbances are noticeable, with atrophy and muscular weakness, beginning also by the lower extremities. In the more advanced stages, patients are confined to a wheel-chair (Andrade 1952). Autonomic neuropathy is particularly severe in Portuguese FAP patients and can constitute the first manifestation in many patients. Autonomic involvement manifests as gastrointestinal disturbances, with alternating periods of constipation and diarrhea, gastric stasis, nausea and vomiting. Loss of weight and asthenia frequently precede gastrointestinal disturbances. Sphincter dysfunction is responsible for both urinary and fecal incontinence as well as impotence. Electrocardiographic abnormalities are common in Portuguese FAP patients and are expressed as conduction disturbances leading to branch blocks which demands pacemaker implantation in some instances. The progression of the disease is slow and relentless, leading to cachexia and death in 10–15 years from its onset. At present, studies on cellular toxicity and neurodegeneration in FAP are emerging. They encompass analyses both on clinical samples and animal models and will guide potential forms of treatment aimed at counteracting toxic cascades generated by deposition of aggregated TTR. We will next describe molecular pathways that have been reported in FAP and how they can guide us in treatments.

12.2

Pathological Features in the Peripheral Nervous System in FAP

TTR amyloid deposits can be found in any part of the peripheral nervous system, including the nerve trunks, plexuses and sensory and autonomic ganglia. In peripheral nerves, deposition occurs extracellularly, particularly in the endoneurium close to Schwann cells (SC) and to collagen fibrils; in the endoneurial space it usually takes the form of globoid deposits (Fig. 12.1a) near the perineurial envelope or near the endoneurial capillaries, or surrounding the capillary wall. Amyloid is also present in the adventicia of the vessels or as small deposits in the epinerve. In severely affected nerves, almost the whole endoneurial contents may be replaced by amyloid and very few nerve fibers survive. FAP is characterized by initial axonal loss affecting unmyelinated and small myelinated fibres and later affecting the larger fibres (Coimbra and Andrade 1971). In the autonomic and sensory nervous system, advanced neuronal degeneration which might cause progressive ascending neuropathy (dying back type) is evident in addition to axonopathy. At the ultrastructural level, the fibrils of amyloid deposits have the usual aspect, sometimes forming bundles that participate in the constitution of globoid deposits, involving and being surrounded by collagen fibers (Fig. 12.1b). In suitable fixed

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Fig. 12.1 Amyloid in the nerve. (a) Anti-TTR immunohistochemistry (scale bar = 0.5 mm); (b) Electron microscopy (Scale bar = 100 nm)

nerve trunks, without shrinkage artifacts, the fibrils are present near the SC basal membrane, but without penetrating the cell membrane or cytoplasm (the same is true for the smooth muscle cells or endothelial cells of blood vessels). The dorsal root ganglia and the sympathetic and parasympathetic intramural ganglia also present progressive loss of neurons and deposition of amyloid in their stroma. There is loss of ganglionic neuronal cells, some of them presenting central chromatolysis and some empty spaces surrounded by satellite cells.

12.3

Early Deposition of TTR in an Asymptomatic Phase in a Nonfibrillar Fashion

12.3.1 Ex Vivo Analyses in Clinical Samples TTR deposition was assessed in nerves from asymptomatic TTR Val30Met carriers and FAP patients in different stages of disease progression. The scoring system of patients’ material was performed by morphometric measurements of myelinated (MF) and unmyelinated (UF) fibers: FAP 0- the least affected nerves (no nerve degeneration, no amyloid deposited) of all individuals were asymptomatic. From stage 1–3, individuals were all presenting symptoms as follows: FAP 1- discrete reduction in the number of nerve fibres and modest fibrillar deposits; FAP 2evident reduction of nerve fibres and amyloid throughout the nerve; and FAP 3severe reduction of nerve fibres and extensive amyloid deposition. It was observed that in the stage prior to loss of UF and MF and major nerve fiber degeneration (FAP 0), despite the absence of Congo red birefringence (the hallmark of amyloid) TTR was present, as revealed by immunohistochemistry with an antiTTR antibody. Thus, TTR deposits in a nonfibrillar, or prefibrillar form in the early stages of FAP, before assembling into mature amyloid fibrils, that give the characteristic green-birefringence by Congo red staining. Furthermore, immunocytolabeling of TTR was observed extracellularly in the proximity of SC, in a nonfibrillar

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form; and very small contiguous fibrillar-like assemblies were noticed, but were most likely too small to give bi-refringence with Congo-red staining on histochemistry. It was concluded that TTR deposits early in the asymptomatic phase in a nonfibrillar fashion (Sousa et al. 2001a).

12.3.2 Ex Vivo Analyses in Animal Models In order to understand the mechanisms underlying fibril formation, deposition and cytotoxic effects caused by aggregates, many questions remain to be investigated. In an attempt to gain insights into the pathogenesis of FAP, several groups generated transgenic mice carrying the human TTR Val30Met gene. Using the human homologous TTR promoter sequences to generate TTR Val30Met transgenics, amyloid was observed starting at 6 months. At the age of 24 months, the pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems (Yi et al. 1991). The same pattern of amyloid deposition was reported when these transgenics were backcrossed to a TTR-null background (Kohno et al. 1997). Animals presented widespread TTR staining as assessed by immunohistochemistry, with particular involvement of the gastrointestinal tract, starting as early as 3 months. At this stage no fibrillar material was detected both by Congo red staining and by immunocytolabeling. Congo red positive deposition was only observed at older ages affecting all mice after 21 months of age and starting at 9 months (Sousa et al. 2002).

12.4

Inflammation and Oxidative Stress Pathways in FAP and Involvement of the Receptor for Advanced Glycation-End Products (RAGE)

The toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates, but not mature fibrils activate caspases, indicative of cytotoxicity by nonfibrillar deposits occurring in early stages of FAP (Sousa et al. 2001b). Increased expression of the receptor for glycation end products (RAGE) has been observed in TTR amyloid - laden tissues. Binding of RAGE by fibrillar TTR triggers activation of the transcription factor NF-kB. RAGE is a member of the immunoglobulin superfamily with a broad repertoire of ligands in addition to amyloid-associated macromolecules, including products of nonenzymatic glyeation (advanced glycation endproducts, AGEs), proinflammatory mediators (S100/ calgranulins) and amphoterin (Schmidt et al. 2000). In each case, the receptor recruits signal-transduction mechanisms, often resulting in a sustained and pathogenic


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inflammatory and stress response. This response might underlie peripheral nerve dysfunction. Analyses of nerve biopsy samples from Portuguese FAP patients at different stages of the disease (including the presymptomatic stage), were compared with age-matched controls by semiquantitative immunohistochemistry and in situ hybridization and showed upregulation of proinflammatory cytokines (tumor necrosis factor and interleukin 1), and the inducible form of nitric oxide synthase (iNOS), as well as increased tyrosine nitration and activated caspase-3 (Sousa et al. 2001b) in axons. Recent work showed that extracellular signalregulated kinases 1/2 (ERK1/2) displayed increased activation in FAP tissues and in TTR transgenic mice. Treatment of a rat Schwannoma cell line with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by RAGE and abrogated by specific ERK1/2 inhibitors. These data suggested that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration (Monteiro et al. 2006).

12.5

The Ubiquitin–Proteasome System in FAP

The ubiquitin-proteasome system (UPS) has been associated with neurodegenerative disorders of intracellular protein aggregation. Studies on the UPS in FAP in TTR synthesizing and nonsynthesizing tissues from affected individuals and in the transgenic Val30Met mice model showed that ubiquitin protein conjugates were upregulated, the proteasome levels were decreased and parkin, and alpha-synuclein expression were both decreased (Santos et al. 2007). On the other hand, the liver, that normally synthesizes a variant of TTR Val30Met, did not show this response. Furthermore, transgenic mice immunized to decrease TTR deposition (Terazaki et al. 2006) showed a significant reduction in ubiquitin levels and an increase in parkin and alpha-synuclein levels in comparison to control mice. When neuronal or Schwannoma cell lines were cultured with TTR aggregates, an increase in ubiquitin and decrease in parkin levels were demonstrated. The overall results indicate that TTR extracellular deposition is an external stimulus to an intracellular UPS response in FAP.

12.6

The Unfolded Protein Response in FAP

12.6.1 Activation of the Heat Shock Response The heat shock proteins (hsp) have been implicated in a variety of neurodegenerative diseases in which the underlying pathology is protein aggregation. The heat shock response was investigated in FAP. Upregulation of hsp27 and hsp70 expression related to the presence of extracellular TTR aggregates in human FAP biopsies, as compared to normal controls, was documented. TTR aggregates did not

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colocalize with hsps, suggesting that extracellular TTR tissue deposits are able to induce an intracellular stress response. Moreover, the heat shock transcription factor 1 (HSF1) was upregulated and localized to the nucleus. Transgenic mice models expressing the Val30Met mutant form of TTR showed a similar response: the presence of TTR deposits induced a stress response with the activation of HSF1 and increased synthesis of hsps. In cell culture, the upregulation of hsp70 and hsp27 in the presence of toxic TTR aggregates was found (Santos et al. 2008a). Taken together, these findings prompt new avenues for the research on pathogenic mechanisms in FAP and the use of the heat shock response as a pharmacological target to treat FAP and other neurodegenerative disorders.

12.6.2 A Compromised Heat Shock Response Increases Systemic Extracellular Transthyretin Deposition and Affects the Peripheral and Autonomic Nervous Systems: Lessons from an Animal Model Protein misfolding diseases result to a certain extent from the incapacity of the cell to stay below the dangerous limits of intra and/or extracellular accumulated aggregated proteins as in Alzheimer’s disease, Parkinson’s disease or amyotrophic lateral sclerosis. The proteins involved are predisposed to aggregate, forming protofilaments and eventually amyloid fibrils. In the case of FAP, extracellular accumulation of aggregated TTR affects mainly the PNS, leading to inflammatory and oxidative stress and ultimately neurodegeneration. It was hypothesized that HSF1 could be involved in FAP pathogenesis as a cellular defense mechanism against the presence of TTR extracellular deposits and that disruption of the heat shock response would aggravate TTR deposition. A mouse model expressing the human TTR- Val30Met in a HSF1 null background was characterized. The lack of HSF1 expression lead to extensive and earlier nonfibrillar TTR, evolving into fibrillar material in distinct organs including the peripheral nervous system. As in the human disease, liver, brain and spinal cord did not present deposition. Furthermore, inflammatory stress and a reduction in unmyielinated nerve fibres were observed, as in human patients, indicating that HSF1 regulated genes are involved in FAP, modulating TTR tissue deposition (Santos et al. 2008b). The TTR/HSF1 model allows different studies, not possible with previous models. Sural nerve biopsies, on which most of the pathological analysis in FAP has been performed, represent a restricted portion of the PNS, and it is clearly possible that TTR deposits in ganglia, or more proximally in nerve trunks, which could be responsible for distal nerve fiber loss. The possibility to analyze dorsal root ganglia (DRG) with deposition in the TTR/HSF1 mice model is invaluable, and permits a close evaluation of events occurring in sensory and autonomic neurons responsible for neurodegeneration.


12.7

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The ER Response

The availability of a transgenic mouse model with extracellular TTR aggregates in DRG is a unique tool to study molecular mechanisms underlying pathogenesis in FAP. One of the first pathways investigated was ER stress. In mouse DRG, BiP expression was only visible in neurons of the ganglia in close contact with TTR extracellular deposits, suggesting a cause–effect relation, first suggesting a connexion between the ER stress response and FAP (Teixeira et al. 2006). Increased levels of the ER-resident chaperone BiP were found, in a consistent way, in human salivary gland biopsies of FAP patients, in TTR transgenic mouse DRG in and cell line models. These results were further confirmed in human salivary gland biopsies, by western blot and immunohistochemistry; increase in BiP level was visible in areas where mutant TTR was deposited. Using an ND7/23 cell line, one of the few cell lines of peripheral neurons, and a human neuroblastoma cell line it was found that extracellular TTR oligomers can induce BiP expression and activation of eIF2a in a way similar to the chemical inducer thapsigargin. This increase in BiP levels involved the mobilization of the secondary messenger Ca2+ from the ER, as it was blocked by Ca2+-channel inhibitors. These inhibitors lowered caspase-3 activation as response to TTR oligomers added to cultures, re-enforcing the notion of ER stress by extracellular TTR deposition as a toxic event in this system.

12.8

Activation of Extracellular Matrix Remodeling Genes

Although inflammation and oxidative stress are already triggered in presymptomatic individuals, clinical disease appears only after amyloid deposition. Gene arrays on clinical material (salivary glands) and control tissues allowed analyses of differential gene expression between pathogenic and healthy tissues. Among the differentially expressed genes, upregulation of genes related to extracellular matrix (ECM) remodeling was evident in patients’ tissues. Matrix metaloproteinase 9 (MMP9) is overexpressed only in amyloid laden tissues, but not in tissues from presymptomatic individuals (Sousa et al. 2005), suggesting that fibrils trigger specific signaling pathways leading to ECM remodeling and that this process causes tissue damage with pathological consequences. In the field of therapeutics it is important to define disease markers, not only to characterize the pathology but also to follow-up and evaluate therapeutic protocols. MMP9 apparently is a good biomarker for the presence of TTR amyloid as suggested by the above described array data but also from studies on TTR transgenic mice. These mice recapitulate the human situation as only mice with amyloid deposits displayed increased levels and activity of MMP-9 as compared to younger animals with nonfibrillar deposits. When treated with doxycycline, a TTR disrupter (Cardoso et al. 2003; Cardoso and Saraiva 2006), tissue MMP-9 expression and activity were decreased to levels comparable to those found in animals

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without TTR deposition. Other amyloid-related components, namely (Serum amyloid protein) SAP, were also decreased, indicating fibril removal.

12.9

Antiapoptotic Treatments

Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent antiapoptotic and antioxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with TTR deposition in FAP, the possible TUDCA therapeutical application in this disease has been investigated. It was shown by semi-quantitative immunohistochemistry that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER chaperone BiP, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most importantly, TUDCA treatment significantly reduced TTR toxic aggregates by as much as 75% (Macedo et al. 2008). Since TUDCA has no effect on TTR aggregation ‘‘in vitro’’, this finding pointed for the ‘‘in vivo’’ modulation of TTR aggregation by oxidative stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutical measures in the FAP population.

Intermediate species

Amyloid

?

ER stress UPR Inflammation Oxidative Stress Matrix remodeling

Fig. 12.2 Identified signaling pathways related to extracellular TTR deposition: a possible contribution to amyloid formation; for explanation, see text


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Concluding Remarks

Protein misfolding occurring in the PNS triggers inflammatory and oxidative stress, matrix remodeling, UPR, and ER pathways that resemble in many aspects (including common molecular players and scenarios) those described in CNS for misfolding disorders, such as Alzheimer Disease. Thus, similarities and dissimilarities between the two systems are very useful to pinpoint and guide us in the treatment of age related neurodegenerative disorders. At this point, the various studies on the molecular pathology associated with FAP suggest that signaling pathways in FAP can contribute to extracellular polymerization of TTR in tissues, as depicted in Fig. 12.2. This avenue of research should be further explored.

References Andrade C (1952) A peculiar form of peripheral neuropathy. Familial atypical generalized amyloidosis with special involvement of the peripheral nerves. Brain 175:408–427. Cardoso I, Merlini G, Saraiva MJ (2003) 40 -iodo-40 -Deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing non-cytotoxic species. Screening for TTR fibril disrupters. FASEB J 17:803–809. Cardoso I, Saraiva MJ (2006) Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model. FASEB J 20:234–239. Coimbra A, Andrade C (1971) Familial amyloid polyneuropathy: and electron microscope study of peripheral nerve in five cases. I. Interstitial changes. Brain 94:199–206. Kohno K, Palha JA, Miyakawa K, Saraiva MJ, Ito S, Mabuchu T, Blaner WS, Iijima H, Tsukahara S, Episkopou V, Gottesman ME, Shimada K, Takahashi K, Yamamura K, Maeda S (1997) Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy. Am J Pathol 150:1497–1508. Macedo B, Batista AR, Ferreira N, Almeida MR, Saraiva MJ (2008) Anti-apoptotic treatment reduces transthyretin deposition in a transgenic mouse model of familial amyloidotic polyneuropathy. Biochem Biophys Acta 1782:517–522. Monteiro F, Sousa MM, Cardoso I, Barbas do Amaral J, Guimara˜es A, Saraiva MJ (2006) Activation of ERK1/2 MAP kinases in familial amyloidotic polyneuropathy. J Neurochemistry 97:151–161. Santos SD, Cardoso I, Magalha˜es J, Saraiva MJ (2007) Impairment of the ubiquitin-proteasome system associated with extracellular transthyretin aggregates in familial amyloidotic polyneuropathy. J Pathology 213:200–209. Santos SD, Magalha˜es J, Saraiva MJ (2008a) Activation of the heat shock response in familial amyloidotic polyneuropathy. J Neuropath Exp Neurol 67:449–455. Santos SD, Fernandes R, Saraiva MJ (2008b) The heat shock response modulates transthyretin deposition in the peripheral and autonomic nervous systems. Neurobiol Aging, May 14 [Epub ahead of print]. Saraiva MJM, Birken S, Costa PP, Goodman DS (1984) Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of a molecular abnormality in transthyretin (prealbumin). J Clin Invest 74:104–119. Schmidt AM, Yan SD, Yan SF, Stern DM (2000) The biology of the receptor for advanced glycation end products and its ligands. Biochim Biophys Acta 1498:99–111. Sousa MM, Cardoso I, Fernandes R, Guimara˜es A, Saraiva MJ (2001a) Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy: evidence for toxicity of non-fibrillar aggregates. Am J Pathol 159:1993–2000.

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Sousa MM, Yan SD, Fernandes R, Guimara˜es A, Stern D, Saraiva MJM (2001b) Familial amyloid polyneuropathy: RAGE-dependent triggering of neuronal inflammatory and apoptotic pathways. J Neuroscience 21:7576–7586. Sousa MM, Fernandes R, Palha JA, Taboada A, Vieira P, Saraiva MJ (2002) Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin Leu55Pro. Am J Pathol 161: 1935–1948. Sousa MM, Barbas do Amaral J, Guimara˜es A, Saraiva MJ (2005) Upregulation of the extracellular matrix remodeling genes, biglycan, neutrophil gelatinase-associated lipocalin and matrix metalloproteinase-9 in familial amyloid polyneuropathy. FASEB J 19:124–126. Teixeira P, Cerca F, Santos SD, Saraiva MJ (2006) Endoplasmic reticulum stress associated with extracellular aggregates: evidence from transthyretin deposition in familial amyloid polyneuropathy. J Biol Chem 281:21998–22003. Terazaki H, Ando Y, Fernandes R, Yamamura K, Maeda S, Saraiva MJ (2006). Immunization in familial amyloidotic polyneuropathy: Counteracting deposition by immunization with a Y78F TTR mutant. Lab Invest 86:23–31. Yi S, Takahashi K, Naito M, Tashiro F, Wakasugi S, Maeda S, Shimada K, Yamamura K, Araki S (1991). Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy, type I. Am J Pathol 138:403–412.

Chapter 13

Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity Klaus Altland and Samantha J. Richardson

Abstract The microheterogeneity of human transthyretin (TTR) is mainly one of ligand and amino acid substitutions. These substitutions modify the conformational stability of monomers, dimers, and tetramers and may eventually result in unfolding– refolding transitions with the endpoint of amyloidosis. In this chapter we focus on a structural peculiarity of human TTR, i.e., a hydrogen bridge between His31 (b-strand B) and Ser46 (b-strand C), which appears to be the vulnerable site for changes of pH within a range (pH 7.4–6.5) observed under conditions of interstitial acidosis. We present arguments in favor of a cooperative interaction of all sites in the TTR monomer in modifying its conformational stability and reversible unfolding-refolding transitions which also affect the dimer and tetramer. We postulate that the unfolded monomer is the pool from which amyloidogenic aggregates are generated. Keywords Cooperative interaction, Histidines, Inflammation, Interstitial acidosis, Unfolding-refolding transition, Wallaby

Abbreviations ATTR CA DTT FAP

Amyloidogenic transthyretin Carrier ampholytes Dithiothreitol Familial amyloidotic polyneuropathy

K. Altland and S.J. Richardson Justus-Liebig-University, Institute of Human Genetics, Schlangenzahl 14, D-35392 Giessen, Germany e-mail: [email protected] School of Medical Sciences (building 223, level 2, rootm 51), RMIT University, PO Box 71, Bundoora 3083, Victoria Australia e-mail: [email protected]


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HEPES IEF MOPS PAGE PIPES RBP SDS TTR

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K. Altland and S.J. Richardson

2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid Isoelectric focusing 3-(N-Morpholino)-propanesulfonic acid Polyacrylamide gel electrophoresis 1,4-Piperazinediethane sulfonic acid Retinol-binding protein Sodium dodecylsulfate Transthyretin

Introduction

More than 100 variants of human transthyretin (TTR) have been identified worldwide and the great majority of these variants have been found to be associated with the inherited forms of TTR amyloidosis (Connors et al. 2003; Benson 2003). No variants have been detected in patients with senile systemic TTR amyloidosis at age >60 years (Cornwell et al. 1983; Westermark et al. 1990, 2003; Kyle et al. 1996). With one exception (Jacobson et al. 1992), inherited TTR amyloidosis is not observed within the first two decades of life. Among carriers of the most frequent amyloidogenic variant ATTR-V30M, the age of onset of the disease significantly varies with the origin of the population (Plante´-Bordeneuve et al. 2003) and even among identical twins (Holmgren et al. 1997). Amyloid has been identified in biopsy specimens from patients with carpal tunnel syndrome (CTS) who are not carriers of a TTR variant (Kyle et al. 1992). CTS is frequently observed in carriers of an amyloidogenic variant years before the onset of systemic amyloidosis. Similarly, TTR amyloid has been detected in tumors from otherwise healthy carriers of an amyloidogenic TTR variant gene (Altland, personal observations). Obviously, these observations require the presence of several causal factors to understand the development of TTR amyloidosis, which include the normal TTR molecule, its inherited heterogeneity, and the affected individuals with their specific developmental, environmental and genetic background. In this chapter we focus on some properties of the human TTR monomer that might support our understanding of the pathway from native TTR to its unfolded conformational state as the source for the development of the disease.

13.2

Microheterogeneity of Human TTR by Ligands and Amino Acid Substitutions

The TTR tetramer has binding sites for three different types of ligands. The channel inside the TTR tetramer contains two binding sites for thyroxine and structurally related drugs, described elsewhere in this book. The outer surface of the TTR

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tetramer has four binding sites for complexes of retinol and retinol-binding protein (RBP). Each of these binding sites covers areas of three TTR monomers. Because of steric hindrance only a maximum of two RBP molecules can bind to a TTR tetramer at a time (Monaco et al. 1995). TTR monomers have one cysteine (Cys10), and its thiol is exposed to the solvent and can be reversibly bound to other sulfhydryls or sulfites from the aqueous environment or may be irreversibly alkylated or oxidized (The´berge et al. 1999; Kishikawa et al. 1999; Altland et al. 1999). The relative quantities of the complexes between monomers and substituents at Cys10 are expected to vary with the affinities, concentrations, and redox states of the free substituents (e.g., sulfhydryls, sulfite) in the solvent under normal and pathological conditions (Mansoor et al. 1992; Nolin et al. 2007; Hack et al. 1998; Mills et al. 2000; Moriarty-Craige et al. 2005; Jones 2006). The quantification of in vivo plasma concentrations of free and protein-bound thiols requires special care to avoid significant deviations by oxidation. Inadequate storage of biological fluids (i.e., plasma, cerebrospinal fluid) appears to be associated with changes of the electrophoretic patterns of human TTR mainly due to a loss of cysteinylated and a gain of sulfonated Cys10 (Feldin and Fex 1984, Altland, personal observation). The genetic microheterogeneity of human TTR mostly consists of amino acid substitutions, but deletions of single amino acids have also been observed (Connors et al. 2003). The great majority of TTR variants were detected by amyloidosis of amyloidogenic transthyretin (ATTR) gene carriers, while non-amyloidogenic variants were identified by electrophoretic screening procedures (Altland et al. 1982; Jenne et al. 1996; Alves et al. 1997), DNA analysis (Fitch et al. 1991), or mass spectrometry (Nepomuceno et al. 2004). Substitutions of amino acids or ligands as such do not provide an understanding of the amyloidogenic pathway(s) without considering the associated changes of the molecular environment or conformation. Efforts failed to identify typical structural differences between amyloidogenic variants and normal TTR or non-amyloidogenic variants (Ho¨rnberg et al 2000).

13.3

Microheterogeneity of Human TTR by Conformational Stabilities

The structural differences between soluble native TTR and insoluble aggregates/ fibrils (Blake and Serpell 1996) require transition pathways that are more or less common to normal and variant TTRs, but favored by amyloidogenic variants. All known amyloidogenic amino acid substitutions, as well as all binding sites for physiological ligands, are located within the ordered (crystal) structure (from Cys10 to Thr123) of the TTR tetramer (Kanda et al 1974; Blake et al. 1978). Exposure of normal and variant TTR to gradients of denaturants such as acid, urea, and guanidinium chloride revealed a reduced conformational stability as a characteristic of all tested amyloidogenic variants (Colon and Kelly 1992; Jenne et al. 1996;

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Lashuel et al. 1998; Altland et al. 2007). Different conformational stabilities were found for normal or variant TTR when different ligands were bound to Cys10 (Altland et al. 1999, 2007; Altland and Winter 1999; Zhang and Kelly 2003). This observation is demonstrated by Figs. 13.1 and 13.2.

Fig. 13.1 Urea titration curves of normal human TTR. The patterns in (a) and (b) were stained with Serva-Violet 17 and achieved by IEF with added carrier ampholytes (CA) in a urea gradient and in the absence (a) and presence (b) of a reducing agent (50 mM dithiothreitol (DTT)) to demonstrate the heterogeneity of monomeric conformers by different ligands at Cys10. The numbers and arrows label the curves of monomers with Cys10 bound to cysteine (1), cysteinylglycine (2), glutathione (7), sulfite (8), and with Cys10 in the reduced (4) and oxidized (9) as well as alkylated (X) form. The grid and the vertical arrow at 2.7 M urea were added to assist comparison of the two patterns. Notice the shift of the curves as an indicator of differential conformational stabilities associated with the substitutions at Cys10. The patterns in (c) and (d) were stained with silver and achieved by short (6 h at 5,000 V or 30 kVh) and long (16 h at 5,000 V or 80 kVh), respectively, exposure of TTR from human plasma to IEF in a linear immobilized pH gradient. Notice the changes of the tetramer pattern in (d) indicating a slow decay of tetramers into monomers as well as a reverse aggregation of monomers into tetramers (T1 and T5). The length of the flat part of the titration curves at the left 0 M end of the urea gradient corresponds to the length of the visible zones T1 and T4 indicating that reaggregation to tetramers requires folded monomers. The pI difference between tetramer T1 (created from the major monomer fraction) and T5 (created from the minor monomer fraction) at 0 M urea corresponds to the pI difference between the major and minor monomer fraction at 8 M urea or about a pI difference created by one charge unit. At 0 M urea, the pI difference between T1 and T5 is about one-half of the pI difference between T1 and the curve of the folded cysteinylated monomer (curve 1) indicating a gain of two charge units by the transition from the (cysteinylated) tetramer to the folded (cysteinylated) monomer. A total gain of three positive charge units appears to be associated with the changes from (cysteinylated) tetramers (t1) to unfolded (cysteinylated) monomers. Reproduced from Altland et al. (1999) by permission


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Fig. 13.2 Demonstration of the additive effects of conformational stabilities from genetic and environmental microheterogeneity. The samples were from heterozygotes for normal TTR and the amyloidogenic variant ATTR-I107V (a) and the amyloidogenic variant ATTR-V30M (b). All informative details of the patterns indicate that conformational monomer stability is dependent on the sum of contributions from amino acid substitutions and substitutions at Cys10. This is a general observation reproduced with samples from carriers of more than 30 different amino acid substitutions. Reproduced by permission from Altland and Winter (1999, 2003)

When TTR isolated from human plasma by polyacrylamide gel electrophoresis (PAGE) is submitted to isoelectric focusing (IEF) in a urea gradient, it migrates first as a tetramer to its isoelectric point (pI) between pH 4.7 and 5. Then the tetramers slowly dissociate into monomers which present as continuous S-shaped so-called urea titration curves. At a urea concentration of >6 M, all curves concentrate in two fractions: one ‘‘major monomer fraction’’ with a pI near 5.7 and one ‘‘minor monomer fraction’’ with a pI near 5.45. At a urea concentration between 1 and 5 M, monomers separate into at least 10 individual monomeric urea titration curves which correspond to the microheterogeneity at the Cys10 thiol (Altland et al. 1999; Altland and Winter 1999). There is general agreement that the S-shaped curves achieved by PAGE or IEF in a linear urea gradient represent the continuous changes of the equilibrium between the folded and the unfolded states of single-domain proteins. In the case of human TTR monomers, the two states have a different pI equivalent with the gain or loss of one charge unit. The inclination points of the curves represent the urea concentration, with about 50% of the monomer species in the folded state and 50% in the unfolded state. The difference between the inclination points of any two monomer species on the urea concentration scale corresponds to the difference of conformational stability towards urea. Thus, one can directly read from the presented patterns that the cysteinylated normal monomer (curve 1) is less stable than the S-sulfonated monomer (curve 8) and that the latter is less stable than the monomer with Cys10 oxidized to cysteic acid (curve 9). Similarly, one can read from the patterns in Fig. 13.2 that the variant monomer ATTR-I107V as well

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as the variant monomer ATTR-V30M is less stable than the normal TTR monomer and that the changes by amino acid substitution and ligand substitutions at Cys10 have an additive effect on the monomer conformational stability. This additive effect of changes at two different sites of the monomer structure is a characteristic of cooperativity in the sense that a change at one site has influence on the change at another site. There is experimental evidence that the effect of changes at Cys10 as a fixed site adds to the effect of amino acid substitutions at many different sites of the molecule (Altland et al. 2007). Thus, cooperativity happens between many, if not all, sites of the molecule. The S-shaped titration curves appear to be caused by changing equilibria between two states with different net charges and require that the reversible transition reactions between the two states are frequent and fast. Another important conclusion is that within the transition range of the urea gradient, monomers are always present in the folded as well as in the unfolded state and that there are no intermediate conformers of significant stability. Any stable intermediate in the transition between the two states would disrupt the continuity of the titration curves achieved by IEF. The observed continuity within the monomer titration curves is a strong argument against the existence of any stable, partially unfolded intermediate within the transition reactions between the folded and unfolded states, as has been postulated by others (Lashuel et al. 1998). However, the concept of a cooperative reversible unfolding-refolding reaction does not exclude that refolding of the unfolded state into the native conformation is incomplete by competing in vivo with alternative refolding transitions into intermediates as a source of toxic and/or amyloidogenic aggregates and/or with proteolytic breakdown of the unfolded material. Another aspect of conformational heterogeneity is demonstrated by comparison of the monomer patterns in Fig. 13.2a, b. At the 0 M urea left end of the gradient, the monomer titration curves of normal TTR and variant ATTR-I107V in Fig. 13.2a have identical pI values, while the visible curves of the variant ATTR-V30M start at a pI value closer to that of normal and variant monomers in the unfolded state. This means that in the absence of urea, more than half of the variant cysteinylated ATTR-V30M monomers are in the unfolded state or that the folded variant ATTRV30M monomer is sensitive to the acid environment. Among 28 tested amyloidogenic variants, 24 showed similar effects indicating another aspect of conformational microheterogeneity. The remaining four variants affected the monomer-monomer (ATTR-I68L, ATTR-I107V, and ATTR-V122I) and dimer–dimer contacts (ATTRV20I) (see Table 1 and Figs. 1 and 6–8 in Altland et al. 2007). A comparison of the patterns in Fig. 13.1c, d reveals that the decay of tetramers into monomers is slow but also reversible. The length of the flat part of the monomer titration curves (1 and 2) in Fig. 13.1c corresponds to the length of tetramer (T1) in Fig. 13.1d. Similarly, the length of the flat part of curves (8 and 9) in Fig. 13.1c corresponds to the length of tetramer (T5) in Fig. 13.1d. It is obvious that by prolonged exposure to the electric field, the tetramers between (T1) and (T5) decrease, while (T1) and (T5) remain or increase in density. The most reasonable interpretation is that reassembly of tetramers from monomers has occurred under


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conditions with most of the monomers in the folded state. This reassembly is not observed with ATTR-V30M. In summary, the patterns achieved by IEF in urea gradients permit detection of most of the heterogeneity of TTR by ligand and/or amino acid substitutions as well as the associated microheterogeneity of conformational stability. The patterns visualize the reversible transitions from the assembled tetramer to the unfolded monomer and support the concept of cooperative interactions between many or possibly all sites within the ordered structure of the molecule.

13.4

The Achilles’ Heel of Human TTR: His31

A gain of two positive charges accompanies the dissociation of the human TTR tetramer into the folded monomer and a gain of a third positive charge occurs which is associated with the unfolding of the monomer (Altland et al 1999). This change of charge can be derived from the pattern shown in Fig. 13.1d: At the 8 M end of the urea gradient, the difference in pI between the major monomer fraction containing monomers with Cys10 in the cysteinylated form and the minor monomer fraction containing monomers with Cys10 in the S-sulfonated form (Cys10-S-SO3 ) and Cys10 in the oxidized form (Cys10-SO3 ) corresponds to a difference of one charge unit. This difference in pI also corresponds to that between the folded and unfolded state of TTR monomers, to the difference between tetramers (T1) and (T5), as well as to the difference between the pIs of tetramer (T1) and the folded state of S-sulfonated monomers. This gain of three positive charges has been derived from the crystal structure of human TTR. Blake et al. (1978) have described His88 to be buried in the monomer-monomer contact. Thus, its protonated imidazole contributes to the net charge only after dissociation of dimers into monomers. A molecular explanation for the gain of a second and third positive charges has been proposed by Altland et al. (1999) using the crystal structure presented by Monaco et al. (1995). The second positive charge was gained when the loss of the positive charge of Arg21 adjacent to a hydrophobic patch contributing to the dimer-dimer contact is reversed in the isolated monomer. The third charge gain was derived from protonation of His31.NE2 after disruption of a hydrogen bridge between His31. NE2 and the hydroxyl of Ser46, as shown by Fig. 13.3. While Arg21 ands His88 are found in the sequence of TTR from many other species including wallaby, TTR from wallaby has His31 (human TTR) replaced by lysine and Ser46 (human TTR) replaced by alanine (Brack et al. 1995). A urea titration pattern of TTR from a wallaby in the absence and presence of a reducing agent is shown in Fig. 13.4a and b, respectively. There are four major differences between the patterns of TTR from wallaby and humans (see Fig. 13.1): (1) There is a dimer pattern (wallaby) instead of a tetramer pattern (human); (2) There are no detectable S-shaped titration curves for monomers from wallaby; (3) The transition from dimers to monomers of wallaby TTR is associated with the gain of only one positive charge which could be derived from His88 after dissociation of the dimer;

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Fig. 13.3 Detail from the crystal structure of human TTR showing the close vicinity of His31 and Ser46. The atomic coordinates were taken from Monaco et al. (1995). The distances between ˚ and between His31.NE2 and Ser46.OG of 2.92 A ˚ imply that Ala29.O and His31.ND1 of 2.79 A hydrogen bridges are formed with the hydrogen presented by His31.ND1 and Ser46.OG, respectively. Separation of b-strands B and C has been expected to be accompanied by protonation of His31.NE2 or the gain of a positive charge (Altland et al. 1999). We conclude that decreasing the pH of the aqueous environment weakens the hydrogen bridge between His31.NE2 and Ser46. OG and adds to the risk factors for human but not for wallaby TTR monomers to unfold. Notice that His31 and Ser46 in human TTR correspond to Lys31 and Ala46 in wallaby TTR (Brack et al. 1995)

(4) Dissociation of wallaby TTR dimers into monomers is observed at urea concentrations >3 M while the human patterns contain monomers even in the absence of urea, indicating resistance of wallaby TTR dimers against proton activity within the tested range of pH 5–6. Together with the above-mentioned observation that most of the variability of human TTR monomers by ligand and/or amino acid substitutions is associated with differences detectable at the 0 M urea end of the monomer titration curves, there appears to be a strong argument in favor of the view that a hydrogen bridge between His31 and Ser46 is the pH-sensitive structural element of the folded human monomer whereas protonated His31 characterizes the unfolded state of monomers. The experiments depicted in Fig. 13.5 were performed to study the pH sensitivity of human TTR within the range 7.4–6.5 as observed in the interstitial volume under inflammatory or ischemic conditions. TTR isolated as tetramers by PAGE was incubated for 1 h in the presence of 2% sodium dodecyl sulfate (SDS) under various conditions and subsequently submitted to SDS-PAGE. SDS inhibits (prevents) unfolded conformers from refolding. In the experiment shown in Fig. 13.5a,


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Fig. 13.4 Urea titration of TTR from wallaby by IEF in linear immobilized pH gradients without (a) and with (b) added reducing agent (50 mM DTT). The pH gradient was the same as used to achieve the patterns in Fig. 13.1c, d. There were similar pI levels of the monomers from both species at 8 M urea. However, there are several obvious differences between the patterns from wallaby (this figure) and human TTR (Figs. 13.1 and 13.2): (1) Instead of a tetramer pattern (human) there is a dimer pattern (zones D1 to D3 in a, wallaby); (2) There is only a shift by one charge unit associating the transition from dimers to monomers; (3) There are no S-shaped titration curves visualizing the transitions between the folded and unfolded states of monomers; (4) Within the range of 0 and 3 M urea monomers are detected for human but not for wallaby TTR

one set of samples was incubated for 1 h at pH 7 and 37 C, while an identical set was incubated for 50 min at 37 C followed by incubation for 10 min at 95 C. As expected, the rise of the temperature resulted in the complete denaturation of TTR into monomers, while TTR incubated at 37 C over the total time span of 1 h resulted in the transition of only a minor proportion of the dimer fraction into monomers. The monomer fractions resulting from incubation at both temperatures were indistinguishable. Even the splitting of the monomer fraction from individuals heterozygous for normal TTR and three different amyloidogenic variants was the same after incubation at both temperatures. This splitting represented differential binding of SDS to the denatured variant monomers and reflected the sensitivity of the procedure for minor differences between the monomeric structures. Therefore, we have concluded that the monomers resulting from the incubation of TTR at 37 C are completely unfolded structures in complex with SDS and that the transition from folded dimers to unfolded monomers does not involve any detectable conformational intermediates. The patterns in Fig. 13.5b, c show the dimer to monomer transition at pH levels between 7.4 and 6.5. It appears obvious that the decay of dimers into monomers is very sensitive to minor changes of pH within this range. This sensitivity is significantly increased for dimers from heterozygotes for normal TTR and variant ATTRV30M. Dimers from a heterozygote for normal TTR and the non-amyloidogenic

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a

b

c

Fig. 13.5 SDS PAGE of TTR after preincubation under various conditions. The TTR fractions were isolated from plasma by PAGE. The gel strips containing the TTR fraction were submitted to SDS PAGE after incubation under conditions given at the bottom of the patterns. The patterns of the left and right half in (a) (from Altland and Winter 2003, Fig. 3, by permission) evidently show that the monomer fraction achieved by incubation at 37 C over 1 h in 2% SDS is indistinguishable from the monomer fraction achieved after a standard procedure for preparing denatured protein– SDS complexes (i.e., incubation at 95 C over 10 min in 2% SDS), indicating complete unfolding at both temperatures. From the patterns in (b) we conclude: (1) The decay from dimers to unfolded monomers significantly increases when the pH drops from the physiological plasma level of 7.4–6.5; (2) Dimers from a heterozygote TTR-N/V30M are much more sensitive to denaturation (unfolding) than dimers from normal individuals; (3) Dimers from heterozygotes for normal TTR and the non-amyloidogenic variant TTR-H90N are as stable/unstable as dimers from normal individuals; (4) Dimers from wallaby are stable at all three studied pH levels. The patterns in (c) present a fine tuning for the decay of dimers into monomer within the pH range 7.2–6.8. Notice that at all tested pH levels known to be associated with various forms of interstitial acidosis there is an increasing decay of dimers into unfolded monomers correlating with increasing proton activity. In separate experiments we found that increasing the ionic strength of the incubation solution to near physiological levels decreased but did not eliminate the demonstrated effect of pH on the decay of dimerss

variant TTR-H90N had the same pH sensitivity as normal dimers. Dimers from wallaby TTR, however, remain stable at all tested pH levels. Mixed dimers from compound heterozygotes for the destabilizing variant ATTR-V30M and the stabilizing variant TTR-T119M were found to be more stable than dimers from heterozygotes for normal TTR and ATTR-V30M, indicating a cooperative interaction between two sites on different monomers. Also, S-sulfonation of Cys10 by sulfite or reduction by dithiothreitol (DTT) was found to stabilize the


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dimer. The decay of dimers into monomers obviously varies not only with the pH of the solvent and with most of the inherited amyloidogenic amino acid substitutions, but also with the ligand substitutions at Cys10, in an additive manner (Altland et al 2004; Gales et al 2007). The risk of human TTR unfolding under minor acidifying conditions requires a molecular explanation. Histidines with a pKa between 6 and 7 and the N-terminal amino group with pKa of about 7 represent the only sites for possible effects by differential protonation. Effects on conformational stability are expected only where these amino acids are involved in the maintenance of the structural integrity of a conformer. The N-terminal glycine of human TTR can be excluded, as it belongs to the disordered N-terminal end of all monomers. His88 is buried in the dimer and therefore has no access to the aqueous environment. His90 can be excluded because substitution by asparagine has no effect on pH sensitivity (see Fig. 13.5b). The structural environment of His56 does not present any argument supporting differential contributions of protonation/deprotonation to structural integrity. What remains is His31, where a hydrogen atom from the hydroxyl of Ser46 competes with protons from the solvent for the contact with His31.NE2.

13.5

Conclusions

This is the first compilation of evidence or arguments for His31 representing the most vulnerable site (‘‘Achilles’ heel’’) of human TTR under conditions of mild acidosis as observed in vivo under inflammatory or ischemic conditions (Tannock and Rotin 1989; Helmlinger et al. 1997; Kubasiak et al. 2002; Trevani et al. 1999; Marzouk et al. 2002; Agullo´ et al. 2002; Street et al. 2001). The sensitivity of the hydrogen bridge between His31 from b-strand B and Ser46 from b-strand C for acid adds to the stabilizing/destabilizing effects of mutations and ligand substitutions. The data presented here are consistent with our view that the invariable endpoint of destabilization is the unfolded TTR monomer, which may be considered as the source of any possible refolding product, including the native tetramer and all aggregates detected in patients with TTR amyloidosis. The demonstrated cooperativity of probably all sites within TTR tetramers, dimers, and monomers participating in the folding/unfolding transitions implies that trials to stabilize TTR at the thyroxine binding site (e.g., treatment by diflunisal), Cys10 (e.g., treatment by sulfite), and His31 (e.g., treatment by base, prevention of inflammation and ischemia) should have synergistic beneficial effects for all TTR amyloidosis patients by reducing the load of unfolded material. Unfolded amyloidogenic polypeptides have been demonstrated to react with (–)-epigallocatechin-3-gallate (EGCG) from green teas to form non-amyloidogenic oligomers (Ehrnhoefer et al. 2008). Curcumin inhibits aggregation of Alzheimer’s amyloid b peptides (Ono et al 2004; Yang et al. 2005). Possibly, treatment by EGCG (Hunstein 2007) and curcumin (both with added piperine to increase the bioavailability (Shoba et al. 1998; Altland and Schreiner, unpublished observation (EGCG)) could help to

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prevent the generation of harmful aggregates of TTR and amyloidosis. Furthermore, His31 is found only in human and rabbit TTRs. Residue 31 in nearly all other species of TTR sequenced (including rat and mouse) is Lys (see Richardson 2007). This may be a reason why TTR amyloidosis has not yet been observed in species other than humans.

Acknowledgment We appreciate the technical assistance of Pia Winter.

References Agullo´ L, Garcia-Dorado D, Escalona N, Inserte J, Ruiz-Meana M, Barrabee´ JA, Mirabet M, Pina P, Soler-Soler J (2002) Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells. Am J Physiol Heart Circ Physiol 383:H917–H925 Altland K, Benson MD, Costello CE, Ferlini A, Hazenberg BPC, Hund E, Kristen AV, Linke RP, Merlini G, Salvi F, Saraiva MJ, Singer R, Skinner M, Winter P (2007) Genetic microheterogeneity of human transthyretin detected by IEF. Electrophoresis 28:2053–2064 Altland K, Kast C, Rauh S, Sgraja T (1982) Demonstration of prealbumin, apolipoprotein AI and 7S-beta1-globulin from human blood by double one-dimensional electrophoresis. In: Peeters H (ed) Protides of the biological fluids. Vol 30. Pergamon Press, Oxford, pp 595–598 Altland K, Winter P (1999) Potential treatment of transthyretin-type amyloidosis by sulfite. Neurogenetics 2:183–188 Altland K, Winter P, Saraiva MJM, Suhr O (2004) Sulfite and base for the treatment of familial amyloidotic polyneuropathy: two additive approaches to stabilize the conformation of human amyloidogenic transthyretin. Neurogenetics 5:61–67 Altland K, Winter P, Sauerborn MK (1999) Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients. Electrophoresis 20:1349–1364 Alves IL, Altland K, Almeida MR, Winter P, Saraiva MJ (1997) Screening and biochemical characterization of transthyretin variants in the Portuguese population. Human Mutat 9: 226–233 Benson MD (2003) The hereditary amyloidoses. Best Practice Res Clin Rheumatol 17:909–927 Blake CCF, Deisow MJ, Oatley SJ, Re´rat B, Re´rat C (1978) Structure of prealbumin: secondary, ˚´ . J Mol Biol tertiary and quarternary interactions determined by Fourier Refinement at 1.8 A 121:339–356 Blake C, Serpell L (1996) Synchroton X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix. Structure 4:989–998 Brack CM, Duan W, Hulbert AJ, Schreiber G (1995) Wallaby transthyretin. Comp Biochem Physiol 110B:523–529 Colon W, Kelly JW (1992) Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31:8654–8660 Connors LH, Lim A, Amareth L, Prokaeva T, Roskens VA, Costello CE (2003) Tabulation of human transthyretin (TTR) variants, 2003. Amyloid: J Protein Folding Disord 10:160–184 Cornwell GG, Murdoch WL, Kyle RA, Wetsermark P, Pitka¨nen P (1983) Frequency and distribution of senile cadiovascular Amyloid. Am J Med 75:618–623 Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Kurz R, Engemann S, Pastore A, Wanker EE (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nature Struct Mol Biol 15:558–566


213

Feldin P, Fex G (1984) The increased negative charge of prealbumin in cerebrospinal fluid is acquired in vitro by oxidation of the cysteinylated residue without formation of disulfides. Scand J Clin Lab Invest 44:231–238 Fitch NJS, Akbari MT, Ramsden DB (1991) An inherited non-amyloidogenic transthyretin variant, [Ser6]-TTR, with increased thyroxine-binding affinity, characterized by DNA sequencing. J Endocrinol 129:309–313 Gales L, Saraiva MJ, Damas AM (2007) Structural basis for the protective role of sulfite against transthyretin amyloid formation. Biochim Biophys Acta 1774:59–64 Hack V, Breitkreuz R, Kinscherf R, Ro¨hrer H, Ba¨rtsch P, Taut F, Benner A, Dro¨ge W (1998) The redox state as a correlate of senescence and wasting and as a target for therapeutic intervention. Blood 92:59–67 Helmlinger G, Yuan F, Dellian M, Jain RK (1997) Interstitial pH and pO 2 gradients in solid tumors in vivo: high resolution measurements reveal a lack of correlation. Nat Med 3: 177–182 Ho¨rnberg A, Eneqvist T, Olofsson A, Lundgren E, Sauer-Eriksson AE (2000) A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 22:649–669 Holmgren G, Ando Y, Wikstro¨m L, Rygh A, Suhr O (1997) Discordant symptoms in monozygotic twins with familial amyloidotic polyneuropathy (FAP) (TTR met30). Amyloid: J Protein Folding Disord 4:178–180 Hunstein W (2007) Epigallocathechin-3-gallate in AL amyloidosis: a new therapeutic option? Blood 110:2216 (letter) Jacobson DR, McFarlin DE, Kane I, Buxbaum JN (1992) Transthyretin Pro55, a variant associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurological involvement. Hum Genet 89:353–356 Jenne DE, Denzel K, Bla¨tzinger P, Winter P, Obermaier B, Linke RP, Altland K (1996) A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dime-dimer contacts and causes systemic amyloidosis. Proc Natl Acad Sci USA 93:6302–6307 Jones DP (2006) Extracellular redox state: refining the definition of oxidative stress in aging. Rejuvenation Res 9:169–181 Kanda Y, Goodman DS, Canfield RE, Morgan FJ (1974) The amino acid sequence of human plasma prealbumin. J Biol Chem 249:6796–6805 Kishikawa M, Nakanishi T, Miyasaki A, Shimizu A (1999) A simple and reliable method of detecting variant transthyretins by mutidimensional liquid chromatography coupled with electrospray ionization mass spectrometry. Amyloid: J Protein Folding Disord 6:48–53 Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA (2002) Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci USA 99:12825–12830 Kyle RA, Gertz MA, Linke RP (1992) Amyloid localized to tenosynovium at carpal tunnel release. Immunohistochemical identification of amyloid type. Am J Clin Pathol 97:250–253 Kyle RA, Spittell PC, Gertz MA, Li CY, Edwards WD, Olson LJ, Thibodeau SN (1996) The premortem recognition of systemic senile amyloidosis with cardiac involvement. Am J Med 101:395–400 Lashuel HA, Lai Z, Kelly JW (1998) Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implication for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 37:17851–17864 Mansoor MA, Svardal AM, Ueland PM (1992) Determination of the in vivo redox status of cysteine, cysteinylglycine, and glutathione in human plasma. Anal Biochem 200:218–229 Marzouk SAM, Buck RP, Dunlap LA, Johnson TA, Cascio WE (2002) Measurement of extracellular pH, K +, and lactate in ischemic heart. Anal Biochem 308:52–60 Mills BJ, Weiss MM, Lang CA, Liu MC, Ziegler C (2000) Blood glutathion and cysteine changes in cardiovascular disease. J Lab Clin Med 135:396–401 Monaco HL, Rizzi M, Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041

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Moriarty-Craige SE, Adkinson J, Lynn M, Gensler G, Bressler S, Jones DP, Sternberg P (2005) Antioxidant supplements prevent oxidation of cysteine/cystine redox in patients with agerelated macular degeneration. Am J Ophthalmol 140:1020–1026 Nepomuceno AI, Mason CJ, Muddiman DC, Bergen III HR, Zeldenrust SR (2004) Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization Fouriertransform ion-cyclotron-resonance mass spectrometry. Clin Chem 50:1535–1543 Nolin TD, McMenamin ME, Himmelfarb J (2007) Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: application to studies of oxidative stress. J Chromatogr B 852:554–561 Ono K, Hasegawa K, Naiki H, Yamada M (2004) Curcumin has potential anti-amyloidogenic effects for Alzheimer’s b-amyloid fibrils in vitro. J Neurosci Res 75:742–750 Plante´-Bordeneuve V, Carayol J, Ferreira A, Adams D, Clerget-Darpoux F, Misrahi M, Said G, Bonaı¨ti-Pellie´ C (2003) Genetic study of transthyretin amyloid neuropathies: carrier risk among French and Portuguese families. J Med Genet 40(11):e120 Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258:137–193 Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PSSR (1998) Influence of Piperine on the pharmacokinetics of Curcumin in animals and human volunteers. Planta Medica 64: 353–356 Street D, Bangsbo J, Juel C (2001) Interstitial pH in human muscle during and after dynamic graded exercise. J Physiol 537:993–998 Tannock IF, Rotin D (1989) Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res 49:4373–4384 The´berge R, Connors L, Skinner M, Skare J, Costello CE (1999) Characterization of transthyretin mutants from serum using immunoprecipitation, HPLC/ electrospray ionization and matrixassisted laser desorption/ionization mass spectrometry. Anal Chem 71:452–459 Trevani AS, Andonegui G, Giordano M, Lo´pez DH, Gamberale R, Minucci F, Geffner JR (1999) Extracellular acidifcation induces human neutrophil activation. J Immunol 162:4849–4857 Westermark P, Berstro¨m J, Solomon A, Murphy C, Sletten K (2003) Thransthyretin-derived senile systemic amyloidosis: clinicopathological and structural considerations. Amyloid: J Protein Folding Disord 10 (Suppl 1):48–54 Westermark P, Sletten K, Johansson B, Cornwell GG (1990) Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA 87:2843–2845 Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, Chen P, Kayed R, Glabe CG, Frautschy SA, Cole GM (2005) Curcumin inhibits formation of amyloid b oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem 280:5892–5901 Zhang O, Kelly JW (2003) Cys-10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions. Biochemistry 42:8756–8761

Chapter 14

New Therapeutic Approaches for Familial Amyloidotic Polyneuropathy (FAP) Yukio Ando, Masaaki Nakamura, Mistuharu Ueda and Hirofumi Jono

Abstract Although liver transplantation is the only therapy to halt the clinical manifestations of transthyretin (TTR) related familial amyloidotic polyneuropathy (FAP), the therapy has given rise to several problems. An alternative treatment is needed. After the precursor protein of amyloid fibrils in FAP was established in 1983, several different approaches have been investigated as an essential therapy for FAP: (1) reduction of variant TTR levels in plasma, (2) down regulation of variant TTR gene mRNA, (3) inhibition of amyloid deposition, (4) stabilization of the tetrameric TTR structure and (5) replacement of the variant TTR gene with the normal TTR gene (which can be achieved by liver transplantation or by gene therapy). In this chapter, we introduce these research strategies and trials and discuss the possibility of the optimal treatment for FAP. Keywords Antibody therapy, Gene therapy, Liver transplantation

14.1

Introduction

After Andrade identified patients with familial amyloidotic polyneuropathy (FAP) in Portugal in 1952 (Andrade 1952), many foci of FAP cases have been reported worldwide. As patients with FAP show various serious systemic symptoms, such as cardiac and renal dysfunction, gastrointestinal disorders, ocular disorders, glandular and autonomic dysfunction, and peripheral neuropathy, many trials have attempted to treat these symptoms (Araki 1984; Benson 1989; Benson and Uemichi 1996; Ando et al. 1993; Ando and Suhr 1998). Although liver transplantation has become a well-established treatment for halting the progression of FAP-related clinical Y. Ando (*) Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, Japan e-mail: [email protected]


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symptoms, no truly effective therapy has been designed (Holmgren et al. 1993; Ericzon et al. 1999; Skinner et al. 1994; Takei et al. 1999; Ando et al. 1995; Suhr et al. 1996; Lendoire et al. 1999), and several problems related to its use have arisen (Ando et al. 2001, 2002). We cannot use liver transplantation for these patients as long-term therapy for the following reasons: (1) Medical care before and after the surgery and the surgery itself are extremely expensive; (2) Patients who have received transplants must continue lifelong administration of immunosuppressants after the surgery, and these agents may have adverse side effects; (3) Carriers of the amyloidogenic transthyretin (ATTR) gene who have no clinical symptoms cannot undergo liver transplantation before the onset of the disease; (4) Clinical symptoms of FAP that were present before the surgery continue to occur after liver transplantation; (5) The worldwide shortage of liver donors means that all FAP patients cannot undergo the surgery; and (6) After liver transplantation, patients with FAP ATTR Val30Met or other types of FAP have been reported to develop ocular disorders, caused by vitreous amyloid deposits, and central nervous system symptoms, because of leptomeningeal amyloidosis, as an effect of continuous TTR production by the retina and choroid plexus, respectively. To overcome all these problems, we must develop a new noninvasive effective treatment. The following methods can be considered potential FAP treatment strategies (Table 14.1): (1) reduction of variant TTR levels in the plasma; (2) down regulation of the variant TTR gene mRNA; (3) inhibition of amyloid deposition; (4) stabilization of the tetrameric TTR structure; and (5) replacement of the variant TTR gene with the normal TTR gene (which can be achieved by liver transplantation or by gene therapy) (Table 14.1). Here, we describe new therapeutic approaches for FAP that our group has recently investigated, as well as previously used therapies. We hope that at least one of these approaches will lead to an effective treatment of FAP. Table 14.1 Strategies for treatment of familial amyloidotic polyneuropathy 1. Reduction of variant TTR levels in plasma A. Plasma exchange B. Affinity column chromatography C. TTR absorption column chromatography 2. Downregulation of TTR gene mRNA A. Injection of a large amount of normal TTR 3. Inhibition of amyloid deposition A. Prevention of amyloid formation BSB Antibody therapy B. Dissolution of amyloid fibrils IDOX 4. Stabilization of the tetrameric TTR structure A. NSAIDs B. Cr3+ 5. Replacement of the variant TTR gene with the normal TTR gene A. Liver transplantation B. Gene therapy

14 New Therapeutic Approaches for Familial Amyloidotic Polyneuropathy (FAP)

14.2

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Elimination of Variant TTR in Blood Circulation

Plasma exchange, affinity column chromatography binding with a monoclonal antibody, and use of a special column having a specific affinity for TTR have been considered as possible methods for elimination of ATTR in blood circulation (Sales-Luis et al. 1991; Ikegawa et al. 1988; Regnault et al. 1992; Costa and Costa 1998; Tokuda et al. 1998). Sales-Luis et al. (1991) performed plasma exchange for 1.5–4.5 years with nine patients with FAP ATTR Val30Met, and the effect of this therapy on the disease was compared with the course of the illness in nontreated FAP patients. Progression of the disease was slightly suppressed, and body weight loss and diarrhea were effectively treated (Sales-Luis et al. 1991, Continho et al. 1988). However, Sales-Luis stopped this plasma exchange because of allergic reactions or virus infection (Continho et al. 1988). Ikegawa and colleagues performed plasma exchange once a month with three FAP patients (Ikegawa et al. 1988). They reported that the levels of variant TTR in the plasma decreased from 39 to 19% of the total TTR levels but returned to the values that were seen before treatment (Ikegawa et al. 1988). Objective and subjective evaluations indicated no improvement in laboratory and clinical data. The treatment was very expensive, and the numbers of treated patients were very small. In addition, no follow-up studies have been reported. Costa and colleagues reported application of an affinity column consisting of a monoclonal antibody for ATTR Val30Met to eliminate the variant TTR from the blood stream (Regnault et al. 1992; Costa and Costa 1998). However, the method was unsatisfactory because the rate of elimination was too slight to halt the progression of FAP, and the elevation of plasma TTR levels returned, which was observed just after treatment (Regnault et al. 1992; Costa and Costa 1998). Moreover, vitamin A levels were decreased, and four patients had the TTR–TTR antibody complex in their plasma because of leakage of the antibody from the column to the blood stream (Costa and Costa 1998). Loss of skin hair throughout the whole body and allergic reactions has been reported. Tokuda et al. used the special column PA-01, which showed a specific affinity for TTR, to eliminate ATTR from the blood (Tokuda et al. 1998). However, this therapy had the same defects as did the two trials just mentioned. Just after treatment, plasma ATTR levels decreased by 50%, but they returned to the same values seen before treatment. From these studies, we can conclude that these methods cannot provide sufficient elimination of plasma ATTR, and that regulation of ATTR mRNA production does not seem to be appropriate because TTR itself is a rapid turnover protein whose plasma half-life is about 2 days (Jouquan et al. 1983; Dickson et al. 1982; Ando et al. 1995). FAP patients who want these treatments must go to a hospital every day or every hour of the day to achieve sufficient reduction of plasma ATTR levels. These results also suggest that ordinary antisense or ribosomal therapies that would target variant TTR mRNA would not be advantageous treatments.

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Down regulation of Variant TTR

It is well documented that FAP patients develop systemic disorders induced by amyloid deposition in the viscera in addition to peripheral nerves. As the disease progresses, FAP patients become emaciated, and hypoproteinemia and hypoalbuminemia are often observed. To compensate for the hypoproteinemia and hypoalbuminemia, intravenous injections of fresh frozen plasma (FFP) are often used. FFP contains a significant amount of TTR in addition to various plasma proteins. A surprising finding has been that the variant TTR levels decreased while levels of total TTR, total protein, and albumin in the plasma increased. It is possible that some genetic regulation occurs in response to the FFP injection. We monitored the changes in plasma TTR levels after injection of a large amount of FFP when they treated FAP patients for hypoproteinemia and hypoalbuminemia (Ando et al. 1997). From the 24th to 48th h after injection, the total plasma TTR levels were elevated, and variant TTR levels decreased, accompanied by an elevation of plasma total protein and albumin levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was injected intravenously into mice and into patients with FAP ATTR Val30Met. Injection of 3 mg of purified TTR into C57Black6 mice resulted in decreased expression of TTR mRNA from the 6th to the 24th h after injection, and then TTR mRNA expression gradually increased up to the 48th h after injection. Injection of 400 mg of normal human TTR into three FAP patients caused elevated total plasma TTR levels and significantly decreased variant TTR levels from the 24th to the 48th h after injection. These results suggested that this method allowed down regulation of the harmful protein by means of replacement with the normal protein. This phenomenon may explain the mechanism for one of the possible methods for decreasing the amount of harmful protein in the circulation. However, because reduction of the amount of variant TTR in the plasma was not sufficient, and as TTR is a rapid turnover protein as described previously (Jouquan et al. 1983; Dickson et al. 1982; Ando et al. 1995), this treatment cannot be applied as an effective therapy for FAP patients.

14.4

Inhibition of Amyloid Deposition in Tissues

14.4.1 Compounds Binding to Amyloid Fibrils 14.4.1.1

Congo Red

Congo red, with the chemical name 3,30 -[(1,10 -biphenyl)-4,40 -diylbis(azo)] bis(4-amino-1-naphthalene acid) disodium salt, is a commonly used histological dye for amyloid detection. At first, Congo red was introduced in Berlin in 1885 as the first of the economically lucrative direct textile dyes (Steensma 2001). As a


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histological stain, Congo red was applied in 1927 by Belgian neuropathologist, Paul Divry, who noted that Congo red gave a green birefringence to the amyloid deposited in the brain tissue under polarized light (Divry 1927). Although the mechanism of interaction of Congo red with amyloid fibrils is not well understood, it is generally believed that Congo red binding depends on the secondary configuration of the amyloid fibril, consisting predominantly of cross b-pleated sheets. Investigations on the inhibitory effects of Congo red on amyloid formation in different, in vitro models of amyloidosis indicated a complexity of involved mechanisms, from inhibition of protein depositions, through blockade of amyloid-cell interaction, to the modulation of the regulation of intracellular homeostasis (Frid et al. 2007). Increasing lines of evidence indicate that Congo red may prevent Ab amyloid fibril formation by binding and stabilizing native monomers or partially folded intermediates, thus reducing the concentration of oligomers (Gellermann et al. 2006). Contrary to that, when the nucleus had already formed for seeding, Congo red might promote further amyloid formation (Fraser et al. 1992). Studies of the concentration-dependent effects of Congo red were undertaken by Kim et al. (2003), who used a variant of the immunoglobulin light chain. They demonstrated that at low concentrations, Congo red binding promoted the generation of partially folded, aggregation prone forms of the proteins into oligomers and protofilament intermediates, resulting in accelerated fibril formation. At higher concentrations, however, Congo red inhibited fibril formation supporting the denatured state, which is much less prone to aggregation. The data proposed that Congo red exhibited antiamyloidogenic properties. However, it is potentially toxic (Giger et al. 1974). It can reduce serum protein concentration and cause platelet aggregation. Especially if administered orally, Congo red is cleaved by enzymes to benzidine, a highly carcinogenic compound, known to induce hepatocellular and urinary bladder carcinomas. Increased efforts aim to produce analogues that will maintain or enhance the antifibrillar aggregation activities of Congo red, but will not be toxic.

14.4.1.2

Congo Red Derivatives

In systemic amyloidoses such as FAP, AA amyloidosis, AL amyloidosis, and dialysis-related amyloidosis (DRA), biopsy samples are often obtained from the gastrointestinal tract and abdominal fat to make the diagnosis, because amyloid deposits are usually found in these tissues at the early stage of the disease (Guy and Jones 2001; Kaplan et al. 1999; Masouye 1997). However, in certain cases it is sometimes difficult to construct the diagnosis by using only these biopsy samples, because the pattern of amyloid deposition in the body varies in each individual. In addition, a biopsy for diagnostic purposes cannot usually be performed in patients with localized amyloidosis, such as amyloidosis in Alzheimer disease and endocrine amyloidosis (Westermark and Westermark 2000). Diagnosis at the early stage of amyloidosis might be possible if a tool were available that incorporated real-time

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amyloid monitoring, such as radioisotope-labeled scintigraphy (Hawkins et al. 1990; Puille et al. 2002). Among several histopathological methods utilizing stains such as Congo red, fast scarlet, and thioflavine S, Congo red staining is one of the most popular for detection of amyloid deposits in tissues. However, judging the positivity of Congo red-positive lesions is sometimes difficult, and false-positive or false-negative results may be obtained. Moreover, Congo red staining cannot be used for in vivo studies because of its toxic effects in the human body. Chrysamine G, a Congo red derivative with a similar molecular structure used for the same purpose as Congo red, has the same limitations (Klunk et al. 1995, 1998; Dezutter et al. 2001a, b). To overcome these problems, we should have a new tool for examination of amyloid deposition in tissues. (trans, trans)-1-Bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (BSB), which has been found to bind to amyloid plaques in postmortem samples of brains from patients with Alzheimer disease, is also a Congo red derivative and has been the focus of recent attention (Dezutter et al. 1999; Skovronsky et al. 2000; Schmidt et al. 2001; Zhuang et al. 2001). As this compound is lipophilic, it can traverse the blood–brain barrier, and bind itself to amyloid fibrils in senile plaques when it is injected intravenously into transgenic mice with Alzheimer disease. Moreover, this compound can detect cell inclusion bodies derived from a-synclein. However, no studies have addressed the question of whether the compound could become a useful tool for detection of amyloid deposits in systemic amyloidosis such as FAP. Various plasma proteins in the systemic circulation may disturb the binding of BSB to amyloid fibrils in tissues.

14.4.1.3

BSB as a Valuable Therapeutic Drug

We used BSB to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial Creutzfeldt-Jakob disease, and systemic amyloidosis, such as FAP, AA amyloidosis, AL amyloidosis, and DRA (Ando et al. 2003). Histopathological methods were employed to examine the reactivity of BSB with amyloid fibrils, and the results were compared with those from studies with immunohistochemical and Congo red staining and polarized light. BSB reactivity in vivo was investigated in mice in which AA amyloidosis had been induced. The authors determined the affinity of BSB and Congo red for purified amyloid fibrils by using a highly sensitive 27-MHz quartz crystal microbalance (Okahata et al. 1998; Matsuno et al. 2001). To test the usefulness of BSB as a possible therapeutic agent in FAP, its inhibitory effect on the formation of TTR amyloid in vitro was examined by means of electron microscopy (Ando et al. 2003). BSB showed a significant affinity for amyloid fibrils purified from FAP patients’ samples and the suppressed formation of TTR amyloid in acidic conditions in vitro. BSB may become a valuable therapeutic drug, although further evaluation should be needed to determine therapeutic effects in vivo.


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14.4.2 Drugs Disrupting Amyloid Fibrils 14.4.2.1

IDOX

40 -Iodo-40 -deoxydoxorubicin (IDOX) has been reported to bind to amyloid and lead to the catabolism of amyloid in deposits.This chemical compound was first reported by Merlini et al. as an agent that would bind to amyloid fibrils found in five different types of amyloidosis (Merlini et al. 1995). A multicenter study attempted to develop a dosing schedule to confirm these results. IDOX was administered to AL amyloidosis patients at 15 mg once per week for four consecutive weeks, and this therapy was repeated every 3 months up to four times (Gertz et al. 2002). However, the results were not clear, and no obvious effect on the patients was seen. The authors concluded that the protocol produced insufficient activity at the dose used in the study. It is possible that IDOX does not show the same attraction for amyloid in FAP as that seen in the AL amyloidosis trial. In an in vitro study, Sebastiao et al. observed an interaction of IDOX with ATTR Leu55Pro and reported that monoclinic ATTR Leu55Pro crystals soaked with IDOX undergo rapid dissociation (Sebastiao et al. 2000). Moreover, under the same conditions, the orthorhombic wild-type TTR crystals were quite stable. This result was explained by the different TTR conformations in the crystals of the two proteins: the ATTR Leu55Pro had the necessary conformation for IDOX binding, but the same structure was not present in the crystallized wild-type protein. This study presented a theoretical model of the interaction of ATTR Leu55Pro with IDOX that is consistent with the dissociation of the amyloid-like oligomer. In this model, the IDOX iodine atom was buried in a pocket located between the two beta-sheets of the ATTR Leu55Pro monomer, with the long axis of the IDOX aromatic moiety nearly perpendicular to the direction of the beta-sheets (Sebastiao et al. 2000). This chemical compound, first developed as an anticancer drug, may be one of the most promising drugs for FAP. However, it has nephrotoxic effects, and an IDOX derivative that is less toxic to the kidneys should be sought, because one of the target organs for amyloid deposition in FAP is the kidney, and renal dysfunction often occurs during the course of the illness. In any case, we must wait for further information from in vivo studies of the effect of IDOX to derive conclusions about the usefulness of IDOX for FAP patients.

14.4.2.2

Tetracycline and Its Derivatives

Cardoso et al. showed that tetracycline and its derivatives, doxycycline, rolitetracycline, and minocycline, interfered with TTR amyloid fibril formation in vitro (Cardoso et al. 2003), acting as fibril disrupters. Doxycycline was revealed as the most effective of the four compounds in that study. Doxycycline affects many mammalian cell functions including proliferation, migration, apoptosis, and matrix remodeling (Bendeck et al. 2002). It was also shown that doxycycline had activity

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against amyloid formation of Ab (Forloni et al. 2001). Cardoso et al. also investigated the effect of doxycycline treatment in vivo using transgenic ATTR Val30Met mice (Cardoso and Saraiva 2006). In that study, tissue analysis by Congo red staining indicated that doxycycline was able to disaggregate TTR amyloid fibrils. However, doxycycline was not able to act on nonfibrillar TTR deposits, as the overall TTR load was similar in both treated and untreated groups of transgenic mice. These in vivo results corresponded with the in vitro findings showing that doxycycline was not capable of inhibiting TTR fibril formation till a certain fibril length (Cardoso et al. 2003). 14.4.2.3

Immunotherapies to Promote Clearance of Amyloid Deposition

Antibody Therapy Against Amyloidogenic TTR Gustavsson et al. used various antigenic mapping procedures to determine whether major antigenic sites differ between normal TTR, ATTR, and in situ amyloid fibrils (Gustavsson et al. 1994). Results of this study suggested that the antigenic sites on normal plasma TTR included the AB loop and the CD loop. The amino acid sequences associated with these loops are present on the outside of the TTR molecule. On the other hand, an antiserum against b-strand H (a peptide TTR115–124, which constitutes the monomer-to-monomer interaction areas in the dimer (Blake et al. 1978), reacted only with ATTR in amyloid fibrils but not with normal plasma TTR. Those results suggested that there was an altered configuration of TTR within amyloid fibrils when compared with plasma TTR. The antiserum to TTR115–124, which so far has turned out to be amyloid specific, may serve as a valuable probe and may be useful in antibody therapies. However, Bergstrom et al. (2006) indicated that the epitope TTR115–124 was hidden in ATTR Tyr114Cys both in vitreous aggregates and in mature amyloid deposits in various tissues, by immunochemical means. Differences in the tertiary structure may exist between amyloid fibrils formed by different ATTR variants. A monoclonal antibody, MAb39–44, reacting with high molecular weight aggregates of TTR but not with tetrameric, TTR was also generated and characterized by Goldsteins et al. (Goldsteins et al. 1999). This antibody recognized a cryptic epitope that was expressed in isolated recombinant amyloidogenic mutants and in ex vivo amyloid. MAb39-44 could selectively detect TTR variants in plasma from carriers and patients with amyloidogenic TTR mutations (Palha et al. 2001). MAb39-44 may be useful for detecting amyloidogenic TTR variants and may be a candidate for the antibody in therapies. To determine the usefulness of these antibodies for the immunotherapy, in vivo studies should be performed. Vaccine Therapy A TTR mutant Tyr78Phe, designed to destabilize the native structure, has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts


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only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils (Redondo et al. 2000). Terazaki et al. demonstrated that immunization with TTR Tyr78Phe was powerful in reducing TTR deposition and clearing amyloid deposition in an FAP rodent model transgenic for the human mutant Val30Met with deposition in the gastrointestinal tract (Terazaki et al. 2006). Immunization with TTR Val30Met, an intrinsic protein found in FAP patients, could not induce a high antibody titer in transgenic (Tg) mice possessing the human ATTR Val30Met gene and did not prevent TTR and amyloid deposition in these mice. By contrast, immunization of Tg mice with TTR Tyr78Phe produced a higher titer of antibodies against Tyr78Phe TTR in the serum, than with ATTR Val30Met, suggesting significant structural changes of the Tyr78Phe mutant comparative to the Val30Met mutant, although some epitopes might be shared. Tissue specimens from the Tg mice immunized with Tyr78Phe demonstrated inflammatory cell infiltration and hyperplasia of lymphoid tissues only in or near the areas of usual TTR deposition which were absent in other areas of the tissue. Most cells in the lymphoid tissues immunoreacted predominantly with anti-CD45 (a surface marker of T cells) antibody but not with anti-CD5 (a surface marker of B cells) antibody. Inflammatory cell-infiltrated lesions displayed a reaction for antiMac-1 antibody, indicating tissue infiltration by macrophages. It was difficult to clearly discern the molecular mechanisms underlying the effects observed that can preclude both prevention and clearance of deposition. However, the infiltration of a large number of B cells and macrophages and the detection of IgG in lesions where TTR and amyloid deposits normally appear suggested a clearance mechanism involving FcR-mediated phagocytosis, as earlier reported in the Alzheimer immunization protocols (Bard et al. 2000). The target for this therapy may be applied in FAP ATTR Val30Met patients with amyloid deposition in tissues. In addition, the therapy could be used as a vaccination for healthy TTR variant gene carriers to prevent TTR amyloid deposition in tissues.

14.5

Stabilization of the Tetrameric Structure of TTR

The amyloid formation working hypothesis established by substantial studies, gave rise to the idea that stabilizing tetrameric TTR is a promising method for prevention of amyloid formation (Kelly and Lansbury 1994; Klabunde et al. 2000; Almeida et al. 2005; Johnson et al. 2005). Colon et al. first demonstrated this concept by using recombinant wild-type TTR and variant TTR (Colon and Kelley 1992; McCutchen et al. 1993). Normal TTR behaves as a tetramer and binds to retinol binding protein (RBP) and thyroxine (T4) in plasma (Monaco et al. 1995). Tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a compact nonnative monomer with low conformational stability can lead to amyloid fibril formation (Quintas et al. 1999). Although several of the 50 FAP-associated TTR single-site mutations have a normal tetrameric structure under physiological

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conditions (Connors et al. 2000), these mutations significantly destabilize the tetramer (McCutchen et al. 1993, 1995). Recent biochemical and pathological studies further revealed that instability of the terameric form of TTR by mutation, especially FAP ATTR Val30Met, leads to amyloid formation in the tissues of FAP patients (Saraiva 2001). It is interesting to note that subjects possessing the TTR Thr119Met gene are asymptomatic carriers, and that compound heterozygotes having ATTR Val30Met and TTR Thr119Met genes show very mild FAP symptoms or have no symptoms (Alves et al. 1993). Alves et al. demonstrated by semi denaturing isoelectric focusing that patients possessing TTR Thr119Met or ATTR Val30Met/TTR Thr119Met genes showed marked TTR tetrameric structural stability (Alves et al. 1997; Almeida et al. 1997). Terazaki et al. also reported an interesting late-onset compound heterozygote patient with FAP ATTR Val30Met/ TTR Arg104His who had very mild and slowly progressive clinical symptoms and whose tetrameric TTR stability was greater than that of the TTR from a compound heterozygote ATTR Val30Met/TTR Thr119Met patient (Almeida et al. 2000; Terazaki et al. 1999). These lines of evidence suggest that stabilizing the tetrameric TTR, as a potential therapeutic strategy, is a prerequisite for prevention of amyloid formation. On the basis of this concept, several treatments were proposed and examined (Fig. 14.1).

14.5.1 Various Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Derivatives As thyroxine (T4) is one of the most important molecules for stabilizing TTR in its tetrameric form, T4-based therapeutic drugs have been proposed. Binding of T4 and their derivatives stabilizes the native conformation of TTR and inhibits amyloid formation in vitro and possibly in vivo (Miroy et al. 1996). In fact, it has been proposed that, in the cerebral spinal fluid (CSF) where a high concentration of TTR with bound T4 is found, the protein is in the stable tetrameric conformation and does not lead to detectable TTR amyloid formation in the brain (Miroy et al. 1996). Baures et al. reported that various NSAIDs have the potential for stabilizing the tetramecic TTR (Baures et al. 1999). Several NSAIDs, which bind and inhibit the cyclooxygenases, also bind to TTR as mimic compounds of T4 (Baures et al. 1998, 1999; Munro et al. 1989). The structure of NSAIDs resembles the structure of T4 and these drugs bind to TTR via a T4 binding site (Baures et al. 1999). Peterson et al. demonstrated that the three-dimensional structure of TTR with flufenamic acid (FLU), a derivative of NSAIDs is a more efficacious TTR amyloid inhibitor than the natural ligand T4 (Peterson et al. 1998). Difulnisal and flufenamic acid exhibit notable inhibition of both acid-mediated aggregation and urea-mediated tetramer dissociation of the most common disease associated variants (Johnson et al. 2005; Hammarstro¨m et al. 2003). Tojo et al. reported that therapeutic serum concentrations of difulnisal (100–200 mM) stabilized serum variant TTR tetramer


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Replacement of the variant TTR gene with the normal TTR gene Liver

Liver transplantaon Gene therapy

Down–regulaon of TTR gene mRNA Injecon of a large amount of normal TTR

Stabilizaon of the tetrameric TTR structure NSAIDs Cr3+ Fx-1006 Dissociaon

Tetrameric TTR Reducon of variant TTR levels in plasma Plasma exchange Affinity column chromatography TTR absorpon column chromatography

Misfolding

Monomeric TTR Prevenon of amyloid formaon Dissoluon of amyloid fibrils IDOX Tetracycline

BSB Anbody therapy Radical scavenger

TTR deposits

Polymerizaon

Amyloid Fibrils

Fig. 14.1 Working hypothesis of amyloid formation in FAP and points of intervention for our treatments. Based on the pathogenesis of FAP, various therapeutic approaches can be considered as potential FAP treatment strategies: (1) reduction of variant TTR levels in plasma, (2) down regulation of variant TTR gene mRNA, (3) inhibition of amyloid Deposition, (4) stabilization of the tetrameric TTR structure, and (5) replacement of the variant TTR gene with the normal TTR gene. TTR transthyretin, NSAID nonsteroidal anti-inflammatory drugs, BSB (trans, trans)-1-Bromo2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene, IDOX 40 -Iodo-40 -deoxydoxorubicin

more effectively than those of flufenamic acid (35–70 mM) (Tojo et al. 2006). Sekijima et al also demonstrated that oral administration of difulnisal mediates kinetic stabilization of TTR in human serum (Sekijima et al. 2006). Furthermore, structurally related derivatives of NSAIDs were also exploited to enhance the selectivity and reduce the adverse effect (Adamski-Werner et al. 2004; Reixach et al. 2006; Julius et al. 2007; Almeida et al. 2004). Various reports confirmed these findings and this concept is now widely accepted (Miller et al. 2004; Kingsbury et al. 2008).

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14.5.2 Cr3+ Recent studies suggested that certain metal ions affect amyloidogenesis in several types of amyloidosis (Sato et al. 2006). In FAP, metal ions may influence the stability of the tetrameric form of TTR. Sato et al. therefore investigated whether various metal ions (e.g., Zn2+, Cu2+, Ca2+, Fe3+, Al3+, Cr3+) affect amyloidogenesis of wild-type TTR and ATTR (Sato et al. 2006). Among the metal ions, Cr3+ increased the tetrameric stability of both wild-type and ATTR Val30Met, and suppressed TTR amyloidogenesis. It is unlikely that Cr3+ binds to T4-binding sites of TTR, because of its chemical structure. Cr3+ may increase the thermodynamic stability of TTR tetramer by facilitating the binding of T4. As Cr3+ is a component of health foods that is widely used throughout the world, administration of this metal ion was thought to be a good candidate for therapy. However, at the serum concentration, Cr3+ had no significant effect on TTR stability in serum (Tojo et al. 2006). While serum Cr3+ concentration will increase after Cr3+ supplementation, it is still very low compared to the concentration of TTR, likely explaining why stabilization is not observed. However, Cr3+ could be useful for treating TTR amyloidosis by enhancing the effects of difulnisal, because the effects of T4 and Cr3+ are not competitive but cooperative. Although no obvious effect of Cr3+ on patients was seen, further in vivo evaluation of the effects is needed.

14.5.3 Fx-1006 Tremendous efforts revealed the possibility of stabilization of the tetrameric form of TTR as a potential therapeutic strategy. Ongoing biochemical and pathological studies explored further potentiality of this therapy. It is noteworthy that Fx-1006A, a potent and selective stabilizer for tetrameric TTR, is underway in a multinational Phase II/III clinical study in patients with FAP worldwide (Labaudinie´re et al. 2006; Bulawa et al. 2006; Devit et al. 2006). Moreover, a number of structurally diverse small molecules that bind to TTR, increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis, have been tested. In fact, several small molecules, such as plant-derived flavones and xanthones (Baures et al. 1998; Green et al. 2005; Maia et al. 2005), the synthetic estrogen diethylstilbestrol (Morais-de-Sa´ et al. 2004), 2,4-dinitrophenol (DNP) as well as 3,5-diiodosalicylic acid were shown to have high affinity towards TTR and to inhibit amyloid formation in vitro (Gales et al. 2008).

14.5.4 Controlling Drugs for Endoplasmic Reticulum (ER) Stress In addition, a recent report also suggested that an endoplasmic reticulum (ER) quality control system may differentially regulate the fate of amyloidogenic TTRs


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and their monomers (Sato et al. 2007). Amyloidogenic TTR variants, which exist as tetramer in the ER, could pass the ER quality control system but form aggregates or amyloid fibrils in the extracellular space. ER quality control systems may monitor amyloidogenecity of TTR at monomer level and regulate the secretion of TTR variants. Thus, inhibiting tetramerization of amyloidogenic TTR variants in the ER thereby reducing the secretion of TTR variants from the cells may become a possible therapeutic strategy of FAP. However, although a certain amount of in vitro evidence has been reported, more clinical studies need to be conducted, and the true therapeutic effect for FAP patients remains to be investigated.

14.6

Gene Therapy

As described above, liver transplantation was originally suggested as a treatment that would halt the production of variant TTR in the liver; FAP does not progress when the variant TTR gene is replaced by the normal TTR gene in the liver by the surgery. This fact led to the suggestion that gene therapy which would suppress the variant TTR mRNA expression or correct the TTR gene mutation could become one of the most important methods for ameliorating the clinical symptoms of FAP.

14.6.1 Gene Silencing Tools Ribozymes, antisense methods and small interfering RNAs (siRNAs) are effective gene silencing tools. Ribozymes are based on catalytic RNA originally found in the protozoan tetrahymena (Inoue et al. 1985) and can cleave a targeted RNA sequence or reverse the mRNA to generate correct sequences (Sullenger and Cech 1994). The antisense oligonucleotides were recognized as the therapeutic tool in the 1970s (Zamecnik and Stephenson 1978) and could cause enzymatic degradation of mRNA by activating ribonuclease H1 (Wu et al. 2004). RNA interference (RNAi) was first discovered in Caenorhabditis elegans as a phenomenon of sequencespecific post-transcriptional gene silencing (Fire et al. 1998). The inhibition of specific gene expression by RNAi has also been achieved in mammalian cells by passing the Dicer step and directly introducing synthesized small interfering RNAs (siRNAs) (Elbashir et al. 2001). These technologies are expected to be a powerful tool for gene therapy. Propsting et al. reported specific cleavage of ATTR Val30Met mRNA in a cell culture system by using hammerhead ribozymes (Propsting et al. 1999). They showed that chemically modified nuclease-stable Inosine (15.1)-Hammerhead ribozymes can target ATTR Val30Met mRNA with high specificity at the RNA level (Propsting et al. 1999). In an FAP gene therapy trial, they used the wild-type human normal TTR-expressing cell line HepG2 and a stable transfected cell line with the ATTR Val30Met gene. They cleaved the ATTR Val30Met and wild-type TTR

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mRNAs with specific nuclease-stable chemically modified Inosine (15.1)-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent Western blotting. They were able to down regulate the TTR concentration by 54.5% (100% = 1.5 mg l1 TTR) and also to specifically target ATTR Val30Met expression in the cell culture system. The therapeutic effect was improved by using cationic liposomes, resulting in a total down regulation of wild-type TTR mRNA and ATTR Val30Met mRNA of 92.1 and 62.7%, respectively. Tanaka et al. reported that another ribozyme targeting a variant TTR (E61 K) degraded the variant mRNA but not the wild-type mRNA (Tanaka et al. 2001). These ribozymes also reduced the amounts of TTR mRNA and protein in HepG2 cells and COS-1 cells transfected with TTR-E61 K cDNA. Benson et al. demonstrated suppression of hepatic TTR mRNA levels and serum TTR levels by as much as 80% in mice transgenic for the human Ile84Ser TTR mutant gene by antisense oligonucleotides (Benson et al. 2006). Kurosawa et al. reported that a siRNA selectively silenced the mutant Val30Met TTR allele in cells expressing both wild type and Val30Met alleles (Kurosawa et al. 2005). Although these data were interesting and the results were clear, this method cannot be applied to treatment of FAP patients, because a perfect ATTR Val30Met gene suppression was not achieved, and, as described previously, TTR is a rapid turnover protein. Continued production of TTR from the liver cannot therefore be sufficiently suppressed. Additional precise in vivo studies and other TTR gene therapy methods should be considered.

14.6.2 Gene Conversion Therapy Chimeric RNA/DNA oligonucleotides (chimeraplasts) have been developed to facilitate correction of single-based mutations of episomal and chromosomal targets in mammalian cells (Yoon et al. 1996; Cole-Strauss et al. 1996). Chimeraplasts consist of short regions of correcting DNA bounded by long stretches of 20 -Omethyl RNA, hairpin loops, and GC clamps. Chimeraplasts were shown to cause a site-specific chromosomal correction or mutation in tissue culture cells and in vivo (Kren et al. 1997, 1998, 1999; Xiang et al. 1997; Alexeev and Yoon 1998). As a permanent and stable gene correction by chimeraplasts was demonstrated by clonal analysis at the level of the genomic sequence, and as shown by protein and phenotypic change (Alexeev and Yoon 1998), chimeraplast-mediated gene repair may be a powerful strategy for treatment of genetic diseases without the use of viral vectors. The strategy of using single-stranded oligonucleotides (SSOs) for gene therapy grew from studies that attempted to characterize and improve chimeraplasty (Gamper et al. 2000a, b). SSOs containing three phosphorothioate bonds at the 30 and 50 termini were more effective than the chimeraplasts in gene repair assays conducted with cell-free extracts and a yeast system (Gamper et al. 2000a, b). SSOs are significantly less expensive than chimeraplasts and far simpler to synthesize and


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purify, so they may be an invaluable resource for treating a variety of genetic diseases. To test the feasibility of gene therapy for FAP, via chimeraplasts and SSOs, that would halt production of variant TTR in the liver and retina, Nakamura et al. first applied chimeraplasts and SSOs to HepG2 cells secreting human wild-type TTR. They then demonstrated gene conversion by SSOs in rabbit eye expressing rabbit wild-type TTR and transgenic murine liver in which the intrinsic wild-type TTR gene was replaced by a TTR Val30Met gene (Fig. 14.2). Their results were promising, although, again, a perfect gene conversion could not be obtained. Although DNA replication plays an important role in achieving significant levels of gene conversion (Olsen et al. 2005; Wu et al. 2005; Engstrom and Kmiec 2007), many potential target tissues in vivo do not have high levels of DNA replication or cell division. To overcome this problem, gene therapy combined with SSOs and partial liver resection might be effective in treating genetic diseases of the liver such as FAP, since modest tissue damage with wound healing would activate DNA replication and cell division processes. These studies have not progressed to the point that a therapeutic agent for FAP can be predicted in the near future. Most important is the development of an animal model to test any new form of therapy including gene therapy in a timely fashion,

Fig. 14.2 Comparison of different gene therapy strategies. (a) Biological activity of antisense oligonucleotides (AS ONs). AS ONs block translation of the mRNA or induce its degradation by RNaseH. (b) Biological activity of ribozyme. Ribozyme possessing catalytic activity cleaves its target RNA. Once the target is cleaved, ribozyme dissociates and recycles itself. (c) Biological activity of siRNA which is produced from cleavage of longer dsRNA precursors by the Dicer or supplied exogenously. The guide strand is incorporated into the RNA-induced silencing complex (RISK), where it guides sequence-specific degradation of the target transcript. (d) Mechanism of targeted gene repair by single-stranded oligonucleotides (SSOs). SSOs create a ‘‘D loop’’ structure with its complement in the target site in the helix. Similar to the chimeraplast, a two-step repair process sequentially converts the targeted base pair

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because FAP is caused by so many different mutations with various phenotypes and usually has a slow but progressive course.

14.7

Other Possible Therapies

14.7.1 Radical Scavengers Advances in immunohistochemistry techniques and the development of new antibodies have increased the possibility of detecting injury caused by oxidative stress (Toyokuni et al. 1994). One of the histopathological hallmarks of Alzheimer’s disease is the occurrence of senile plaques that are found in the neocortex and hippocampus. The main constituent of the core of these plaques is a 40- to 43peptide sequence called ß-amyloid (Aß), which is capable of destabilizing calcium homeostasis and exhibits neurotoxic properties. In a number of studies, the cytotoxicity of Aß(1–40) and its active fragment Aß(25–35) could be suppressed by catalase and antioxidants, e.g., vitamin E and melatonin. Treatment of both cultured hippocampal neurons and synaptosomes with Aß(25–35) produced substantially increased concentrations of the lipid per oxidation product 4-hydroxy-2-nonenal (HNE). The neurotoxic effects of both Aß(25–35) and HNE could be prevented by a treatment with glutathione ethyl ester, whereas the antioxidant propyl gallate was effective against only Aß(25–35)-induced neurotoxicity (Mark et al. 1997). Aßresistant subclones of PC12 cells, unlike the parent cells, do not accumulate peroxides after Aß(25–35) treatment, because of an over expression of antioxidant enzymes (Sagara et al. 1996). Taken together, these data suggest that the neurotoxic effects of Aß are mediated by hydrogen peroxide and HNE. Proteases from polymorphonuclear leukocytes and macrophages are present in amyloid fibrils found in all types of systemic amyloidoses, and the release of free radicals from these cells may be involved in amyloid formation (Skinner et al. 1986; Stone et al. 1993). Hydroxyl radicals readily induce lipid peroxidation, so that products of this reaction should be present in amyloid deposits if free radicals are involved in the process. Several reports of accumulation of aldehydic lipid pe oxidation products during oxidative stress conditions have been published (Benedetti et al. 1980; Siems et al. 1989). HNE is a lipid peroxidation product that exhibits several toxic properties: cell and enzyme stimulation, and enzyme inactivation and modification. By using a purified polyclonal antibody to HNE, this toxic metabolite can be detected immunohistochemically in tissues (Toyokuni et al. 1994; Uchida et al. 1995). We reported that the HNE adduct was present in amyloid deposits in tissues from FAP patients, and that levels of thiobarbituric acid reactive substances (TBARS) and protein carbonyl were elevated in amyloid-rich biopsy samples from FAP patients compared with levels in nonamyloidotic samples (Ando et al. 1997, 1998). Nyhlin et al. reported the presence of advanced glycation end products,


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some of which were formed via oxidative stress (Nyhlin et al. 2000). These results suggest that oxidative stress is significantly involved in the amyloid formation process. In Sweden, 20 FAP Va130Met patients completed the 6-month study period of scavenger treatment (vitamin C, 1 g, three times daily, vitamin E, 0.1 g, three times daily and acetylcysteine, 0.2 g, three times daily). They were evaluated clinically and by immunohistochemical measurement of hydroxynonenal (HNE), a product of lipid peroxidation, in biopsy specimens. Clinically, no differences were found for FAP patients, but an increased nutritional status, measured by a modified body mass index (mBMI) was noted for transplanted patients (Suhr et al. 2001). The results were inconclusive regarding the effect of radical scavengers because the reasonable duration of administration and doses of radical scavengers could not be determined.

14.7.2 Tauroursodeoxycholic Acid (Tudca) TUDCA is a unique natural compound that acts as a potent antiapoptotic and antioxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with TTR deposition in FAP, Macedo et al. investigated the possible TUDCA therapeutical application in this disease. Semi-quantitative immunohistochemistry and western blotting revealed that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2a, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most importantly, TUDCA treatment significantly reduced TTR toxic aggregates by as much as 75%. Since TUDCA has no effect on TTR aggregation in vitro, this finding points to the in vivo modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis. They concluded that TUDCA is a candidate as a safe drug in prophylactic and therapeutic measures in FAP.

14.8

Summary

For an optimal effective therapy for TTR related FAP, several projects based on TTR metabolism and amyloid formation mechanisms are now ongoing both in basic research and in clinical trials. While several projects may be promising, we must wait for a while because some are just now beginning and in vivo experiments require more than a year to judge the changes in TTR and amyloid deposition. Although each therapeutic project is progressing independently, combined therapies may have the greatest potential.

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Acknowledgments The authors’ work was supported by grants from the Amyloidosis Research Committee, the Pathogenesis, Therapy of Hereditary Neuropathy Research Committee, the Surveys and Research on Specific Disease, the Ministry of Health and Welfare of Japan, and the Charitable Trust Clinical Pathology Research Foundation of Japan; Research for the Future Program Grant; and Grants-in-Aid for Scientific Research (B) 15390275, (B) 17458276, and from the Ministry of Education, Science, Sports and Culture of Japan.

References Adamski-Werner SL, Palaninathan SK, Sacchettini JC et al. (2004) Diflunisal analogues stabilize the native state of transthyretin. Potent inhibition of amyloidogenesis. J Med Chem 47:355–374 Alexeev V and Yoon K (1998) Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA/DNA oligonucleotide. Nat Biotechno1 16:1343–1346 Almeida MR, Alves IL, Terazaki H et al. (2000) Comparative studies of two transthyretin variants with protective effects on familial amyloidotic polyneuropathy: TTR R104H and TTR T119M. Biochem Biophys Res Commun 270:1024–1028 Almeida MR, Damas AM, Lans MC et al. (1997) Thyroxine binding to transthyretin Met 119. Comparative studies of different heterozygotic carriers and structural analysis. Endocrine 6:309–315 Almeida MR, Gales L, Damas AM et al. (2005) Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses. Curr Drug Targets CNS Neurol Disord 4:587–596 Almeida MR, Macedo B, Cardoso I et al. (2004) Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative. Biochem J 381:351–356 Alves IL, Divino CM, Schussler GC, Altland K, Almeida MR, Palha JA, Coelho T, Costa PP and Saraiva MJ (1993) Thyroxine binding in a TTR Met 119 kindred. J Clin Endocrinol Metab 77:484–488 Alves IL, Hays MT and Saraiva MJ (1997) Comparative stability and clearance of [Met30] transthyretin and [Met119]transthyretin. Eur J Biochem 249:662–668 Ando E, Ando Y and Haraoka K (2001) Ocular amyloid imvolvement after liver transplantation for polyneuropathy. Ann Intern Med 135:931–932 Ando Y, Araki S and Ando M (1993) Transthyretin related amyloidosis. Intern Med 32:920–922 Ando Y, Haraoka K and Terazaki H (2003) A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo. Lab Invest 83:1751–1759 Ando Y, Nyhlin N, Suhr O et al. (1997) Oxidative stress is found in amyloid deposits in systemic amyloidosis. Biochem Biophys Res Commun 232:497–502 Ando Y and Suhr O (1998) Autonomic dysfunction in familial amyloidotic polyneuropathy (FAP). Amyloid 5:288–300 Ando Y, Suhr O and El-Salhy M (1998) Oxidative stress and amyloidosis. Histol Histopathol 13:845–850 Ando Y, Tanaka Y, Ando E et al. (1995) Effect of liver transplantation on autonomic dysfunction in familial amyloidotic polyneuropathy type I. Lancet 345:195–196 Ando Y, Terazaki H, Haraoka K et al. (2002) Presence of autoantibody against ATTRVal30Met after sequential liver transplantation. Transplantation 73:674–675 Ando Y, Yamashita T, Nakamura M et al. (1997) Down regulation of a harmful variant protein by replacement of its normal protein. Biochim Biophys Acta 1362:39–46 Andrade C (1952) A peculiar form of peripheral neuropathy: Familial generalized amyloidosis with special involvement of the peripheral nerves. Brain 75:408–427


233

Araki S (1984) Type I familial amyloidotic polyneuropathy (Japanese type). Brain Dev 6: 128–133 Bard F, Cannon C, Barbour R et al. (2000) Peripherally administered antibodies against amyloid b-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 6:916–919 Baures PW, Oza VB, Peterson SA et al. (1999) Synthesis and evaluation of inhibitors of transthyretin amyloid formation based on the non-steroidal anti-inflammatory drug, flufenamic acid. Bioorg Med Chem 7:1339–1347 Baures PW, Peterson SA and Kelly JW (1998) Discovering transthyretin amyloid fibril inhibitors by limited screening. Bioorg Med Chem 6:1389–1401 Bendeck MP, Conte M, Zhang M et al. (2002) Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury. Am J Pathol 160:1089–1095 Benedetti A, Comporti M and Esterbauer H (1980) Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim Biophys Acta 620:281–296 Benson MD (1989) Familial amyloidotic polyneuropathy. Trends Neurosci 12:88–92 Benson MD, Kluve-Beckerman B and Zeldenrust SR (2006) Targeted suppression of an amyloidogenic transthyretin with antisense oligonucleotides. Muscle Nerve 33:609–618 Benson MD and Uemichi T (1996) Transthyretin amyloidosis. Amyloid Int J Clin Invest 3:44–56 Bergstrom J, Engstrom U, Yamashita T et al. (2006) Surface exposedepitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils. Biochem Biopys Res Commun 348:532–539 Blake CCF, Geisow MJ, Oatley SJ et al. (1978) Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A. J Mol Biol 121:339–356 Bulawa C, Wang L, Coelho T et al. (2006) Stabilization of amyloidogenic transthyretin variants in plasma by Fx-1006A. Amyloid 13(S1):21 Cardoso I, Merlini G and Saraiva MJ (2003) 40 -Iodo-40 -Deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing noncytotoxic species. Screening for TTR fibril disrupters. FASEB J 17:803–809 Cardoso I and Saraiva MJ (2006) Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model. FASEB J 20:234–239 Cole-Strauss A, Yoon K, Xiang Y et al. (1996). Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273:1386–1389 Colon W and Kelley JW (1992) Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31:8654–8660 Connors LH, Richardson AM, The´berge R et al. (2000) Tabulation of transthyretin (TTR) variants as of 1/1/2000. Amyloid 7:54–69 Continho P, Macedo E, Estacio A et al. (1988) Periodic plasma exchanges in treatment of familial amyloid polyneuropathy: Preliminary results. In: Isobe T, Araki S, Uchino F, Kito S, and Tsubura E (eds) Amyloid and Amyloidosis, pp 845–849 (New York: Plenum Press) Costa PMP and Costa PP (1998) The future of selective apheresis in FAP. Amyloid 5:143–144 Devit M, Wang L, Weigel C et al. (2006) In vitro profile of Fx-1006A, a novel, potent and selective transthyretin (TTR) stabilizer. Amyloid 13(S1):26 Dezutter NA, Dom RJ, de Groot TJ et al. (1999) 99mTc-MAMA-chrysamine G, a probe for betaamyloid protein of Alzheimer’s disease. Eur J Nucl Med 26:1392–1399 Dezutter NA, Landman WJ, Jager PL et al. (2001a) Evaluation of 99mTc-MAMA-chrysamine G as an in vivo probe for amyloidosis. Amyloid 8:202–214 Dezutter NA, Sciot RM, de Groot TJ et al. (2001b) In vitro affinity of 99Tcm-labelled N2S2 conjugates of chrysamine G for amyloid deposits of systemic amyloidosis. Nucl Med Commun 22:553–558 Dickson PW, Howlett GJ and Schreiber G (1982) Metabolism of prealbumin in rats and changes induced by acute inflammation. Eur J Biochem 129:289–293 Divry P (1927) Etude histochimique des plaques seniles. J Belge Neurol Psychiatr 27:643–657

234

Y. Ando et al.

Elbashir SM, Harborth J, Lendeckel W et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498 Engstrom JU and Kmiec EB (2007) Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair. BMC Mol Biol 8:9 Ericzon BG, Holmgren G, Lundgren E et al. (1999) New structural information and update on liver transplantation in transthyretin-associated amyloidosis. Report from the 4th International Symposium on Familial Amyloidotic Polyneuropathy and Other Transthyretin Related Disorders & the 3rd International Workshop on Liver Transplantation in Familial Amyloid Polyneuropathy, Umea Sweden, June 1999. Amyloid 7:145–147 Fire A, Xu S, Montgomery MK et al. (1998) Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 391:806–811 Forloni G, Colombo L, Girola L et al. (2001) Anti-amyloidogenic activity of tetracyclines: studies in vitro. FEBS Lett 487:404–407 Fraser PE, Nguyen JT, Chin DT et al. (1992) Effects of sulfate ions on Alzheimer beta/A4 peptide assemblies: implications for amyloid fibril-proteoglycan interactions. J Neurochem. 59:1531–1540 Frid P, Anisimov SV and Popovic N (2007) Congo red and protein aggregation in neurodegenerative diseases. Brain Res Rev 53:135–160 Gales L, Almeida MR, Arsequell G et al. (2008) Iodination of salicylic acid improves its binding to transthyretin. Biochim Biophys Acta 1784:512–517 Gamper HB Jr, Cole-Strauss A, Metz R et al. (2000a) A plausible mechanism for gene correction by chimeric oligonucleotides. Biochemistry 39:5808–5816 Gamper HB, Parekh H, Rice MC et al. (2000b) The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts. Nucleic Acids Res 28:4332–4339 Gellermann GP, Ullrich K, Tannert A et al. (2006) Alzheimer-like plaque formation by human macrophages is reduced by fibrillation inhibitors and lovastatin. J Mol Biol 360:251–257 Gertz MA, Lacy MQ, Dispenzieri A et al. (2002) A multicenter phase II trial of 40 -iodo-40 deoxydoxorubicin (IDOX) in primary amyloidosis (AL). Amyloid 9:24–30 Giger M, Baumgartner HR and Zbinden G (1974) Toxicological effects of Evans blue and Congo red on blood platelets. Agents Actions 4:173–180 Goldsteins G, Persson H, Andersson K et al. (1999) Exposure of cryptic epitopes on transthyretin only in amyloid and in amyloidogenic mutants. Proc Natl Acad Sci USA 96:3108–3113 Green NS, Foss TR and Kelly JW (2005) Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc Natl Acad Sci USA 102:14545–14550 Gustavsson A, Engstro¨m U and Westermark P (1994) Mechanisms of transthyretin amyloidogenesis. Antigenic mapping of transthyretin purified from plasma and amyloid fibrils and within in situ tissue localizations. Am J Pathol 144:1301–1311 Guy CD and Jones CK (2001) Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: Specificity, positive predictive value, and diagnostic pitfalls. Diagn Cytopathol 24:181–185 Hammarstro¨m P, Wiseman RL, Powers ET et al. (2003) Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299:713–736 Hawkins PN, Lavender JP and Pepys MB (1990) Evaluation of systemic amyloidosis by scintigraphy with 123I-labeled serum amyloid P component. N Engl J Med 323:508–513 Holmgren G, Ericzon BG, Groth CG et al. (1993) Clinical improvement and amyloid regression after liver transplantation in hereditary transthyretin amyloidosis. Lancet 341:113–116 Ikegawa S, Tanase S, Morino Y et al. (1988) Evaluation of serum variant prealbumin levels, and its behavior after plasma exchange for familial amyloidotic polyneuropathy (type 1). In: Isobe T, Araki S, Uchino F, Kito S, and Tsubura E (eds) Amyloid and Amyloidosis, pp 839–844 (New York: Plenum Press)


235

Inoue T, Sullivan FX and Cech TR (1985) Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 50 exons. Cell 43:431–437 Johnson SM, Wiseman RL, Sekijima Y et al. (2005) Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc Chem Res 38:911–921 Jouquan J, Pennec Y, Mottier D et al. (1983) Retinol carrier protein, prealbumin and transferrin. Influence of age and sex on the serum concentration of proteins with a short half-life. Presse Med 12:1877 Julius RL, Farha OK, Chiang J et al. (2007) Synthesis and evaluation of transthyretin amyloidosis inhibitors containing carborane pharmacophores. Proc Natl Acad Sci USA 104:4808–4813 Kaplan B, Vidal R, Kumar A et al. (1999) Immunochemical microanalysis of amyloid proteins in fine-needle aspirates of abdominal fat. Am J Clin Pathol 112:403–407 Kelly JW and Lansbury PT Jr (1994) A chemical approach to elucidate the mechanism of transthyretin and b-protein amyloid formation. Amyloid 1:186–205 Kim YS, Randolph TW, Manning MC et al. (2003) Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation. J Biol Chem 278:10842–10850 Kingsbury JS, Laue TM, Klimtchuk ES et al. (2008) The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescencedetected analytical ultracentrifugation. J Biol Chem 283:11887–11896 Klabunde T, Petrassi HM, Oza VB et al. (2000) Rational design of potent human transthyretin amyloid disease inhibitors. Nat Struct Biol 7:312–321 Klunk WE, Debnath ML, Koros AM et al. (1998) Chrysamine-G, a lipophilic analogue of Congo red, inhibits A beta-induced toxicity in PC12 cells. Life Sci 63:1807–1814 Klunk WE, Debnath ML and Pettegrew JW (1995) Chrysamine-G binding to Alzheimer and control brain: Autopsy study of a new amyloid probe. Neurobiol Aging 16:541–548 Kren BT, Bandyopadhyay P and Steer CJ (1998) In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotide. Nat Med 4:285–290 Kren BT, Cole-Strauss A, Kmiec EB et al. (1997) Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25:1462–1468 Kren BT, Parashar B, Bandyopadhyay P et al. (1999) Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide. Proc Natl Acad Sci USA 96:10349–10354 Kurosawa T, Igarashi S, Nishizawa M et al. (2005) Selective silencing of a mutant transthyretin allele by small interfering RNAs. Biochem Biophys Res Commun 337:1012–1018 Labaudinie´re R, Bulawa C, Wang L et al. (2006) Fx-1006A, a novel, potent and selective TTR stabilizer for the treatment of TTR amyloidosis. Amyloid 13(S1):11 Lendoire J, Trigo P, Aziz H et al. (1999) Liver transplantation in transthyretin familial amyloid polyneuropathy: First report from Argentina. Amyloid 6:297–300 Maia F, Almeida Mdo R, Gales L et al. (2005) The binding of xanthone derivatives to transthyretin. Biochem Pharmacol 70:1861–1869 Mark RJ, Lovell MA, Markesbery WR et al. (1997) A role for 4 hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid ß-peptide. J Neurochem 68:255–264 Masouye I (1997) Diagnostic screening of systemic amyloidosis by abdominal fat aspiration: An analysis of 100 cases. Am J Dermatopathol 19:41–45 Matsuno H, Niikura K and Okahata Y (2001) Direct monitoring kinetic studies of DNA polymerase reactions on a DNA-immobilized quartz-crystal microbalance. Chemistry 7:3305–3312 McCutchen SL, Colon W and Kelley JW (1993) Transthyretin mutation Leu-55-Pro alters tetrameric stability and increases amyloidogenicity. Biochemistry 32:12119–12127 McCutchen SL, Lai Z, Miroy GJ et al. (1995) Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease. Biochemistry 34:13527–13536

236

Y. Ando et al.

Merlini G, Ascari E, Amboldi N et al. (1995). Interaction of the anthracycline 40 -iodo-40 -deoxydoxorubicin with amyloid fibrils: Inhibition of amyloidogenesis. Proc Natl Acad Sci USA 92:2959–2963 Miller SR, Sekijima Y and Kelly JW (2004) Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants. Lab Invest 84:545–552 Miroy GJ, Lai Z, Lashuel HA et al. (1996) Inhibiting transthyretin amyloid fibril formation via protein stabilization. Proc Natl Acad Sci USA 93:15051–15056 Monaco HL, Rizzi M and Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041 Morais-de-Sa´ E, Pereira PJ, Saraiva MJ et al. (2004) The crystal structure of transthyretin in complex with diethylstilbestrol: a promising template for the design of amyloid inhibitors. J Biol Chem 279:53483–53490 Munro SL, Lim CF, Hall JG et al. (1989) Drug competition for thyroxine binding to transthyretin (prealbumin): comparison with effects on thyroxine-binding globulin. J Clin Endocrinol Metab 68:1141–1147 Nyhlin N, Ando Y, Nagai R et al. (2000) Advanced glycation end product in familial amyloidotic polyneuropathy (FAP). J Intern Med 247:485–492 Okahata Y, Niikura K, Sugiura Y et al. (1998) Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance. Biochemistry 37:5666–5672 Olsen PA, Randol M and Krauss S (2005) Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides. Gene Ther 12:546–551 Palha JA, Moreira P, Olofsson A et al. (2001) Antibody recognition of amyloidogenic transthyretin variants in serum of patients with familial amyloidotic polyneuropathy. J Mol Med 78:703–707 Peterson SA, Klabunde T, Lashuel HA et al. (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95:12956–12960 Propsting MJ, Blaschke M, Haas RE et al. (1999) Inosine(15.1)-Hammerhead ribozymes for targeting the transthyretin-30 mutation. Biochem Biophys Res Commun 260:313–317 Puille M, Altland K, Linke RP et al. (2002) 99mTc-DPD scintigraphy in transthyretin-related familial amyloidotic polyneuropathy. Eur J Nucl Med 29:376–379 Quintas A, Saraiva MJ and Brito RM (1999) The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis. J Biol Chem 274: 32943–32949 Redondo C, Damas AM, Olofsson A et al. (2000) Search for intermediate structures in transthyretin fibrillogenesis: soluble tetrameric Tyr78Phe TTR expresses a specific epitope present only in amyloid fibrils. J Mol Biol 304:461–470 Regnault V, Costa PM, Teixeira A et al. (1992). Specific removal of transthyretin from plasma of patients with familial amyloidotic polyneuropathy: Optimization of an immunoadsorption procedure. Int J Artif Organs 15:249–255 Reixach N, Adamski-Werner SL, Kelly JW et al. (2006) Cell based screening of inhibitors of transthyretin aggregation. Biochem Biophys Res Commun 348:889–897 Sagara Y, Dargusch R, Klier FG et al. (1996) Increased antioxidant enzyme activity in amyloid ß protein-resistant cells. J Neurosci 16:497–505 Sales-Luis ML, Galvao M, Carvalho M et al. (1991) Treatment of familial amyloidotic polyneuropathy (Portuguese type) by plasma exchange. Muscle Nerve 14:377–378 Saraiva MJ (2001) Transthyretin amyloidosis: a tale of weak interactions. FEBS Lett 498:201–203 Sato T, Ando Y, Susuki S et al. (2006) Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation. FEBS Lett 580:491–496 Sato T, Susuki S, Suico MA et al. (2007) Endoplasmic reticulum quality control regulates the fate of transthyretin variants in the cell. EMBO J 26:2501–2512 Schmidt ML, Schuck T, Sheridan S et al. (2001) The fluorescent Congo red derivative, (trans, trans)-1-bromo-2,5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene(BSB), labels diverse


237

beta-pleated sheet structures in postmortem human neurodegenerative disease brains. Am J Pathol 159:937–943 Sebastiao MP, Merlini G, Saraiva MJ et al. (2000) The molecular interaction of 40 -iodo-40 deoxydoxorubicin with Leu-55Pro transthyretin ‘amyloid-like’ oligomer leading to disaggregation. Biochem J 351:273–279 Sekijima Y, Dendle MA and Kelly JW (2006) Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 13:236–249 Siems W, Kowalewski J, Werner A et al. (1989) Radical formation in the rat small intestine during and following ischemia. Free Radic Res Commun 7:347–353 Skinner M, Lewis WD, Jones LA et al. (1994) Liver transplantation as a treatment for familial amyloidotic polyneuropathy. Ann Intern Med 120:133–134 Skinner M, Stone P, Shirahama T et al. (1986) The association of an elastase with amyloid fibrils. Proc Soc Exp Biol Med 181:211–214 Skovronsky DM, Zhang B, Kung MP et al. (2000) In vivo detection of amyloid plaques in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 97:7609–7614 Steensma DP (2001) ‘‘Congo’’ red: out of Africa? Arch Pathol Lab Med 125:250–252 Stone P, Camistol J, Abraham CR et al. (1993) Neutrophil proteases associated with amyloid fibrils. Biochem Biophys Res Commun 30:130–136 Suhr O, Danielsson A, Rydh A et al. (1996) Impact of gastrointestinal dysfunction on survival after liver transplantation for familial amyloidotic polyneuropathy. Dig Dis Sci 41:1909–1914 Suhr OB, Lang K, Wikstrom L et al. (2001) Scavenger treatment of free radical injury in familial amyloidotic polyneuropathy: a study on Swedish transplanted and non-transplanted patients. Scand J Clin Lab Invest 61:11–18 Sullenger BA and Cech TR (1994) Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing. Nature 371:619–622 Takei Y, Ikeda S, Hashikura Y et al. (1999) Partial-liver transplantation to treat familial amyloid polyneuropathy: Follow-up of 11 patients. Ann Intern Med 131:592–595 Tanaka K, Yamada T, Ohyagi Y et al. (2001) Suppression of transthyretin expression by ribozymes: A possible therapy for familial amyloidotic polyneuropathy. J Neurol Sci 183:79–84 Terazaki H, Ando Y, Fernandes R et al. (2006) Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant. Lab Invest 86: 23–31 Terazaki H, Ando Y, Misumi S et al. (1999) A novel compound heterozygote (FAP ATTR Arg104His/ATTR Val30Met) with high serum transthyretin (TTR) and retinol binding protein (RBP) levels. Biochem Biophys Res Commun 264:365–370 Tojo K, Sekijima Y, Kelly JW et al. (2006) Diflunisal stabilizes familial amyloid polyneuropathyassociated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci Res 56:441–449 Tokuda T, Kondo T, Hanaoka N et al. (1998) A selective transthyretin-adsorption column for the treatment of patients with familial amyloid polyneuropathy. Amyloid 5:111–116 Toyokuni S, Uchida K, Okamoto K et al. (1994) Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc Natl Acad Sci USA 91:2616–2620 Uchida K, Itakura K, Kawakishi S et al. (1995) Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies. Arch Biochem Biophys 324:241–248 Westermark GT and Westermark P (2000) Endocrine amyloid—a subject of increasing interest for the next century. Amyloid 7:19–22 Wu H, Lima WF, Zhang H et al. (2004) Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J Biol Chem 279:17181–17189 Wu XS, Xin L, Yin WX et al. (2005) Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression. Proc Natl Acad Sci USA 102:2508–2513 Xiang Y, Cole-Strauss A, Yoon K et al. (1997) Targeted gene conversion in a mammalian CD34enriched cell population using a chimeric RNA/DNA oligonucleotide. J Mol Med 75:829–835

238

Y. Ando et al.

Yoon K, Cole-Strauss A and Kmiec EB (1996) Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA DNA oligonucleotide. Proc Natl Acad Sci USA 93:2071–2076 Zamecnik PC and Stephenson ML (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 75:280–284 Zhuang ZP, Kung MP, Hou C et al. (2001) IBOX (2-(40 -dimethylaminophenyl)-6-iodobenzoxazole): A ligand for imaging amyloid plaques in the brain. Nucl Med Biol 28:887–894

Chapter 15

Liver Transplantation for Transthyretin Amyloidosis Bo-Go¨ran Ericzon, Erik Lundgren and Ole B. Suhr

Abstract Liver transplantation has until now proved to be the only treatment available that halts the progression of hereditary transthyretin (TTR) associated amyloidosis. The rationale behind the procedure is to replace the liver producing variant TTR with one that produces wild type TTR only, and thereby cease the production of amyloidogenic TTR (ATTR). Even though the transplantation does not improve the patient’s symptoms, the progression of the disease comes to a halt for a majority of patients. However, unforeseen complications after the transplantation have emerged, in particular a continuous amyloid formation in the heart observed in non-ATTR Val30Met mutations. Thus, combined liver and heart transplantation has been performed in selected cases. Since the ATTR liver functions normally apart from a synthesis of the variant TTR, utilisation of ATTR-amyloid patients’ livers for transplantation of liver disease patients has been performed. In a few patients, development of amyloid disease has been reported, but the procedure remains an important source of organs, especially for patients with hepatocellular cancer. Keywords Transthyretin, Liver transplantation, Heart complication, Amyloidosis

Abbreviations TTR ATTR FAP PND-score

Transthyretin Amyloidogenic variant transthyretin Familial amyloidotic polyneuropathy Polyneuropathy disability score

O.B. Suhr (*) Department of Medicine, Section for Gastroenterology and Hepatology, Umea˚ University Hospital, SE 901 85 Umea˚, Sweden e-mail: [email protected]


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B.-G. Ericzon et al.

FAPWTR mBMI ECG MIBG 99 Tc-DPD SSA SAP

15.1

Familial amyloidotic polyneuropathy world transplant registry Modified body mass index Electrocardiogram Iodine-123 metaiodobenzylguanidine scintigraphy 99 Tc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy Senile systemic amyloidosis Serum amyloid P component

Background

The identification of mutated transthyretin (TTR) as the amyloidogenic protein in hereditary TTR amyloidosis (Saraiva et al. 1984), and the knowledge that the circulating TTR was mainly synthesised by the liver initiated the proposal that a liver transplantation should cease the production of the amyloidogenic mutant TTR and replace it with the wild type TTR, and thereby halt amyloid formation. The suggestion to try liver transplantation as a treatment for a familial amyloidotic polyneuropathy (FAP) patient was put forward by the late Prof. Go¨sta Holmgren and Dr Steen from Umea˚ University Hospital, Sweden to Prof. Carl Gustaf Groth and Bo-Go¨ran Ericzon at Karolinska Institute in Stockholm, Sweden in 1989. Several Swedish FAP-patients suffering from the most common neuropathic TTR mutation, ATTR Val30Met, were considered as candidates for the procedure, and the first patient was accepted and underwent liver transplantation in 1990. Later the same year, a FAP patient of Portuguese descent underwent transplantation at Sahlgrenska Hospital, Go¨teborg, Sweden. Both transplantations were successful. Several patients also underwent transplantation over the following years with encouraging results (Holmgren et al. 1993), including almost complete disappearance of mutated TTR from the circulation (Holmgren et al. 1991). Until 1995, practically all patients with the FAP ATTR Val30Met who presented without any contraindications for the operation were accepted for transplantation. However, it soon became obvious that patients in a depleted nutritional status with severe gastrointestinal complications were poor candidates with an unacceptable high mortality and morbidity (Suhr et al. 1995) as serious circulatory complications during the transplantation accounted for several fatalities (Suhr et al. 1997b). Therefore the transplantation policy changed, and patients in a depleted nutritional status were not accepted, and patients with severe autonomic dysfunction were regarded as high risk patients especially if they suffered from advanced disabling disease. The initial hope of an improvement of the patients’ neuropathy and gastrointestinal complications after transplantation was not fulfilled. Several investigations disclosed that no improvement of the patient’s symptoms was noted after transplantation. Therefore, an early intervention was advocated, before the patient’s neurological status had deteriorated and other complications had supervened

15 Liver Transplantation for Transthyretin Amyloidosis

241

(Adams et al. 2000; Bittencourt et al. 2002; Parrilla et al. 1997; Perdigoto et al. 2003; Sharma et al. 2003; Suhr et al. 1995; Yamamoto et al. 2007b). In addition, it soon became obvious that the outcome after transplantation varied with genotype, and thus cardiomyopathy appeared to progress in spite of the transplantation in several ATTR-mutations (Pomfret et al. 1998; Stangou et al. 1998). A combined heart and liver transplantation was favorable for some genotypes to halt a progressing cardiomyopathy (Sharma et al. 2003). Today, 18 years after the first transplantation, the outcome for an individual patient is still difficult to predict. Even though the initial hopes of improvement for ATTR-amyloidosis after liver transplantation have not been fulfilled, the procedure has proved to be the only available treatment in which the progression of the disease in the majority of patients has been halted. With time, better selection of patients and better understanding of the special complications encountered during and after transplantation of ATTR-amyloid patients have also improved survival (Fig. 15.1), with a five year survival time approaching 90% according to the FAP World Transplant Registry (FAPWTR,: www.fapwtr.org). Long-term follow-up and the initial experience of transplanting patients with multiple complications, has clearly indicated that patients should be transplanted at an early stage, before they are marked by multiple complications, not only to ensure a reasonable chance of survival, but also to ensure an acceptable quality of life (Jonseń et al. 2001).

15.2

Patient Selection and Outcome After Transplantation

Since TTR amyloidosis is a systemic amyloidosis, a pre-transplant evaluation of the patient needs to include an examination of several organ functions in addition to those normally performed. Thus, patients with predominantly peripheral neurological

Actuarial survival, %

100 80 60

1990-94 (n=146) 1995-99 (n=463) 2000-04 (n=555) 2005-2007 (n=277) Overall (n=1441)

40 20 0 0

1

2

3

4

5

Years after transplantation

Fig. 15.1 Patients’ survival after liver transplantation. Overall survival and survival in relation to era. From the survival curves it is evident that results have improved with time, reflecting an initial learning phase. Factors contributing to the improvements are probably better patient selection as well as an improved post-transplant care (with permission from the FAPWTR)

242


symptoms will often also have other organ systems such as the autonomic nervous system, the heart, gastrointestinal tract, kidneys and bladder affected. Serious unexpected complications can arise after transplantation if the pre-transplant examinations have failed to take the systemic aspects of the disease into account.

15.2.1 Demographic Data and ATTR-Mutation Expectedly, the age of the patient was clearly related to long-term, but not to shortterm (7yrs (n=133)

20 0 0

2

4 6 8 Years after transplantation

10

Fig. 15.2 Patient survival in relation to duration of symptoms. All mutations (with permission from the FAPWTR)


100 80 60

p 60 PND score ³ IIIB No transplant

Consider combined or sequential LHT

No

Yes

No

Age < 60 PND score £ IIIB Vigilant annual monitoring Consider LT but make provisions for possible sequential HTx Consider heart Tx (Ile 122)

Fig. 15.5 Adopted from Algorithm of the King’s College Hospital proposed guidelines for liver transplantation in the management of familial amyloid polyneuropathy. Presented at the VII International symposium on Familial Amyloid polyneuropathy, London, UK 2008 (with permission from Arie Stangou). TTR transthyretin, ATTR amyloidogenic transthyretin variant, BNP B-natriuretic peptide, PND polyneuropathy disability score, LT liver transplantation, LHT liver and heart transplantation


251

septal thickness below 15 mm), and well preserved systolic and diastolic function) may well be candidates for liver transplantation. Besides, cardiomyopathy may develop after transplantation in patients under the age of 50, even without signs of heart amyloidosis before transplantation; this has been the case for ATTR Val30Met patients and several other patients carrying non-ATTR Val30Met mutations such as the ATTR Glu42Gly and Gly47Glu (Olofsson et al. 2002; Otto et al. 2008; Stangou et al. 1998). With careful selection of patients for transplantation, their expected survival is better than that of non-transplanted historical controls (Suhr et al. 2005). The identification of different types of amyloid fibrils in ATTR Val30Met patients and their relationship with age at onset and presence of cardiomyopathy raises the hope, that the type of amyloid fibril in the individual patient could be linked to the development of post-transplant cardiomyopathy, and that it may be an important tool for prediction of the outcome of transplantation. However, this needs to be substantiated in future studies (Ihse et al. 2008).

15.3

The Transplantation Procedure

Most commonly, liver transplantation is performed in patients with end stage liver disease having typical complications of liver cirrhosis such as; portal hypertension with varices, ascites and failing metabolic liver function. Common indications are hepatitis C cirrhosis, primary biliary cirrhosis and metabolic liver disorders causing damage to the liver such as alfa-1 antitrypsin deficiency, Wilson’s disease and thyrosinemia. Some of the metabolic liver disorders do not cause end stage liver disease, but may result in death of the patient due to complications elsewhere, thus familial hypercholesterolemia may cause early and significant cardiovascular disease and primary hyper oxalosis may result in end stage renal failure. Familial amyloidotic polyneuropathy belongs to the latter. As mentioned above, liver transplantation for FAP should be performed early in the course of the disease in order to optimise survival after transplantation, and for the patient to benefit more from the procedure since liver transplantation mainly halts the progress of the disease (Ericzon et al. 1995; Suhr et al. 2005). In the early era of liver transplantation for FAP, the negative impact on the progress of the disease during waiting time was often underestimated, and the patient was usually waiting longer than other patients with end stage liver disease. This was in particular seen when allocating organs according to the MELD-system (Forman and Lucey 2001; Wiesner et al. 2003). The FAP patient would not easily qualify for transplantation since they would lack signs of progressing end stage liver disease. Therefore, it is generally accepted that patients suffering from FAP need to be evaluated for timing of transplantation separately from patients with cirrhosis, for instance. Surgically, liver transplantation for FAP is straight forward and a relatively easy procedure, since signs of cirrhosis with portal hypertension and severe coagulopathy is absent. The intra-operative challenge is rather related to the FAP patient’s

252


autonomic dysfunctions such as arrhythmia and cardiomyopathy, and the difficulties to maintain an adequate myocardial function during the procedure (Suhr et al. 1997b; Viana Jda et al. 1999). These patients often lack the ability to compensate for hypotension with increasing heart-rate and most commonly a pacemaker is used temporarily during the operation procedure in order to be able to increase the pace of the heart. Some patients already have a permanent pacemaker placed preoperatively. In traditional liver transplantation i.e. orthotopic liver transplantation the native diseased liver is removed together with a segment of the retro-hepatic vena cava. With this technique the inferior vena cava is clamped during the anhepatic phase of the transplant procedure. This can readily be done in patients with pre-existing portal hypertension with several collaterals developed for the return of the blood from the lower part of the body to the heart. In order to minimise the effect of this caval clamping in recipients with minimal or no collaterals, a veno-venous by-pass pump is routinely used at many centres, pumping the blood from the splanicus via a portal vein catheter and from the inferior vena cava via the saphenous vein into the jugular or auxiliary vein and the heart. An alternative technique is to leave the retro-hepatic vena cava in place when removing the native liver, thus maintaining the caval blood flow during the anhepatic phase (Lerut et al. 1989). In many patients today and especially in patients without end stage liver disease such as TTR-amyloidosis, this technique is preferred. With this technique the supra hepatic vena cava of the graft is anastomosed to the hepatic vein entrance of the recipient, or alternatively the donor vena cava is anastomosed side to side to the recipient’s vena cava using temporary partial occlusion of the recipient’s vena cava. With this so called ‘piggy back’ technique no veno-veno by-pass pump is necessary. However, if the FAP liver is going to be used in a domino procedure (see below) it is generally believed that the use of a by pass pump is preferred. Normally the portal vein anastomoses is performed end to end as is the arterial anastomoses. The bile duct reconstruction is usually performed by an end to end cholodocho-choledocho stomi with or without a temporary stent in the biliary duct. The gallbladder is routinely removed from the transplanted liver. Post-operatively the patient will need to remain on life long immunosuppressant based on either Tacrolimus or Ciclosporin A in combination with steroids and sometimes Azatioprin or Mycofenylate Mofetyl. Rejection usually occurs in about 1/3 of the patients during the first months after transplantation. It is most often successfully treated by elevation of the base line immuno-suppression, and/or a bolus dose of steroids. The patients are usually sent home at the end of the first month after transplantation. The recovery process after liver transplantation for end stage liver disease is usually experienced very positively by the patients with a feeling of new strength and well-being, with improved capacity to handle daily work. The situation for the FAP patient is different since he or she first will suffer from a major surgical procedure, and later on only be able to return to the pretransplant condition, with the hope that progression of the disease symptoms has been halted.


15.4

253

Domino Liver Transplantation

The domino procedure means that a liver that has to be removed from a patient who needs to undergo liver transplantation, in turn can be used for another patient with a liver disease necessitating liver transplantation. This procedure was first carried out in Portugal in 1995 (Furtado et al. 1997; Furtado 2003). In FAP the liver morphology and function are normal apart from the production of variant TTR. In spite of being inherited, FAP clinical symptoms never develop before the age of 15 and often only after the age of 30–50. In some individuals with the genetic trait, the disease will never manifest during the normal life span (Hellman et al. 2008; Holmgren et al. 1994). Once the disease starts to develop, the survival is often 10–13 years. Therefore, in theory, by correct selection of liver recipients, many patients should benefit from being transplanted with a domino FAP liver with minimal or no risk to have the disease transferred during their expected life span. In the early era of domino liver transplantation using FAP livers, mainly patients with extended liver tumours were considered. Today approximately 50% of domino recipients have a malignant disorder and the remaining have end stage liver disease. The survival of Domino recipients transplanted for liver tumour or other liver diseases are displayed in Fig. 15.6. As can be seen from the figure, survival mainly depends on whether the patient was transplanted for a malignant disease or not. Apart from the risk of transferring the disease, there are several advantages of using FAP livers for domino transplantation. First of all it is the advantage of a whole organ live donor graft. Minimal ischemic damage therefore takes place during the organ retrieval procedure, since the donor is well evaluated, the circulation is stable and cold ischemia time can be kept to a minimum, when the domino transplant is performed shortly after organ retrieval. One of the challenges in using the domino technique is that the donor and the recipient of the domino liver graft have to share a short segment of the suprahepatic caval vein. Several suggestions on how to handle this problem have been proposed (Furtado et al. 1997; Nishida and Tzakis 2002). At our institution we prefer to leave the longer caval segment in the domino donor,


100 80 60

p

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