Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing (Molecular Biology Intelligence Unit)

M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T 2 Vivian Y.H. Hook Proteolytic and Cellular Mechanisms in Prohormo...

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M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T

2

Vivian Y.H. Hook

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

R.G. LANDES C O M P A N Y

MOLECULAR BIOLOGY INTELLIGENCE UNIT

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing Vivian Y.H. Hook Department of Medicine University of California, San Diego La Jolla, California, U.S.A.

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MOLECULAR BIOLOGY INTELLIGENCE UNIT Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-553-4

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Proteolytic and cellular mechanisms in prohormone processing / [edited by] Vivian Y.H. Hook. p. cm. -- (Molecular biology intelligence unit) ISBN 1-57059-553-4 (alk. paper) 1. Peptide hormones--Metabolism. 2. Proteolytic enzymes. 3. Peptide hormones-Physiological transport. 4. Protein precursors. 5. Post-translational modifications. I. Hook, Vivian Yuan-Hen Ho, 1953- II. Series. QP572.P4P767 1998 572'.76--dc21 98-28730 CIP

PUBLISHER’S NOTE Landes Bioscience produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published within 90 to 120 days of receipt of the manuscript. We would like to thank our readers for their continuing interest and welcome any comments or suggestions they may have for future books. Judith Kemper Production Manager R.G. Landes Company

CONTENTS 1. Targeting and Activation of Peptide Hormones in the Secretory Pathway ........................................................................... 1 Ken Teter and Hsiao-Ping H. Moore Introduction ............................................................................................. 1 Trafficking and Modification of Peptide Hormone Precursors ........... 3 Prohormone Sorting Mechanisms ......................................................... 8 Site of Prohormone Sorting .................................................................. 10 Prohormone Activation ........................................................................ 12 Summary and Future Perspectives ....................................................... 15 2. The Mechanism of Sorting Proopiomelanocortin to Secretory Granules and Its Processing by Aspartic and PC Enzymes................... 29 Niamh X. Cawley, David R. Cool, Emmanuel Normant, Fu-Sheng Shen, Vicki Olsen and Y. Peng Loh General Introduction ............................................................................ 29 Mechanism of Sorting POMC to the Regulated Secretory Pathway ................................................. 31 Endoproteolytic Processing of Proopiomelanocortin ......................... 34 Future Directions ................................................................................... 42 3. The Mammalian Precursor Convertases: Paralogs of the Subtilisin/ Kexin Family of Calcium-Dependent Serine Proteinases ..................... 49 Nabil G. Seidah, Majambu Mbikay, Mieczyslaw Marcinkiewicz, Michel Chrétien Introduction ........................................................................................... 49 Subtilisin/Kexin-like Precursor Convertases (PCs): Structural and Cellular Considerations ............................................ 51 Ontogeny, Tissue Expression and Subcellular Localization ................ 59 Structure, Loci, and Evolution of PC Genes ........................................ 62 Antisense Transgene Inhibition ............................................................ 63 Heritable Deficiency of PC in Human and Mouse .............................. 65 Inhibitors of PCs .................................................................................... 66 Enzymatic Cascades: ADAM Family and PCs ..................................... 67 Conclusions ........................................................................................... 68 4. The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5 ... 77 Margery C. Beinfeld Introduction ........................................................................................... 77 The Discovery of the Subtilisin Family of Prohormone Convertases ............................................................. 77 Distribution of PC1, PC2 and PC5 ....................................................... 79 Biosynthesis and Activation of PC1, PC2 and PC5 ............................. 79 Regulation of PC Expression ................................................................ 80 Experimental Systems Used to Study Processing ................................ 80 Enzymatic Activity of PC1, PC2, and PC5 ........................................... 81

Antisense PC1 and PC2 Strategies to Study Proneuropeptide Processing .............................................. Endoproteases in CCK Processing, a Case in Point ............................. Processing Enzyme Knockouts and Mutations ................................... Future Challenges ..................................................................................

81 82 82 83

5. ‘Prohormone Thiol Protease’ (PTP), a Novel Cysteine Protease for Proenkephalin and Prohormone Processing ................................... 89 Vivian Y.H. Hook, Yuan-Hsu Kang, Martin Schiller, Nikolaos Tezapsidis, Jane M. Johnston and Ada Azaryan Introduction ........................................................................................... 89 The Novel ‘Prohormone Thiol Protease’ (PTP): A Major Proenkephalin Processing Enzyme in Chromaffin Granules ......... 92 Participation of PC1/3 and PC2 Subtilisin-Like Proteases, and 70 kDa Aspartyl Protease (PCE) in Proenkephalin Processing in Chromaffin Granules ............................................... 100 Conclusions ......................................................................................... 100 6. Regulation of Prohormone Conversion by Coordinated Control of Processing Endopeptidase Biosynthesis with That of the Prohormone Substrate ............................................................... 105 Terence P. Herbert, Cristina Alarcon, Robert H. Skelly, L. Cornelius Bollheimer, George T. Schuppin and Christopher J. Rhodes Introduction ......................................................................................... 105 Coordinated Regulation of Prohormone and Processing Enzyme mRNA Levels ........................................... 106 Coordinated Translational Regulation of Specific Prohormone and Processing Enzyme Biosynthesis ............................................. 110 7. Carboxypeptidase and Aminopeptidase Proteases in Proneuropeptide Processing ............................................................ 121 Vivian Y.H. Hook and Sukkid Yasothornsrikul Introduction ......................................................................................... 121 Neuroendocrine-specific Carboxypeptidase E/H .............................. 122 Molecular Genetic Analysis of Mutant Carboxypeptidase E/H in fat/fat Obese Mice: Effects of Inactive CPE/H on Prohormone Processing ............................................................ 129 Mutant CPE/H in fat/fat Mice Leads to Discovery of Novel Carboxypeptidase D and Carboxypeptidase Z ............... 130 Evidence for CPE/H as a Sorting Receptor for the Intracellular Routing of POMC and Possibly Other Prohormones to the Secretory Vesicle ................................................................... 132 Aminopeptidase(s) for Prohormone Processing ............................... 133 Conclusions and Future Perspectives ................................................. 134

8. The Neuroendocrine Polypeptide 7B2 as a Molecular Chaperone and Naturally Occurring Inhibitor of Prohormone Convertase PC2 .......................................................... 141 A. Martin Van Horssen and Gerard J.M. Martens Introduction ......................................................................................... 141 History of 7B2 ...................................................................................... 141 The 7B2 Gene and Its Regulation ....................................................... 142 Evolutionary Aspects ........................................................................... 144 7B2 is a Neuroendocrine-Specific Polypeptide .................................. 144 Biochemical Characteristics of 7B2 .................................................... 145 Posttranslational Modifications of 7B2 .............................................. 145 Regulated Secretion of 7B2 ................................................................. 146 The Quest for the Role of 7B2 ............................................................. 146 Model of the Interaction Between 7B2 and PC2 ............................... 149 Implications and Future Prospects ..................................................... 151 9. Neuroendocrine α1-Antichymotrypsin as a Possible Regulator of Prohormone and Neuropeptide Precursor Processing .................. 159 Shin-Rong Hwang and Vivian Y.H. Hook Introduction ......................................................................................... 159 Biochemical Evidence for α1-Antichymotrypsin (ACT) as an Endogenous Regulator of the ‘Prohormone Thiol Protease’ (PTP) and Other Prohormone Processing Proteases .................... 160 Molecular Cloning Reveals Multiple Isoforms of Bovine ACT Expressed in Neuroendocrine Tissues ........................................... 164 10. Proteolytic Inactivation of Secreted Neuropeptides ........................... 173 Eva Csuhai, Afshin Safavi, Michael W. Thompson and Louis B. Hersh Introduction ......................................................................................... 173 Neprilysin ............................................................................................. 174 Aminopeptidases ................................................................................. 176 Angiotensin Converting Enzyme ........................................................ 178 Pyroglutamyl Peptidase II ................................................................... 178 Proline Specific Peptidases .................................................................. 179 Soluble Neuropeptidases ..................................................................... 180 Endopeptidase 24.15 and Endopeptidase 24.16. ................................ 181 Summary .............................................................................................. 182 11. Stimulation of Peptidergic Receptors by Peptide Hormones and Neurotransmitters: Studies of Opioid Receptors ......................... 191 George Bot, Allan D. Blake and Terry Reisine Introduction ......................................................................................... 191 Opioid Receptor Types ........................................................................ 191 Endogenous Opioids ........................................................................... 192 Endogenous Peptide Receptor Selectivity .......................................... 192 Opioid Ligands .................................................................................... 194

Opioid Cellular Activity ...................................................................... 195 Opioid Receptor Cloning .................................................................... 196 ORL1 and Nociceptin/Orphanin FQ .................................................. 196 Structure-Function Analysis of Cloned Opioid Receptors ............... 198 µ Receptor Knockout Mice Model ..................................................... 201 Agonist Regulation of Cloned Opioid Receptors .............................. 201 G Protein Role in Differential Agonist Activity ................................. 204 Conclusion ........................................................................................... 204 Index ................................................................................................................ 213

EDITORS Vivian Y.H. Hook Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapters 5, 7, 9

CONTRIBUTORS Cristina Alarcon Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Cornelius Bollheimer Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Ada Azaryan Department of Pharmacology Uniformed Services University of the Health Sciences Bethesda, Maryland, U.S.A. Chapter 5

George Bot Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11

Margery C. Beinfeld Department of Pharmacology and Experimental Therapeutics Tufts University School of Medicine Boston, Massachusetts, U.S.A. Chapter 4

Niamh X. Cawley Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2

Allan D. Blake Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11

Michel Chrétien J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3

David R. Cool Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Eva Csuhai Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10 Terence P. Herbert Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Louis B. Hersh Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10 Shin-Rong Hwang Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapter 9 Jane M. Johnston Department of Neurological Surgery Albert Einstein College of Medicine Bronx, New York, U.S.A. Chapter 5

Yuan-Hsu Kang Naval Medical Research Institute Bethesda, Maryland, U.S.A. Chapter 5 Y. Peng Loh Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Mieczyslaw Marcinkiewicz J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Gerard J.M. Martens Department of Animal Physiology University of Nijmegen, Nijmegen Toernooiveld, The Netherlands Chapter 8 Majambu Mbikay J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Hsiao-Ping H. Moore University of California at Berkeley Department of Molecular and Cell Biology Berkeley, California, U.S.A. Chapter 1

Emmanuel Normant Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Vicki Olsen Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Terry Reisine Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11 Christopher J. Rhodes Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Afshin Safavi Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10

Martin Schiller Department of Neuroscience Johns Hopkins University Baltimore, Maryland, U.S.A. Chapter 5 George T. Schuppin Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Nabil G. Seidah J.A. De Sève Laboratories of Biochemical Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Fu-Sheng Shen Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Robert H. Skelly Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Ken Teter University of California at Berkeley Department of Molecular and Cell Biology Berkeley, California, U.S.A. Chapter 1 Nikolaos Tezapsidis Department of Psychiatry Mt. Sinai Medical Center New York, New York, U.S.A. Chapter 5 Michael W. Thompson Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10

A. Martin Van Horssen Department of Animal Physiology University of Nijmegen, Nijmegen Toernooiveld, The Netherlands Chapter 8 Sukkid Yasothornsrikul Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapter 7

PREFACE

P

roteolysis of proneuropeptides is key for the production of bioactive neuropeptides that mediate cell-cell communication in the endocrine and nervous systems. The conversion of inactive precursor to active peptide hormone or neurotransmitter is required for neuroendocrine functions. This text is designed to provide the reader with an understanding of current knowledge concerning proteases involved in prohormone processing, and cellular aspects that must be considered for proper processing, storage, and secretion of bioactive peptides. It is well known that prohormone processing occurs in well-defined subcellular compartments of the regulated secretory pathway. Knowledge of the cell biology of prohormone processing is required in the search for processing enzymes that are colocalized with prohormone substrates and neuropeptide products. Therefore, important cellular aspects of the targeting and activation of peptide hormones in the secretory pathway are discussed in the first two chapters. The next several chapters (chapters 2-6) present evidence for endoproteases that have been demonstrated to be involved in prohormone processing. These endoproteases include the large family of subtilisin/kexin prohormone convertases, a novel cysteine protease known as ‘prohormone thiol protease’ (PTP), and an aspartyl protease that has been termed “POMC converting enzyme” (PCE). These studies provide evidence for three different mechanistic classes of endoproteases that participate in prohormone processing. Subsequent to the actions of endoproteases, carboxypeptidase and aminopeptidase enzymes (discussed in chapter 7) that remove basic amino acids from the COOH- and NH2-termini of peptide intermediates are needed to complete the proteolytic processing of precursors into peptide forms. Moreover, recent molecular genetic studies illustrate the role of prohormone convertases 1 and 2, as well as the carboxypeptidase E/H, in obesity and conditions related to diabetes. The processing pathway is critical for generating active peptides. Therefore, it is likely that endogenous regulators exist that control the prohormone processing pathway. Evidence for the 7B2 polypeptide as a molecular chaperone and inhibitor of a prohormone convertase is discussed in chapter 8. Also, the role of endogenous isoforms of the protease inhibitor α1-antichymotrypsin in regulating prohormone processing enzymes is presented in chapter 9. Upon secretion of neuropeptides into the extracellular environment, the actions of these active peptides can be terminated by proteolytic inactivation. Therefore, chapter 10 discusses extracellular proteases involved in inactivation of secreted peptides. However, before the extracellular proteolysis is complete, the essential function of the released peptide is to activate its specific receptor on the target cell to initiate certain physiological responses. Thus, chapter 11 presents the manner in which peptide hormones and neurotransmitters stimulate peptidergic receptors, with discussion of the opioid receptors as the main example.

The authors have presented the latest developments in this field. A wealth of knowledge has been gained over the last few years concerning the identity, regulation, and molecular and cell biology of proteases and protease inhibitors involved in prohormone processing. However, there are still many open areas to investigate in this field. It is likely that there are still, as yet, unknown processing proteases to be discovered. Importantly, future knowledge of the key proteases and regulatory components required in prohormone and proneuropeptide processing may provide future design of clinical therapeutics that modify the processing pathway in health and disease.

ACKNOWLEDGMENTS I wish to thank the authors who participated in this volume for their expertise and enthusiasm in providing discussions of the current status of knowledge in the prohormone and proneuropeptide processing field. In addition, support from the National Institutes of Health is appreciated. Finally, this book is dedicated to my family, who have shared with me the excitement of science and the continuous effort that has allowed this scientific endeavor to be achieved.

CHAPTER 1

Targeting and Activation of Peptide Hormones in the Secretory Pathway Ken Teter and Hsiao-Ping H. Moore

Introduction

P

rofessional secretory cells—generally cells of neuronal, endocrine, or exocrine origin— utilize two divergent secretory pathways with distinct temporal and spatial characteristics (reviewed in refs. 1-4). The first pathway of constitutive secretion mediates the continual and unstimulated transport of lipids, membrane proteins, and soluble cargo to the cell surface. This pathway thus provides the plasma membrane with a steady supply of protein and lipid components while it simultaneously releases secretory proteins into the extracellular space. Most cells are capable of constitutive secretion, but it was commonly thought that the second pathway of regulated secretion was limited to professional secretory cells. Only these cells were believed to express the specialized machinery required to sort and store specific cargo into a subpopulation of vesicles which accumulate intracellularly until a secretagogue triggers release. Yet recent evidence suggests that many other cell types utilize this process as well, and the mechanism of regulated exocytosis may in fact be used for such additional purposes as membrane repair during wound healing and the regulation of membrane permeabilities to water, ions and nutrients. As summarized in Table 1.1, regulated exocytosis has been detected by a number of techniques in many cell types which have previously been considered to possess only the constitutive secretory pathway. Insights gained from the study of peptide hormone trafficking can thus be generalized to similar events occurring in a variety of cells. Regulated secretory vesicles can be divided into two classes based on morphology, origin and content: Dense core secretory granules (SGs) arise from the trans-Golgi network (TGN) and contain an electron dense peptide hormone aggregate packaged in a 100-200 nm vesicle,5-8 whereas the synaptic vesicles are electron translucent 50 nm structures which derive from the endosomal system and carry nonpeptide neurotransmitters as cargo. After budding from the TGN, SGs are transported to the cell periphery in a microtubule-dependent process.8,9 Peptide hormone precursors are converted to a bioactive state en route by a family of enzymes called prohormone convertases (PCs, reviewed in refs. 10-13) and are incorporated into a highly condensed protein aggregate. Because the soluble constituents of SGs are delivered via the secretory pathway, this route of regulated secretion is referred to as the biosynthetic pathway. Synaptic vesicles, which recruit membrane proteins from the endosomal system and soluble cargo from the cytoplasm, utilize what has been termed the recycling pathway for biogenesis. The function and generation of synaptic vesicles has been reviewed elsewhere. 4,14-17 This chapter will focus on the sorting and activation of Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

stimulated release of lysosomal enzymes and pre-internalized BSA-gold complex

membrane wound/disruption

perfusion of cytosol with Ca2+

Ca2+ ionophore

Bovine endothelial cells

CHO fibroblasts

NRK fibroblasts

stimulated release of sulfated GAG chains

Ca2+ ionophore

L cells; CHO fibroblasts

229

227, 228

226

225

224

223

221, 222

Mechanisms of regulated secretion are widespread in animal cells. Although regulated secretion has historically been viewed as a specialized property of professional secretory cells, this phenomenon has recently been characterized in other cell types and may be used for a variety of physiological processes. One such purpose appears to be the transient modification of cell surface permeability. This facilitates the uptake of water, ions, or nutrients and is accomplished by the stimulated translocation of membrane channels and pumps from a specialized intracellular pool to the plasma membrane. The same general mechanism may be widely used for membrane repair during wound healing.

release of pre-loaded acetylcholine detected by patch clamp measurements of an abutting myocyte

Ca2+ influx; membrane depolarization

targeting of transfected Glut4 to a unique vesicle population, presumably a storage organelle similar to the Glut4-containing vesicle in adipocytes and muscles

vesicle accumulation and microvilli formation at wound site visualized by electron microscopy; heightened release of pre-internalized dye in wounded cells; increase in cell size

induction of numerous exocytic ‘pores’ visualized with fluorescent dyes and confocal microscopy; heightened release of pre-loaded dye in wounded cells

Amphibian myocytes and fibroblasts; CHO fibroblasts

CHO fibroblasts; not determined 3T3 fibroblasts

increase in membrane capacitance, reflecting increased cell surface area

membrane wound/disruption

Sea urchin eggs and embryos; NIH fibroblasts

220

mobilization of H+/K+-ATPase to the cell surface

histamine

Gastric cells

15, 218, 219

15, 217

References

mobilization of Glut4 glucose transporter to the cell surface

mobilization of water channels to the cell surface

vasopressin

insulin

Kidney ductules

Observation/Method of Detection

Adipocytes

Stimulus

Cell Type

Table 1.1. Nonclassical regulated secretion in animal cells

2 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Targeting and Activation of Peptide Hormones in the Secretory Pathway

3

prohormones and prohormone converting enzymes along the biosynthetic regulated secretory pathway.

Trafficking and Modification of Peptide Hormone Precursors Proteins destined for either regulated or constitutive release are transported and modified together as they migrate from the ER, through the Golgi and to the TGN. Targeting to the regulated pathway then begins in the TGN with the preferential packaging of materials into a budding immature secretory granule (ISG),18,19 the intermediate vesicle that is biochemically distinct from the mature SG.7,20-25 Sorting appears to continue during the period of granule maturation, since the lysosomal enzymes and constitutively secreted proteins that are initially incorporated into the nascent SG are found in substantially reduced quantities in the mature SG. As such, the ISG has sometimes been regarded as the functional extension of the TGN by completing the sorting function that is initiated at that site (reviewed in ref. 26). In most cases, prohormone conversion begins in the TGN but takes place predominantly in the ISG. This organelle therefore represents a key intermediate station for prohormone sorting and cleavage (Table 1.2). The extent of prohormone sorting and activation occurring in the TGN vs. the ISG appears to vary for different proteins and cell types, but in all cases the maturation process eventually generates a homogeneous SG vesicle population with highly concentrated bioactive peptides. The production of mature peptide hormones thus involves numerous sorting and processing events.

Role of ER and Golgi Prohormone sorting and modification begins in the ER. Peptide hormones enter the secretory pathway by virtue of a hydrophobic signal sequence which directs cotranslational passage through a ‘translocon’ complex into the ER lumen (reviewed in refs. 27-29). Once positioned in the ER, preprohormones are modified by a series of reactions: The signal sequence and/or prosequence are cleaved, N-linked core carbohydrates are added, and the prohormones are transiently associated with molecular chaperones. In the case of thyroglobulin, sequential interactions with the BiP and calnexin chaperones are required for its proper folding.30 BiP also prevents proinsulin degradation during folding and dimerization.31 This process, as well as proinsulin disulfide bond formation, is facilitated by the oxidizing lumenal environment of the ER.32 Since proper folding is a requisite for ER export, the time required to complete these modifications determines, in part, the exit rate for each protein (reviewed in ref. 33). Until recently, secretory proteins were thought to leave the ER by default. This ‘bulk flow’ mechanism would allow transport and constitutive secretion of proteins with no targeting information other than the initial signal sequence (reviewed in refs. 34, 35). According to this model, only residents of organelles would require specific targeting signals in order to be retained within their respective compartments, and a variety of organelle targeting motifs have indeed been identified (Table 1.3). However, another prediction of the bulk flow model—lack of sorting and concentration of migrant proteins upon exit from the ER—is not supported by recent observations. Many yeast proteins, for instance, are concentrated in ER-derived carrier vesicles (reviewed in refs. 36, 37). Quantitative immunoelectron microscopy has also established that albumin and VSV G are concentrated in ER-derived vesicles in mammalian cells.38,39 These observations indicate that exit of migrant proteins is facilitated by an active sorting mechanism. In the case of VSV G, this process requires a cytoplasmic, di-acidic anterograde targeting signal.40 A phenylalanine-containing anterograde transport signal has also been found on two putative ER cargo sorting receptors, p24 and ERGIC-53.41-45 Although cognate transport signals in prohormones have yet to be

tubulo-cisternal

+++

yes

mildly acidic/neutral

yes

limited

no

no

morphology

lysosomal enzymes

clathrin coat

pH

sorting compartment

processing compartment

stimulated release

unstimulated release yes

yes

yes

yes

5.0-7.0 average 5.7-6.3

partial

++

vesicles of irregular shape and size, 80 nm average diameter

perinuclear to peripheral

ISG

limited

yes

limited

no

5.0-5.5

no

+/–

100-200 nm vesicles

peripheral

SG

23, 82, 118, 153, 155

8, 118, 148, 153, 155

20, 82, 107, 108, 121, 153, 174, 178-180, 182, 183, 188, 200, 201, 230-234

18, 19, 24, 118, 145, 146, 149, 154, 157, 158

9, 108, 188, 200-202, 204-206

7, 20-22, 24

149, 158, 159

6, 8, 22

22, 24, 118

References

Serving as an intermediate between the TGN and SG, the ISG shares characteristics of both organelles. Like the TGN, the ISG is decorated with γ-adaptin and clathrin, contains lysosomal enzymes, maintains an average pH of approximately 6.2, and acts as sorting station for routing secretory traffic to multiple sites. Prohormone processing also takes place in both the TGN and ISG, although in most cases cleavage in the TGN is fairly limited. Another major difference between the two organelles is the response to secretagogues: Only the ISG is capable of stimulated exocytosis. The ISG resembles the mature SG in this respect, although stimulation usually results in the preferential exocytosis of young granules. In addition, the contents of newly formed ISGs exhibit a higher rate of unstimulated release. In some cases, this secretion has been shown to result from the budding of transport vesicles from ISGs. With time, however, the maturation process places release from ISGs under tight regulation and eventually transforms the vesicles into a uniform population of SGs. Note that the characteristics summarized in the table above are derived from numerous studies involving different cell types, marker proteins and model systems. This variability may account for some of the inconsistencies present in the literature.

perinuclear

location

TGN

Table 1.2. Relationship between the TGN, ISG and SG



5

identified, trafficking of these proteins may also involve ER export signals and cargo sorting receptors. By contrast, bulk flow may apply to intra-Golgi transport since further concentration of migrant proteins does not occur as trafficking continues across the Golgi stacks.38,39,46 While the exact mechanism of intra-Golgi transport is currently under debate,47-49 it is clear that a number of protein modifications occur at this site. Proinsulin hexamerization is initiated in an early Golgi compartment by the addition of zinc,32,50 and a subset of regulated secretory proteins are phosphorylated in the trans-Golgi.51 A prevalent modification is the addition of N- and O-linked carbohydrate chains, generated by the coordinate action of a characteristic set of enzymes in each Golgi cisternae (reviewed in refs. 52,53). The multiple Golgi cisternae also appear to act as a “molecular sieve”54 which allows repeated opportunities for the capture and return of missorted ER resident proteins. This retrieval process is mediated by a set of carrier vesicles bearing the coat complex COPI (reviewed in refs. 36,55,56). Proteins destined for secretion appear to be excluded from these retrograde transport carriers, as recent studies indicate that proinsulin and VSV G protein are segregated into distinct vesicles from those containing the ER retrieval KDEL receptor.46 Interestingly, the proinsulin-containing vesicles are also COPI-coated. The possibility that COPI functions in both anterograde and retrograde transport remains an issue that requires further clarification.56-59

Role of TGN, ISG and SG The TGN serves as a major sorting station for secretory traffic, diverting proteins to regulated, constitutive, lysosomal, and (in polarized cells) apical or basolateral destinations (reviewed in refs. 60, 61). Recent studies indicate that in nonpolarized mammalian cells and in yeast, different classes of constitutive vesicles, each with a distinct set of cargo, are also generated by the TGN.62-64 Its central role in these trafficking events has made the TGN a subject of numerous studies. Originally defined as the site at which newly synthesized plasma membrane proteins accumulate at 20°C, the TGN could be visualized by electron microscopy as a tubulocisternal network directly apposed to the trans-Golgi cisternae.65-67 Later, it was defined by two biochemical reactions—sialation and tyrosine sulfation—which occur at this site.68,69 The intracellular distributions of TGN38, furin and the mannose 6-phosphate receptor have also been used to delineate the compartment.70-74 Thus, over time the TGN has been defined by many different parameters and markers. These definitions have been used interchangeably, but a close examination of the literature reveals considerable inconsistencies regarding the response of the TGN to drug treatment (Table 1.4). This raises the possibility that the organelle known as the “TGN” may in fact be referring to more than one compartment. Because models for prohormone sorting and activation rely critically on the exact locations in which these events occur, the definition of the TGN needs to be revisited in order to avoid confusion caused by inconsistent usage of the term “TGN”. In addition to sulfation and sialation, one other major prohormone modification— processing to a bioactive state—is initiated in the TGN. Functional peptide hormones are usually quite small but are often synthesized as larger, inactive precursors which are eventually cleaved by the PC enzymes. Each prohormone convertase recognizes different dibasic consensus sequences, so the generation of a specific bioactive peptide relies upon the activity of one or more PCs. As a result, the same prohormone can yield different products depending on which PC(s) it encounters.75-78 Prohormone cleavage also exposes C-terminal basic amino acids which are recognized and removed by another modifying enzyme, carboxypeptidase E (CPE, reviewed in ref. 79). In some instances, alpha-amidation may also follow prohormone cleavage (reviewed in ref. 80).

241 241 144, 242, 243 244 242, 243

intercalates into glycolipid ‘rafts’ destined for apical membrane interaction with the lectin-like, carbohydrate binding receptor VIP36 unknown mechanism

238 166, 167, 239 166, 167, 240

unknown retention mechanism;may involve “kin recognition” or an interaction with the Golgi lipid bilayer unknown retention mechanism unknown retention mechanism;involves phosophorylation state retrieval mechanism most likely involving cytoplasmic coat (clathrin and AP-2) proteins addition of phosphate to a mannose residue in the cis Golgi pH-dependent sorting mechanism involving a receptor in the TGN

55, 237

236

235

36, 55, 56

36, 55

References

Protein targeting requires specific localization signals. Organelle residence is established by a combination of retention and retrieval. The retention mechanism prevents most resident proteins from continuing along the secretory pathway, while an auxillary retrieval mechanism captures errant proteins in distal secretory compartments and returns them to the proper organelle. Targeting is most often due to either a unique physical property of the protein or to a receptor/motif interaction. Variations on these two themes are used throughout the secretory pathway, including diversion to the SG, lysosome, or polarized plasma membrane.

transmembrane domain (TGN38) cytoplasmic acidic cluster (furin) cytoplasmic tyrosine tight-turn motif (TGN38 and furin) Lysosomes specific conformational motif mannose 6-phosphate residue Epithelial plasma membrane apical surface GPI lipid anchor, transmembrane domain N-linked glycans basolateral surface cytoplasmic tyrosine tight turn motif or dileucine sequence

TGN

transmembrane domain

unknown retrieval mechanism

cytoplasmic N-terminal RR motif

Golgi

unknown retention mechanism

transmembrane domain

type II membrane proteins

retrieval mechanism effected by cytoplasmic coat (COPI) proteins

pH-dependent retrieval mechanism involving a KDEL receptor

TargetingMechanism

cytoplasmic C-terminal KK motif

lumenal C-terminal KDEL tag

Targeting Determinant

type I membrane proteins

ER soluble proteins

Destination

Table 1.3. Targeting signals for organelles along the secretory path


large vesicular structures ER ER cofractionates with TGN38 ER not determined MTOC MTOC MTOC

20° block site VSV G viral protein SFV viral proteins secreted GFP fusion construct radiolabeled proinsulin Sialyltransferase compartment Site for sulfation TGN38 compartment Furin compartment MPR compartment blocked blocked blocked blocked not applicable blocked enhanced not inhibited not inhibited

Export in the Presence of BfA

245 246 247 248 249-252 82, 148, 182, 245, 253 254, 255 73, 256 257

References

Serving as a major sorting station for secretory traffic, the TGN has been the subject of numerous studies and can be defined by a number of parameters. Yet a survey of the literature reveals that these parameters produce conflicting results when subjected to Brefeldin A (BfA) treatment. In the presence of BfA, a fungal metabolite which induces the redistribution of Golgi residents to the ER,258 the TGN (as defined by TGN38, furin, or MPR) did not collapse to the ER but was instead found at a tubularized perinuclear site which also contained internalized transferrin. This established the TGN as a functionally and physically distinct organelle, separate from the trans-Golgi. However, the TGN does redistribute to the ER when it is defined by other criteria (i.e., the 20° block site or the sialyltransferase compartment). This inconsistency and the differential effect of BfA on TGN transport leave open the possibility that two distinct compartments are both being defined as the TGN. Further examination of the nature of the TGN and the relationship between the various definitions of the compartment may thus help to clarify some of the discrepancies regarding the site of prohormone sorting and activation.

Location in the Presence of BfA

Definition

Table 1.4. Definitions of the TGN and responses to Brefeldin A

Targeting and Activation of Peptide Hormones in the Secretory Pathway 7

8


Prohormone cleavage and activation continues in the ISG, the vesicular intermediate linking the TGN and SG. Concomitant with this event are other steps of granule maturation: Translocation to the cell periphery, progressive lumenal acidification, homotypic fusion between two or more ISGs, and loss of the clathrin coat with the simultaneous removal of missorted proteins and excess membrane (reviewed in refs. 25,26,81). Many of the missorted proteins are lysosomal and constitutive secretory proteins, but membrane proteins of the constitutive fusion machinery may also be incorporated into the budding ISG. Mistargeting of components of this fusion machinery could explain the high level of unstimulated release of ISGs early in the maturation process.82 In contrast, other proteins required for regulated exocytosis are apparently added to the ISG. The SG membrane protein VAMP-2, for example, is delivered to the nascent SG from a post-Golgi site (B. Eaton and H.-P. Moore, manuscript in preparation). The delayed delivery of components of the regulated fusion machinery could also account for the short lag period in which newly formed ISGs are refractory to stimulated release in pituitary AtT20 cells (M. Haugwitz and H.-P. Moore, manuscript in preparation). SG biogenesis is thus a multi-step process involving numerous budding and fusion events which serve to alter both the soluble and membrane composition of an ISG.

Prohormone Sorting Mechanisms The mechanisms for targeting peptides to the regulated secretory pathway appear to have been conserved within the family of professional secretory cells: Regulated proteins which are not endogenously expressed by a particular professional secretory cell are still recognized and stored in SGs when introduced into that cell by DNA transfection.83 This indicates that the regulated sorting machinery recognizes common determinants present in many secretory proteins and explains why multiple secretory products can often be found within the same granule.18,84-88 As with other intracellular targeting events, there are two conceptually distinct mechanisms that may explain sorting to the regulated pathway. Sorting may be accomplished by a receptor which recognizes a targeting motif on specified proteins and in turn delivers them to the proper compartment. Typically, the receptor would cycle between the sorting and target compartments to mediate multiple rounds of binding and dissociation. An alternative mechanism involves the formation of specific macromolecular complexes between molecules destined for the same organelle. This would entail a conformational transition that initiates the formation of these molecular aggregates at the site of sorting. This mechanism thus allows multiple components to be sorted synchronously without the need of a cycling receptor. Numerous studies in recent years have uncovered specific interactions between various protein components of the regulated secretory pathway. To date, a cycling receptor that mediates multiple rounds of binding and dissociation has not been found. Instead, the available data support a model in which the formation of specific macromolecular aggregates of granule components dictates their coordinate sorting into regulated secretory granules. These interactions can be divided into two types: content-to-content interactions, and content-to-membrane interactions.

Interactions between SG Contents Homotypic interactions Proteins targeted to the regulated pathway are highly concentrated in the mature SG (reviewed in refs. 1, 2, 89). Electron microscopic studies have shown that the concentration process begins at the dilated rim of the trans-Golgi (reviewed in refs. 89, 180), thus leading to the hypothesis that selective aggregation of soluble SG content plays a key role in sorting


9

to the regulated pathway.2,4 In support of this, the aggregation and condensation of regulated secretory proteins can be triggered in vitro by a combination of high (1-10 mM) Ca2+ and low (6.5) (G. Giorgi, S. Lin, G. Chandy, H.-P. Moore and T. Machen, manuscript in preparation). The near neutral pH of the TGN raises important questions about whether peptide hormone sorting can indeed occur at this site. DAMP does not accumulate in the Golgi apparatus of either fibroblasts or professional secretory cells, suggesting that the Golgi cisternae may be neutral.108,179,200,204,206 However, refined in vivo techniques using retrograde transport of labeled verotoxin have found the average Golgi pH to be mildly acidic, in the vicinity of pH 6.5.207 A liposome fusion method which targeted pH-sensitive dyes to the trans-Golgi was used to obtain a pH of 6.2-6.3,208,209 although these studies have been widely misquoted as TGN pH. It remains to be established whether the trans-Golgi of peptide hormone secreting cells is also acidic, and whether the level of acidity in the trans-Golgi is sufficiently low to promote aggregation of hormones. Measurements of cis- and medial-Golgi pH have yet to be reported. The in vivo pH of the ER has been determined via specific targeting of a pH-sensitive dye; in this case, an avidinKDEL fusion construct was used to generate a resident ER marker which could then target


15

a membrane-permeant biotin derivative to this site. J. Llopes, M. Wu, K. Teter, R.Y. Tsien, T. Machen and H.-P. Moore (manuscript in preparation) were thus able to record an ER pH of ~7.6 in living HeLa cells. Compartmental Ca2+ Levels According to the models of prohormone sorting and activation, lumenal pH should drop and free Ca2+ levels should rise as prohormones pass through the distal organelles of the secretory pathway. Thus far, this prediction is only supported in part by actual experimental evidence. The ER indeed maintains a neutral pH and contains low levels of free Ca2+. The free Ca2+ concentration of the ER has been determined in vivo with the use of ERtargeted fusion constructs, initially with the Ca2+-activated photoprotein aqueorin210,211 and more recently with Ca2+-sensing derivatives of green fluorescent protein “cameleons”.212 A free Ca2+ concentration of 0.3-400 µM has been recorded for this compartment. Thus far, estimates of Golgi Ca2+ have been limited to total rather than free Ca2+ levels. These measurements, obtained with fixed cells, have shown that the Golgi apparatus contains a significant store of total intracellular Ca2+.213-215 Surprisingly, the Ca2+ concentration within the TGN of PC12 cells is below the threshold of detection.215 Professional secretory cells do, however, maintain a major repository of organellar Ca2+ within the ISG and SG,213,215,216 and a free Ca2+ concentration of 24 µM has been estimated for the mature SG.216 Further application of the improved techniques for measuring free Ca2+ and pH in specific compartments of living cells is needed to clarify the pH and free Ca2+ concentrations of the compartment(s) in which prohormone sorting occurs.

Summary and Future Perspectives Prohormone sorting and modification is a continuous process which occurs throughout the secretory pathway. Proper folding and core glycosylation are executed in the ER, after which prohormones are likely to be sorted and concentrated in COPII transport vesicles. Posttranslational modifications continue as hormone precursors move through the Golgi stack. Endoproteolytic cleavage and homo- or heterotypic aggregation then begin at the TGN as (pro)hormones are sorted and concentrated in ISGs by a “sorting for entry” process. Sorting continues as the “sorting by retention” mechanism retains regulated secretory proteins in the ISG while CLS removes missorted constitutive proteins and lysosomal enzymes. Efficient prohormone processing is concomitantly facilitated by the progressive acidification of an ISG, and the granule membrane undergoes significant remodeling during maturation to attain a regulated exocytotic state. The molecular details of each of the aforementioned steps require further investigation. It will be important to determine if active sorting occurs during export of prohormones from the ER, and to identify such ER export signals and the cognate transport receptor. Identification of putative membrane attachment proteins has already generated a number of interesting questions: 1. Does the membrane association occur at the TGN or ISG? 2. How is the membrane anchor itself targeted to the SG? 3. How does a receptor like CPE perform both sorting and enzymatic functions? 4. Are there multiple membrane anchors? and 5. Does the membrane anchor associate with other components to form a raft? The process of CLS also deserves further attention. Is the main function of CLS to remove missorted proteins, or is this process a byproduct of granule membrane remodeling during the transformation of ISG to a functional SG? The exocytotic state of an ISG exhibits characteristics of both constitutive and regulated secretory vesicles, and it is possible that the multiple fusion and fission events which accompany granule maturation serve to modify


16

the composition of the granule fusion machinery. Budding of constitutive-like vesicles may help to remove protein components of the constitutive fusion machinery from the ISG. Finally, given the role of pH and Ca2+ in prohormone sorting and activation, an understanding of how an individual organelle establishes and maintains precise pH and Ca2+ levels will be a major challenge in the future.

Acknowledgment The authors thank Michael Haugwitz, Ben Eaton and other members of the Moore lab for critical reading of the manuscript. This work was supported by NIH grant GM (35239) and a grant from the Cystic Fibrosis Foundation to HPM. KT was supported by the MCB training grant.

Abbreviations BfA CgB CLS CPE DTT ER GFP ISG MPR MTOC PC POMC SG TGN VSV

Brefeldin A chromogranin B constitutive-like secretion carboxypeptidase E dithiothreitol endoplasmic reticulum green fluorescent protein) immature secretory granules mannose 6-phosphate receptor microtubule organization center prohormone convertase proopiomelanocortin secretory granules trans-Golgi network vesicular stomatitis virus

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218. Slot JW, Geuze HJ, Gigengack S et al. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 1991; 113(1):123-135. 219. Smith RM, Charron MJ, Shah N et al. Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxy-terminal epitope of intracellular GLUT4. Proc Natl Acad Sci 1991; 88:6893-6897. 220. Forte JG, Hanzel DK, Urushidani T et al. Pumps and pathways for gastric HCl secretion. Ann NY Acad Sci 1989; 574:145-158. 221. Steinhardt RA, Guoqiang B, Alderton JM. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 1994; 263:390-393. 222. Bi GQ, Alderton JM, Steinhardt RA. Calcium-regulated exocytosis is required for cell membrane resealing. J Cell Biol 1995; 131(6 Pt 2):1747-58. 223. Miyake K, McNeil PL. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol 1995; 131(6 Pt 2):1737-45. 224. Coorssen JR, Schmitt H, Almers W. Ca2+ triggers massive exocytosis in Chinese hamster ovary cells. Embo J 1996; 15(15):3787-91. 225. Rodriguez A, Webster P, Ortego J et al. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J Cell Biol 1997; 137(1):93-104. 226. Herman GA, Bonzelius F, Cieutat AM et al. A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4. Proc Natl Acad Sci USA 1994; 91(26):12750-4. 227. Girod R, Popov S, Alder J et al. Spontaneous quantal transmitter secretion from myocytes and fibroblasts: Comparison with neuronal secretion. J Neurosci 1995; 15(4):2826-38. 228. Morimoto T, Popov S, Buckley KM et al. Calcium-dependent transmitter secretion from fibroblasts: Modulation by synaptotagmin I. Neuron 1995; 15(3):689-96. 229. Chavez RA, Miller SG, Moore HPH. A biosynthetic regulated pathway in constitutive secretory cells. J Cell Biol 1996; 133:1177-1191. 230. Tooze J, Hollinshead M, Frank R et al. An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage. J Cell Biol 1987; 105(1):155-162. 231. Steiner DF, Michael J, Houghten R et al. Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: Demonstration of cleavage within clathrin-coated (pro)secretory vesicles. Proc Natl Acad Sci USA 1987; 84:6184-6188. 232. Tanaka S, Nomizu M, Kurosumi K. Intracellular sites of proteolytic processing of proopiomelanocortin in melanotrophs and corticotrophs in rat pituitary. J Histochem Cytochem 1991; 39(6):809-821. 233. Tanaka S, Kurosumi K. A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs. Endocrinol 1992; 131(2):779-786. 234. Xu H, Shields D. Prohormone processing in the trans-Golgi network: Endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells. J Biol Chem 1993; 122(6):1169-1184. 235. Tang BL, Low SH, Hong W. Endoplasmic reticulum retention mediated by the transmembrane domain of type II membrane proteins Sec12p and glucosidase I. Eur J Cell Biol 1997; 73(2):98-104. 236. Schutze M, Peterson PA, Jackson MR. An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J 1994; 13(7):1696-1705. 237. Bretscher MS, Munro S. Cholesterol and the Golgi apparatus. Science 1993; 261(5126): 1280-1281. 238. Ponnambalam S, Rabouille C, Luzio JP et al. The TGN38 glycoprotein contains two nonoverlapping signals that mediate localization to the trans-Golgi network. J Cell Biol 1994; 125(2):253-68.


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239. Jones BG, Thomas L, Molloy SS et al. Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO J 1995; 14(23):5869-5883. 240. Luzio JP, Banting G. Eukaryotic membrane traffic: Retrieval and retention mechanisms to achieve organelle residence. Trends Biochem Sci 1993;18(10):395-8. 241. Kornfeld S, Mellman I. The biogenesis of lysosomes. Ann Rev of Cell Biol 1989; 5:483-525. 242. Mostov KE, Cardone MH. Regulation of protein traffic in polarized epithelial cells. BioEssays 1995; 17(2):129-138. 243. Matter K, Mellman I. Mechanisms of cell polarity: Sorting and transport in epithelial cells. Curr Op Cell Biol 1994; 6:545-554. 244. Fiedler K, Simons K. The role of N-glycans in the secretory pathway. Cell 1995; 81(3):309-12. 245. Miller SG, Carnell L, Moore HPH. Post-Golgi membrane traffic: Brefeldin A inhibits export from the distal Golgi compartment to the cell surface but not recycling. J Cell Biol 1992; 118(2):267-283. 246. Sariola M, Saraste J, Kuismanen E. Communication of post-Golgi elements with early endocytic pathway: regulation of endoproteolytic cleavage of Semliki forest virus p62 precursor. J Cell Sci 1995; 108(Pt 6)):2465-75. 247. Teter K, Hacker J, Moore HPH. Unpublished observations. 248. Huang XF, Arvan P. Formation of the insulin-containing secretory granule core occurs within immature beta-granules. J Biol Chem 1994; 269(33):20838-44. 249. Tang BL, Wong SH, Qi X et al. Golgi-localized beta-galactoside alpha 2,6-sialyltransferase in transfected CHO cells is redistributed into the endoplasmic reticulum by brefeldin A. Eur J Cell Biol 1992; 59(1):228-31. 250. Berger EG, Burger P, Hille A et al. Comparative localization of mannose-6-phosphate receptor with alpha-2,6-sialyltransferase in HepG2 cells: An analysis by confocal double immunofluorescence microscopy. Eur J Cell Biol 1995; 67(2):106-11. 251. Strous GJ, van Kerkhof P, van Meer G et al. Differential effects of brefeldin A on transport of secretory and lysosomal proteins. J Biol Chem 1993; 268(4):2341-7. 252. Nakamura N, Rabouille C, Watson R et al. Characterization of a cis-Golgi matrix protein, GM130. J Cell Biol 1995; 131(6 Pt 2):1715-26. 253. Rosa P, Barr FA, Stinchcombe JC et al. Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules for the trans-Golgi network. Euro J Cell Biol 1992; 59(2):265-274. 254. Ladinsky MS, Howell KE. The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A. Eur J Cell Biol 1992; 59(1):92-105. 255. Reaves B, Banting G. Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: Redistribution of a TGN-specific integral membrane protein, TGN38. J Cell Biol 1992; 116(1):85-94. 256. Lin S, Moore HPH. Unpublished observations. 257. Wood SA, Park JE, Brown WJ. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 1991; 67(3):591-600. 258. Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A: Insights into the control of membrane traffic and organelle structure. J Cell Biol 1992; 116(5):1071-80.

CHAPTER 2

The Mechanism of Sorting Proopiomelanocortin to Secretory Granules and Its Processing by Aspartic and PC Enzymes Niamh X. Cawley, David R. Cool, Emmanuel Normant, Fu-Sheng Shen, Vicki Olsen and Y. Peng Loh

General Introduction

P

roopiomelanocortin (POMC, Fig. 2.1) is a 31 kDa precursor of a number of hormones/ neuropeptides, which include adrenocorticotropin (ACTH), β-endorphin (β-END), α-melanocyte stimulating hormone (α-MSH), β-lipotropin (β-LPH) and N-POMC1-48.1-3 These POMC-derived peptide products have different functions such as opiate-like activity (β-END), mitogenic activity (N-POMC1-48) and steroidogenic activity (ACTH). The precursor is expressed abundantly in the melanotrophs of the intermediate lobe of the pituitary, the site of α-MSH and β-END synthesis and in the corticotrophs of the anterior lobe of the pituitary where the main products generated are ACTH and β-LPH.3,4 In addition, POMC is expressed in the arcuate nucleus of the hypothalamus where POMC products are similar to that of the melanotrophs.5,6 POMC gene expression is under the negative control of dopamine in the intermediate lobe, and under the positive control of corticotropin releasing hormone (CRH) and negative control of glucocorticoids in the anterior lobe.7 Similar to other precursors, POMC is processed at paired basic residues by various endoproteases, e.g., prohormone convertases, PC1, PC2 and proopiomelanocortin converting enzyme (PCE), to yield the active peptides (Fig. 2.1). These cleaved peptides are then subsequently modified by the removal of basic residues at the N and/or C-terminus by amino-8 and carboxypeptidases (carboxypeptidase H/E)9,10 respectively, followed by C-terminal amidation, and/or N-terminal acetylation for α-MSH and β-END peptides. The biosynthesis and processing of POMC involves routing of the prohormone from the rough endoplasmic reticulum (RER), the site of synthesis, to the trans-Golgi network (TGN), similar to that of other secretory proteins (Fig. 2.2). At the TGN, POMC is segregated from other proteins, such as lysosomal enzymes, plasma membrane proteins and constitutively secreted proteins, which have traversed a common pathway thus far.11,12 POMC, like other prohormones, is specifically sorted at the TGN and packaged into immature Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

Fig. 2.1. Schematic diagram of bovine pro-opiomelanocortin (POMC). This figure illustrates the POMC domains that are delineated by dibasic cleavage sites. These sites are cleaved in a tissue specific manner to produce a variety of peptide hormones depending on the complement of processing enzymes present. ACTH and β-LPH are primarily found in the anterior pituitary while the smaller POMC derived peptides, N-POMC1-48, γ3-MSH, α-MSH and β-END, are primarily found in the intermediate lobe of the pituitary and brain. ACTH, adrenocorticotropin hormone; LPH, lipotropin hormone; MSH, melanocyte stimulating hormone; END, endorphin. and represent N- and O-linked glycosylation sites, respectively.


Sorting Proopiomelanocortin to Secretory Granules and Its Processing

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Fig. 2.2. Schematic diagram illustrating the presence of the regulated and constitutive secretory pathways in endocrine cells. Immature secretory granules (ISG) of the regulated secretory pathway bud from the TGN. Prohormones (open stars) in the ISGs are processed and the mature secretory granule (MSG) appears to have a dense core. MSGs are stored in this region, awaiting an extracellular stimulus (secretagogue) that will cause them to fuse with the plasma membrane and release their contents of peptide hormone (closed stars) to the blood. In the constitutive secretory pathway, smaller vesicles containing proteins to be secreted, such as serum albumin or immunoglobulins (open arrowheads), receptors, ion channels, membrane transporters and other plasma membrane proteins, are rapidly segregated away from the regulated secretory proteins and shunted to the plasma membrane, where they fuse without the need for a secretagogue and release their contents to the blood. Reprinted with permission from Loh YP et al, Trends in Endocrinology and Metabolism 1997; 8:130.

secretory granules of the regulated secretory pathway and processed within these organelles.13 The processed POMC derived peptides in the granules form a dense core, from which, upon stimulation, they are released by exocytosis in a calcium-dependent manner into the circulation or extracellular space.

Mechanism of Sorting POMC to the Regulated Secretory Pathway Introduction The mechanism by which prohormones, proneuropeptides and other secretory proteins are sorted at the trans-Golgi network into the regulated secretory pathway has been debated for a long time. Two hypotheses have been raised since the 1980s.14,15 One is the sorting signal ligand-receptor active sorting hypothesis in which a sorting signal in the prohormone binds to the receptor located in the lumen of the TGN, followed by budding of the TGN and formation of an immature granule encapsulating the proteins destined for the regulated secretory pathway. The other is an aggregation passive sorting hypothesis in which the proteins, by forming a condensing aggregate, are trapped and packaged into the granules.

32


Attempts have been made to search for consensus sorting signals in the primary sequence of regulated secretory pathway proteins but none were found that were proven experimentally. 16,17 On the other hand, evidence accumulated that various hormones, 18 and chromogranins19 aggregate at the TGN under conditions of acidic pH and high Ca2+. Hence, the aggregation hypothesis became more plausible. However, in 1993, with the identification of a sorting signal domain in chromogranin A20 and chromogranin B,21 the ACTH/ endorphin prohormone, proopiomelanocortin (POMC),22 and proenkephalin,23 the tide began to turn. Recent studies have provided strong evidence in support of a sorting signalreceptor mediated mechanism for targeting POMC to the regulated secretory pathway. However, passive sorting of other hormones and regulated secretory pathway proteins cannot be discounted at this point.

Identification of the POMC Sorting Signal Deletion and mutagenesis studies have identified a POMC sorting signal for the regulated secretory pathway within the N-terminus of the prohormone.24 This signal consists of a loop between residues Cys8-Cys20 (Fig. 2.3). Proof that this sorting signal is sufficient and necessary for sorting POMC to the regulated pathway came from two lines of evidence: 1. transfection experiments in Neuro2a cells, an endocrine cell line, showing that deletion of N-POMC1-26, which contains the sorting signal, resulted in the constitutive secretion of POMC;25 and 2. fusion of N-POMC1-26 to a reporter protein, chloramphenicol acetyltransferase (CAT), caused the transport of this bacterial protein to the regulated secretory granules.22 Molecular modeling studies have identified the POMC sorting signal motif as an amphipathic loop structure comprised of residues Cys8-Cys20 (Fig. 2.3). The disulfide bridge between Cys8-Cys20 is essential to stabilize the loop.24 A key feature in this motif is the exposure of two aliphatic hydrophobic residues (Leu11 and Leu18) and two acidic residues (Asp10 and Glu14), with specific molecular distances apart, at the surface of the amphipathic loop (Fig. 2.3). Similar sorting signal motifs have also been identified in proenkephalin and proinsulin in modeling studies but not in chromogranin A, another regulated secretory pathway protein (Snell and Loh, unpublished data). Future studies will determine the commonality of the motif found in POMC for prohormone sorting to the regulated secretory pathway.

Identification of the POMC Sorting Receptor Identification of the regulated secretory pathway sorting signal in POMC led to the search for a sorting receptor. Using N-POMC1-26, which contains the POMC sorting signal (N-POMC8-20), as the ligand in binding studies, a sorting receptor has recently been demonstrated in bovine pituitary secretory granule and Golgi-enriched membranes.26 In crosslinking studies followed by purification, amino acid sequencing and Western blot, the receptor has been identified as the membrane form of Carboxypeptidase E (CPE, also known as Carboxypeptidase H). The optimum pH of binding the POMC sorting signal to CPE was between 5.5-6.5, consistent with the pH of the trans-Golgi network where sorting occurs.27 Equilibrium binding studies and Scatchard analysis revealed a Kd = 6 µM, IC50 = 65 µM and a Bmax = 580pmoles/mg protein,26 characteristic of low affinity first-order kinetics of binding, similar to that of enzyme-substrate interactions. Proinsulin and proenkephalin also bound CPE-containing vesicle membranes which were displaced by N-POMC1-26.26,28 This indicated that CPE is a sorting receptor for other prohormones and proneuropeptides as well. The N-POMC1-26 binding was highly specific for the lumenal side of the secretory granule membranes, since the POMC sorting signal did not bind to plasma membranes and bound minimally to Golgi membranes from L cells, a nonendocrine fibroblast cell line.


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Fig. 2.3. Schematic diagram illustrating the sorting signal of human proopiomelanocortin. The three-dimensional computer model of the N-terminal region shows the predicted conformation of the sorting signal (POMC8-20) stabilized by a disulfide bridge. The conserved charged or hydrophobic amino acids exposed to the surface are shown (Asp10, Leu11, Glu14, Leu18). Paired- and tetra-basic cleavage sites are shown with the single amino acid abbreviations K=Lys, R=Arg. ± indicates that these sites are not always glycosylated. Reprinted with permission from Loh YP et al, Trends in Endocrinology and Metabolism 1997; 8:130.

Moreover, only full length POMC, but not mutant POMC with the sorting signal or other domains of the POMC molecule deleted, bound to CPE in the secretory granule membranes.26 Also, constitutively secreted proteins such as immunoglobulins and proalbumin did not bind to CPE. A role for CPE as a sorting receptor was further supported by the finding that membrane CPE was tightly associated with secretory granule membranes and was resistant to depletion by high salt and detergent.26 CPE in pituitary secretory granules is primarily membrane-associated and exhibits poor enzymatic activity.29 Interaction of the POMC sorting signal with CPE appears to be at a different site than the active site, since the specific inhibitor for CPE, guanidino-ethylmercaptosuccinic acid (GEMSA) did not inhibit binding (Cool, D.R. unpublished).

34


Evidence that CPE is a sorting receptor in vivo came from studies in the neuroendocrine cell line, Neuro2a, which was downregulated in the expression of CPE by CPE antisense RNA.26 POMC transfected into these cells was secreted unprocessed, through the constitutive pathway. Also, immunocytochemistry showed no POMC-containing secretory granules in these cells. The role of CPE as a regulated secretory pathway sorting receptor in vivo was further demonstrated in a mouse model, Cpefat/Cpefat, which has a natural mutation in the Cpe gene.30 CPE in these mice has a Ser202 → Pro202 substitution in the enzyme, rendering it unstable, resulting in rapid degradation after synthesis.31 Secretion studies showed that intact POMC was the major component released from primary cultures of intermediate and anterior pituitary cells of Cpefat mice.26,32 Moreover, it was secreted at a high basal level, constitutively, in an unregulated manner. There was very little ACTH1-39 released from anterior pituitary cells in the Cpefat mice, and most of the ACTH were C-terminal basic residue-extended forms.32 These data are consistent with the misrouting of POMC to the constitutive pathway in the pituitary of Cpefat mice. CPE performs a dual role, as a membrane bound sorting receptor for the regulated secretory pathway and as an exopeptidase to remove basic residues from the C-terminal of peptide hormones subsequent to cleavage from the prohormone9,10(see also chapter 7). Recently, CPE has been implicated as a regulated secretory pathway sorting receptor for a number of other prohormones as well, including proinsulin, proenkephalin and growth hormone.26,28,32

Summary Current studies have provided strong evidence in support of a receptor-mediated mechanism for the sorting of POMC to the regulated secretory pathway, although aggregation also plays a role in enhancing the efficiency of sorting. Thus, we propose a regulated secretory pathway sorting mechanism for POMC which involves aggregation of POMC18 as a concentration step, followed by binding of the aggregate via the sorting signal to the receptor, CPE, at the TGN. Subsequently, the granule buds off from the TGN to form an immature granule in which processing takes place (Fig. 2.4).

Endoproteolytic Processing of Proopiomelanocortin Introduction During transport through the Golgi to mature secretory granules, posttranslational modifications of the secretory proteins occur, including glycosylation, phosphorylation, sulfation and endoproteolytic processing. POMC is processed by specific endoproteases that recognize paired-basic cleavage sites (e.g., Lys-Arg), and it is likely that the variety of bioactive peptides generated in the observed tissue-specific manner depends on the complement of enzymes present. POMC therefore represents a complicated prohormone to study in light of its mechanism of tissue-specific proteolytic processing and multiple levels of regulation. The search for the physiologically relevant enzymes capable of processing POMC and other prohormones in vivo has been a long and ongoing endeavor. It is the goal of this section to give a historical perspective and to highlight the significant experiments up to the current time that have advanced our understanding of the endoproteolytic processing of POMC. For this purpose the section has been divided into two parts covering the processing of POMC by two classes of enzymes that have been most extensively studied: 1. the prohormone converting aspartic proteases; and 2. the prohormone convertases.


35

Fig. 2.4. Working model for POMC sorting by binding to membrane associated carboxypeptidase E. In this model, POMC aggregates in the TGN and binds via its sorting signal to CPE (1). The TGN buds, carrying the CPE/POMC complex (2). The immature secretory granule becomes acidified; processing enzymes begin to cleave POMC and CPE (3). The more enzymatically active soluble CPE begins to process the C-terminal basic residues from the peptide cleavage products of POMC (4).

36


Processing of POMC by Prohormone Converting Aspartic Proteases Proopiomelanocortin converting enzyme (PCE) The definition of a prohormone processing enzyme is one that can specifically cleave the prohormone substrate to the correct products under conditions similar to those that are found in vivo and is localized to the same cellular compartment where processing occurs. While knowledge of the cleavage sites33,34 predicted the processing enzymes to be trypsinlike, it was surprising to find that the first activity which fulfilled these criteria was characterized as an aspartic protease from bovine and rat pituitary secretory vesicles.35-37 The enzyme was subsequently purified from bovine pituitary intermediate lobe secretory vesicles, the site of POMC processing, and was capable of cleaving the Lys-Arg pairs of POMC in vitro under acidic conditions, i.e., at a pH consistent with the intragranular pH of dense core secretory vesicles38,39 to yield 21-23 kDa ACTH, β-LPH, 13 kDa and 4.5 kDa ACTH, β-END, and 16 kDa NH2-terminal glycopeptide (see Fig. 2.5).40 The enzyme was characterized as a 70,000 dalton glycoprotein with an acidic pH optimum. Its enzymatic activity was specifically inhibited by pepstatin A and diazoacetyl-norleucine, both aspartic protease inhibitors, but not by other class-specific inhibitors such as phenylmethanesulfonyl fluoride, p-chloromercuribenzoate and ethylenediamine tetraacetic acid.40 Its ability to cleave POMC specifically under conditions similar to those in vivo rendered this enzyme a candidate prohormone processing enzyme and was named proopiomelanocortin converting enzyme, PCE (EC 3.4.23.17). A similar enzyme was also purified from bovine pituitary neural lobe secretory vesicles. In addition to its ability to cleave mouse POMC in a similar manner to that of the intermediate lobe PCE, this enzyme was shown to cleave proinsulin and provasopressin to generate insulin and vasopressin-Lys-Arg, respectively.41,42 PCE exhibited coordinate secretion from the intermediate lobe with α-MSH and an aminopeptidase B-like enzyme.43 The role that PCE plays in POMC processing in vivo was assessed by analyzing the processing of POMC in mouse neuro-intermediate lobe cells in the presence of pepstatin A.44 POMC processing decreased by 36.4% in the pepstatin A treated cells, indicating a role for PCE in POMC processing in vivo. PCE was able to cleave not only the intact POMC molecule but also its fragments, specifically human β-LPH and bovine N-POMC1-77. Human β-LPH was cleaved first at the Lys-Lys junction within γ-LPH generating an intermediate of β-MSH/β-END1-31. This intermediate was further cleaved primarily between the Lys-Arg cleavage site to release βMSH and Arg-β-END1-31 (Fig. 2.5).45 Bovine N-POMC1-77 was also cleaved by purified soluble PCE and a membrane bound form of PCE from bovine intermediate lobe secretory vesicle membranes to generate N-POMC1-49 and Lyso-γ3-MSH.46 This cleavage, however, depended on the glycosylation state of Thr45 of POMC, which is an ‘O’-linked glycosylation site. Processing at this site by PCE occurred only when Thr45 was not glycosylated, or only partially glycosylated, and the sugar chains lacked the terminating sialic acid residues.47 It is thought that the sialic acid forms a salt bridge with the Arg49 of POMC, providing steric hindrance and thereby preventing cleavage by PCE. PCE did not cleave the Lys28-Lys29 of β-END1-31 or the tetrabasic residues Lys15-Lys16-Arg17-Arg18 of ACTH1-39 to generate des-acetyl-α-MSH.40,45 Chromaffin granule aspartic protease (CGAP) In addition to other mammalian aspartic proteases that have been characterized by their ability to cleave prohormone substrates,48,49 the recently characterized 70 kDa chromaffin granule aspartic protease (70 kDa CGAP) from bovine adrenal medulla50,51 was also shown to process POMC in vitro at similar sites to PCE (Fig. 2.5). The physical properties,


37

Fig. 2.5. Schematic diagram of bovine proopiomelanocortin illustrating its potential peptide products. The basic residue cleavage sites are indicated by shaded and hatched bars representing lysine and arginine residues, respectively. The sites of POMC that can be cleaved by the aspartic and PC enzymes are summarized and the corresponding references where such processing was identified are listed. The asterisk indicates that the products were generated by endogenous convertases and have been included in the table within the context of the enzymes that were being studied.

inhibitor profile and specificity are very similar to PCE, rendering the designation of the 70 kDa CGAP as the adrenal form of pituitary PCE. Interestingly, this 70 kDa CGAP was also shown to cleave proenkephalin at three sites, one of which was identified by peptide microsequencing as being on the carboyxl side of Lys172-Arg173,50 making this enzyme a candidate processing enzyme of proenkephalin in vivo, along with the prohormone thiol protease (PTP) and the prohormone convertases PC1 and PC2 also present in these granules.52,53 Analysis of the relative cleaving activity of these enzymes for proenkephalin, proneuropeptide Y and POMC was carried out. It was found that PTP preferred proenkephalin while 70 kDa CGAP and the PCs preferred POMC; however, 70 kDa CGAP appeared to be more efficient than the PCs at cleaving POMC.54

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Yeast aspartic protease 3 (Yapsin 1) The first enzyme to be characterized biochemically and genetically as a prohormone processing enzyme was yeast Kex2, a subtilisin-like serine protease responsible for the processing of α-mating factor precursor at Lys-Arg cleavage sites in yeast.55-57 Since its discovery, a vast amount of information has been discovered about this class of enzymes which include the mammalian prohormone convertases (PCs), (see Seidah et al, this volume). However, it is of special interest in this section to note that not long after the discovery and characterization of Kex2, an aspartic protease was cloned in Kex2-deficient yeast.58 The aspartic protease was cloned on the basis of its ability to suppress the Kex2 deficient phenotype observed in these cells and was initially named yeast aspartic protease 3 (Yap3). Yap3 was also cloned independently a few years later based on its ability to cleave the heterologously expressed anglerfish prosomatostatin II at the monobasic cleavage site to generate somatostatin-28,59 a result that was supported by in vitro experiments.60 At that time Yap3p was proposed to be a homologue of the anglerfish somatostatin-28 generating enzyme which was also characterized as an aspartic protease.61 Since the initial characterization of PCE, efforts to clone the enzyme have been unsuccessful and, as a result, this class of prohormone converting aspartic proteases had remained somewhat obscure. However, with the cloning of yeast aspartic protease 3, now named yapsin 1,62 a new era in the characterization of prohormone processing aspartic proteases began. Yapsin 1 was overexpressed, purified and characterized with respect to its physical and chemical properties.63-65 It was initially characterized as a 68,000 dalton glycoprotein with an acidic pH optimum and demonstrated many similarities to bovine PCE. In fact, the antiyapsin 1 serum MW283 crossreacted with PCE on Western blot.66,67 The purified enzyme was able to cleave mouse POMC in vitro to generate ACTH and β-LPH63 (Fig. 2.5). It also cleaved human β-LPH to generate β-MSH and β-END1-31, and bovine N-POMC1-77 to generate Lyso-γ3-MSH and γ3-MSH. Differently from the action of PCE, yapsin 1 cleaved the Lys-Lys site of β-END1-31 to generate β-END1-28 and β-END1-29 and cleaved ACTH1-39 very efficiently to generate ACTH1-15, which is currently used as the standard assay of yapsin 1 activity.65 Recently, yapsin 1 was cotransfected with POMC into the rat pheochromocytoma cell line, PC12 cells, and was found to be correctly activated and targeted to the secretory pathway in these cells to generate ACTH1-39 and ACTH1-14, providing evidence that a yeast aspartic protease could be expressed and functionally active in the secretory pathway of mammalian cells.68 PCE: A mammalian homologue of Yapsin 1 PCE shares many properties with yapsin 166,69 which include size, specificity, inhibitor profile and localization to the secretory pathway. Since an antibody to yapsin 1 crossreacted with purified PCE,67 it is likely that PCE represents a mammalian homologue of yapsin 1. Based on these data, PCE has been renamed yapsin A to represent the first mammalian member of this novel subclass of prohormone converting aspartic proteases. Two yeast members currently include yapsin 1 and yapsin 2, previously called Yap358 and MKC7,70 respectively. To further explore the existence of mammalian homologues of yapsin 1, the antiserum MW283 was used for immunocytochemistry (ICC) in combination with in situ hybridization of peptide hormones.67 The results of this work showed a distinct distribution of yapsin 1-like immunoreactive cells in mammalian brain and pituitary. In the brain the immunostaining was distributed in neuropeptide-rich regions and was specifically colocalized with cholecystokinin mRNA in rat hippocampus and cortex and (Arg)vasopressin mRNA in the supraoptic nucleus of the hypothalamus. Yapsin 1-like immunoreactivity was also colocalized with POMC mRNA in the melanotrophs of the intermediate lobe by ICC in


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combination with in situ hybridization, and with POMC immunoreactivity in the corticotrophs of the anterior lobe by double ICC. These results support the hypothesis that mammalian prohormone converting aspartic proteases (mammalian yapsins) are likely to play a role in the processing of POMC, and potentially other prohormones or proneuropeptides, in vivo. The extent to which these mammalian yapsins function in this capacity has yet to be determined once their molecular structures have been elucidated.

Processing of POMC by Prohormone Convertases The explosion of information within the ten years following the cloning and characterization of Kex255-57 has slowly settled and has since been thoroughly reviewed.71-74 Of particular interest were the initial experiments of Thomas et al,75 who successfully cotransfected Kex2 and mouse POMC into a number of mammalian cell lines and documented the generation of β-LPH, γ-LPH and β-END1-31 (Fig. 2.5). These experiments were the first of their kind and further stimulated interest in the search and eventual cloning of the mammalian prohormone convertases. Mammalian prohormone convertases (PCs) were cloned using a PCR strategy with primers based on the predicted conserved homology of the active site of Kex2 and furin, a human gene product with homology to Kex2 and previously thought to be an oncogene.76 Since then, a family of PCs has emerged that have been shown to play important roles in the processing of proproteins (see Seidah et al, this volume). Two members of this family, PC177-79 (also called PC380) and PC277,81 were found to be expressed primarily in endocrine and neuroendocrine tissues,71,77,78,82-84 implicating a specialized function for these enzymes in prohormone and proneuropeptide processing. In particular, the expression of PC1 as the predominant PC in anterior pituitary, and the presence of PC2 and PC1 in the intermediate lobe of the pituitary82,85 implicated even further a role for PC1 and PC2 in the differential processing of POMC. Indeed, experiments demonstrating coordinate regulation of PC1 and PC2 mRNA with that of POMC mRNA by the dopamine agonist and antagonist, bromocryptine and haloperidol, in the rat pituitary intermediate lobe suggests a significant relationship between POMC and PC1 and PC2.85,86 Transfection experiments BSC-40 cells The ability of PC1 and PC2 to cleave POMC correctly was investigated primarily by cotransfection studies of their cDNAs using vaccinia virus expression vectors into cell lines from endocrine and nonendocrine origins. The POMC gene was expressed either alone or in conjunction with PC1 and/or PC2 cDNA in the African green monkey kidney epithelial cell line BSC-40, which does not contain a regulated secretory pathway. The transfected POMC alone was secreted in a constitutive manner in an unprocessed form,87-89 indicating the absence of endogenous convertase activity in this nonendocrine cell line. When cotransfected with PC1, POMC was processed primarily to 13 kDa ACTH and β-LPH88-90 and to a lesser extent γ-LPH and β-END1-3188,89 (Fig. 2.5). When PC2 was cotransfected with POMC in these cells, β-END1-31, but very little or no ACTH, was identified. However, an intermediate ACTH immunoreactive peak was identified by one group as JP-ACTH88 and as ACTH linked to γ-LPH by another group.89 Expression of PC2 together with PC1 resulted in the efficient conversion of β-LPH to γ-LPH and β-END1-31.89 Rin m5F cells In higher order mammalian species, there are two Lys-Lys cleavage sites present in the POMC molecule giving rise to β-MSH in one case and resulting in the formation of β-END1-26

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and β-END1-27 in the other case. These latter two products are found in the intermediate pituitary and arcuate nucleus of the hypothalamus and act as potential antagonists91,92 of β-END1-31 in vivo. In the insulinoma cell line Rin m5F, which contains PC2 as the predominant PC enzyme,93 transfected mouse or monkey POMC were processed to γ-LPH and desacetyl β-END1-31 and secreted in a regulated manner.87,94 β-END1-27 was only observed when the Lys-Lys cleavage site of β-END was mutated to Lys-Arg.87 In fact when the Lys-Arg site between ACTH and β-LPH and the Lys-Lys site within β-END of mouse POMC were permutated to every variation of a paired-basic cleavage site, (e.g., Arg-Lys, Arg-Arg, LysLys, Lys-Arg), it was found that cleavage sites containing Lys as the second basic residue were not cleaved. This indicated a strict requirement by the endogenous processing enzymes for an Arg in this position.95 In contrast, however, monkey POMC was processed at the Lys-Lys site within γ-LPH to generate a significant amount of β-MSH,94 implicating either the existence of a specific Lys-Lys cleaving enzyme for this site in these cells or a significant role for the secondary structure surrounding these sites in the regulation of processing by PC2. BAM cells The specificity of endogenous processing enzymes was further assessed in primary cultures of bovine adrenomedullary chromaffin cells, which were shown to contain primarily PC1. Thorne et al95 transfected these cells with the wild type and the mutant POMC constructs mentioned above, and analyzed the products formed. These cells produced ACTH and β-LPH in addition to γ-LPH and β-END, a pattern similar to anterior pituitary corticotrophs. When cotransfected with PC2, the products generated were more of an intermediate lobe pattern, with the identification of α-MSH and the complete conversion of β-LPH to γ-LPH and β-END.89 The same order of cleavage preferences for the POMC mutants were observed as that of the Rin m5F cells, with inefficient processing of Lys-Lys and Arg-Lys sites. PC12 cells In the pheochromocytoma cell line PC12, cotransfection of mouse POMC (mPOMC) or porcine POMC (pPOMC) with PC1 or PC2 generated products similar to those observed when these molecules were coexpressed in BSC-40 cells. However, an additional product was identified as des-acetyl α-MSH from the PC2 cotransfected PC12 cells88 and N-POMC1-80 was tentatively identified from the PC1 cotransfected cells, indicating the release of joining peptide (JP) by PC190 (Fig. 2.5). N-POMC1-80 was not observed, nor was the ACTH/β-LPH site cleaved by PC2 cotransfected into these cells. In addition, N-POMC1-49 was not observed in either experiment, indicating that the Arg-Lys site of N-POMC1-49/γ3MSH was not a readily cleaved site by PC1 or PC2 in these cells90 (Fig. 2.5). Neuro2a cells When monkey POMC was transfected into Neuro2a cells, a cell line with no measurable PC1 mRNA and only low levels of PC2 mRNA, β-END immunoreactivity was observed to be released from POMC without the intermediate production of β-LPH,93 implying that PC2 cleaved β-END directly from POMC. What is interesting to note here is that while the Lys-Lys site of γ-LPH was not cleaved, a significant amount of β-END1-27 was generated in these cells, a result that is reversed in Rin m5F cells, which contain abundant PC2.93,94 This cell-specific differential processing of the Lys-Lys sites of monkey POMC suggests the existence of a different enzyme capable of processing these sites.


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AtT20 cells In AtT20 cells, a mouse corticotroph cell line, POMC is expressed endogenously and processed into ACTH biosynthetic intermediates (ABI), 13 kDa and 4.5 kDa ACTH and β-LPH. A significant amount of β-END was observed in these cells96 and may be a result of the high levels of expression of PC1 in these cells, estimated at a 1:5 ratio with that of POMC,97 or the low levels of PC2, estimated at a 1:20 ratio with that of PC1.88 When stably transfected with PC2, ACTH1-39 was converted to ACTH1-13(NH2) and β-LPH was converted to β-END which was shown to contain primarily β-END1-31.96 In addition, after a longer pulse/ chase paradigm, a Lyso-γ3-MSH peak was identified (see Fig. 2.5). The absence of significant amounts of β-END1-27 generated in these PC2 overexpressing cells supports further the existence of a different enzyme more suited for this type of cleavage site. It was also reported that JP levels remained the same as that in wild type cells, indicating that PC1 was responsible for these cleavages. In similar experiments with overexpression of rat PC1 in AtT20 cells, two observations were made: 1. the rate of synthesis of the normal cleavage products was enhanced; and 2. the endogenous ACTH decreased significantly with the corresponding increase of ACTH1-13(NH2), demonstrating that PC1 could cleave at the tetra-basic site within ACTH.97 The role that PC1 plays in POMC processing in vivo was further assessed by expression of its antisense mRNA in these cells. AtT20 cells stably expressing antisense PC1 were analyzed for the extent of processing of the endogenous POMC. The results showed a significant decrease in ACTH-containing cleavage products both intra- and extra-cellularly, with a concomitant increase of intact POMC being secreted.98 This demonstrated a significant correlation between PC1 expression and the early steps in POMC processing in AtT20 cells. GH3 cells The role that PC2 plays in the processing of POMC was investigated by transfection experiments in GH3 cells.99 In this cell line, PC2 but no PC1 was detected by RT-PCR. When bovine POMC was transfected into this cell line after secretory granules were induced to form by β-estradiol and insulin, the products generated were identified as predominantly ACTH1-15, β-END and Lyso-γ3-MSH (Fig. 2.5), a result that demonstrated the ability of PC2 to cleave POMC to the smaller peptides in the absence of PC1. In the GH3 cells stably expressing antisense PC2 RNA, a significant decrease of POMC products was observed in a dose dependent level of PC2 knockout.99 In vitro experiments In addition to cotransfection experiments, the in vitro processing of mouse POMC has been investigated. In the first case,100 a lysate of purified insulin secretory granules, a source of type I and type II proinsulin processing endopeptidases, was shown to contain an activity capable of cleaving mPOMC into products similar to those generated when Rin m5F cells were transfected with mPOMC87 and in BSC-40 cells cotransfected with mPOMC, PC1 and PC2.89 The proinsulin processing endopeptidases I and II were subsequently identified as PC1 and PC2, respectively.101,102 Recombinant PC1 was also shown to cleave mPOMC into 16 kDa N-POMC, ACTH and β-LPH, while other products were tentatively identified as N-POMC1-74 and JP-ACTH103 (see Fig. 2.5). When the partially purified PC1 was tested against bovine N-POMC1-77, Lyso-γ3-MSH and γ3-MSH were generated and ACTH1-15 was generated from ACTH1-39 (see Fig. 2.5). These last two processing events are not normally observed in corticotroph cells of the anterior pituitary but are biologically active peptides presumably generated by PC2 in the intermediate lobe. The ability of PC1 to generate Lyso-γ3-MSH in vitro is different from the results obtained from overexpression of PC1 in

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AtT20 cells, since even when PC1 was in molar excess of POMC, no Lyso-γ3-MSH was seen,97 indicating cellular regulation by glycosylation in vivo. The significance of PC1’s ability to cleave this site is highlighted by the observation of mitogenic activity of N-POMC1-49 from anterior pituitary for adrenal regeneration.104

Summary From the present literature, it would seem that PC1, PC2 and the aspartic protease PCE play direct roles in the processing of POMC, as well as other prohormones in vivo (Fig. 2.5). The combined results of cotransfection and in vitro experiments have demonstrated the ability of PC1 to cleave POMC primarily at the dibasic cleavage sites flanking ACTH with some processing at the γ-LPH/β-END junction, a pattern found in anterior pituitary corticotrophs where PC1 is present. PC1 is also capable of cleaving the N-POMC/γ3-MSH junction and the γ3-MSH/JP junction. In addition, overexpression of PC1 in AtT20 cells and in vitro studies show that PC1 can cleave at the tetra-basic cleavage site of ACTH to generate α-MSH as well. PC2 generated Lyso-γ3-MSH, α-MSH and β-END1-31 and depending on the cell line β-END1-31 was further processed to β-END1-27. This pattern of POMC products are found in the intermediate lobe where PC2 is abundant and PC1 is present in significant amounts. PCE, found in anterior and intermediate lobes of the bovine and rat pituitary, appears to be able to cleave POMC at the ACTH/β-LPH junction as well as other sites cleaved by PC1 and PC2, with the exception of the tetra-basic site of ACTH and the Lys-Lys site of β-END1-31. Thus, there is redundancy in the enzymes capable of processing POMC. Future kinetic studies to determine the efficiency of cleavage of each of the sites of POMC by recombinant enzymes will determine which is likely to be the primary enzyme performing each of the cleavages in vivo. Other enzymes have also been found in bovine pituitary intermediate lobe secretory vesicles, which have a more specific function. This includes an acidic ACTH converting enzyme that generates ACTH1-17 from ACTH1-39105 and a mono- and dipeptidyl serine carboxypeptidase involved in the removal of Glu and Glu-Phe, respectively, from the carboxyl terminal of ACTH1-39,106 which may have significant effects on the steroidogenic properties of ACTH, an important neurotransmitter in the central nervous system107 in addition to being a source of Glu.

Future Directions While major advances have been made in the understanding of the mechanism of sorting POMC to the regulated secretory granules, how the processing enzymes are cosorted into these organelles remains an enigma. Sorting domains have been found in PC5A108 and PC2.109 Future studies on determining whether these sorting domains interact directly with POMC, which in turn is bound to CPE, the sorting receptor, or are sorted by directly binding to CPE, or other regulated secretory pathway proteins, will shed light on this very important question. With regards to the processing of POMC, much remains to be understood with respect to the interplay of the roles of PC1, PC2 and PCE, since these enzymes share some common cleavage specificity for the various cleavage sites of POMC. Kinetic studies using recombinant enzymes to evaluate the efficiency of each enzyme for the different POMC cleavage sites will further determine the redundancy of PC1, PC2 and PCE in POMC processing. Suggestions of redundancy were evident in the patient with hyperproinsulinemia who had a defect in the PC1 enzyme, yet showed significant processing of POMC to ACTH.110 The continued search and identification of patients with genetic defects in their prohormone processing enzymes will be a major focus of this field and lead to a better understanding of the level of redundancy of the enzymes for the processing of different prohormones.


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65. Cawley NX, Chen H-C, Beinfeld MC et al. Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3. J Biol Chem 1996; 271:4168-4176. 66. Loh YP, Cawley NX, Friedman TC et al. Yeast and mammalian basic residue-specific aspartic proteases in prohormone conversion. Adv Exp Med Biol 1995; 362:519-527. 67. Cawley NX, Pu L-P, Loh YP. Immunological identification and localization of yeast aspartic protease 3-like prohormone processing enzymes in mammalian brain and pituitary. Endocrinology 1996; 137:5135-5143. 68. Cool DR, Louie DY, Loh YP. Yeast aspartic protease 3 (YAP3p) is sorted to secretory granules and activated to process pro-opiomelanocortin in PC12 cells. Endocrinology 1996; 137:5441-5446. 69. Loh YP, Cawley NX. Processing enzymes of the pepsin family: Yeast aspartic protease 3 and pro-opiomelanocortin converting enzyme. Meth Enzym 1995; 248:136-146. 70. Komano H, Fuller RS. Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast. Proc Natl Acad Sci USA 1995; 92:10752-10756. 71. Seidah NG, Day R, Benjannnet S et al. The prohormone and proprotein processing enzymes PC1 and PC2: Structure, selective cleavage of mouse POMC and human renin at pairs of basic residues, cellular expression, tissue distribution, and mRNA regulation. Nida Res Monogr 1992; 126:132-150. 72. Van de Ven WJM, Van Duijnhoven HLP. Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases. Crit Rev Oncogen 1993; 4:115-136. 73. Seidah NG, Chretien M, Day R. The family of subtilisin/kexin like pro-protein and prohormone convertases: Divergent or shared functions. Biochimie 1994; 76:197-209. 74. Rouille Y, Duguay SJ, Lund K et al. Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: The subtilisin-like proprotein convertases. Front Neuroendocrinol 1995; 16:322-361. 75. Thomas G, Thorne BA, Thomas L et al. Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science 1988; 241:226-230. 76. van den Ouweland AMW, van Duijnhoven HLP, Keizer GD et al. Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2[published erratum appears in Nucleic Acids Res 1990 Mar 11;18(5):1332]. Nucleic Acids Res 1990; 18:664. 77. Seidah NG, Gaspar L, Mion P et al. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: Tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA and Cell Biol 1990; 9:415-424. 78. Seidah NG, Marcinkiewicz M, Benjannet S et al. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: Distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 1991; 5:111-122. 79. Nakayama K, Hosaka M, Hatsuzawa K et al. Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites. J Biochem 1991; 109:803-806. 80. Smeekens SP, Avruch AS, LaMendola J et al. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci USA 1991; 88:340-344. 81. Smeekens SP, Steiner DF. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem 1990; 265:2997-3000. 82. Hakes DJ, Birch NP, Mezey E et al. Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats. Endocrinol 1991; 129:3053-3063.


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83. Schafer MK-H, Day R, Cullinan WE et al. Gene expression of prohormone and proprotein convertases in the rat CNS: A comparative in situ hybridization analysis. J Neurosci 1993; 13:1258-1279. 84. Pu L-P, Ma W, Barker JL et al. Differential coexpression of genes encoding prothyrotropinreleasong hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: Implications for differential processing of pro-TRH. Endocrinology 1996; 137:1233-1241. 85. Day R, Schafer MK-H, Watson SJ et al. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 1992; 6:485-497. 86. Birch NP, Tracer HL, Hakes DJ et al. Coordinate regulation of mRNA levels of proopiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in the rat pituitary intermediate lobe. Biochem Biophys Res Commun 1991; 179:1311-1319. 87. Thorne BA, Caton LW, Thomas G. Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for β -endorphin processing. J Biol Chem 1989; 264:3545-3552. 88. Benjannet S, Rondeau N, Day R et al. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-3568. 89. Thomas L, Leduc R, Thorne BA et al. Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-5301. 90. Seidah NG, Fournier H, Boileau G et al. The cDNA structure of the porcine pro-hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N-terminal glycopeptide segment of porcine POMC. FEBS Lett 1992; 310:235-239. 91. Nicholas PR, Hammonds G, Li CC. β-Endorphin-induced analgesia is inhibited by synthetic analogs of β-endorphin. Proc Natl Acad Sci USA 1984; 81:3074-3077. 92. Bals-Kubik R, Herz A, Shippenberg TS. β-Endorphin(1-27) is a naturally occurring antagonist of the reinforcing effects of opioids. Naunyn Schmiedebergs Arch Pharmacol 1988; 338:392-396. 93. Day NC, Lin H, Ueda Y et al. Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA. Neuropeptides 1993; 24:253-262. 94. Lin H-L, Day NC, Ueda Y et al. Tissue-specific and substrate-specific endoproteolytic cleavage of monkey pro-opiomelanocortin in heterologous endocrine cells: Processing at LysLys dibasic pairs. Neuroendocrinology 1993; 58:94-105. 95. Thorne BA, Thomas G. An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes. J Biol Chem 1990; 265:8436-8443. 96. Zhou A, Bloomquist BT, Mains RE. The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 1993; 268:1763-1769. 97. Zhou A, Mains RE. Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 and 2. J Biol Chem 1994; 269:17440-17447. 98. Bloomquist BT, Eipper BA, Mains RE. Prohormone-converting enzymes: Regulation and evaluation of function using antisense RNA. Mol Endocrinol 1991; 5:2014-2024. 99. Friedman TC, Cool DR, Jayasvasti V et al. Processing of pro-opiomelanocortin in GH3 cells: Inhibition by prohormone convertase 2 (PC2) antisense mRNA. Mol Cell Endocrinol 1996; 116:89-96. 100. Rhodes CJ, Thorne BA, Lincoln B et al. Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases. J Biol Chem 1993; 268:4267-4275. 101. Bailyes EM, Shennan KIJ, Seal AJ et al. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochem J 1992; 285:391-394. 102. Bennett DL, Bailyes EM, Nielsen E et al. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem 1992; 267:15229-15236.

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103. Friedman TC, Loh YP, Birch NP. In vitro processing of proopiomelanocortin by recombinant PC1 (SPC3). Endocrinology 1994; 135:854-862. 104. Estivariz FE, Morano MI, Carino M et al. Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary. J Endocrinol 1988; 116:207-216. 105. Estivariz FE, Friedman TC, Chikuma T et al. Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. J Biol Chem 1992; 267:7456-7463. 106. Friedman TC, Chen H-C, Loh YP. Generation of (1-37) and (1-38) forms of adrenocorticotropin by mono- and di-peptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles. Endocrinol 1993; 133: 2951-2961. 107. Dani JA, Mayer ML. Structure and function of glutamate and nicotinic acetylcholine receptors. Curr Opin Neurobiol 1995; 5:310-317. 108. De Bie I, Marcinkiewicz M, Malide D et al. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments. J Cell Biol 1996; 135:1261-1275. 109. Creemers JW, Usac EF, Bright NA et al. Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem 1996; 271: 25284-25291. 110. O’Rahilly S, Gray H, Humphreys PJ et al. Brief report: Impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function. New Engl J Med 1995; 333:1386-1390. 111. Vieau D, Seidah NG, Mbikay M et al. Expression of the prohormone convertase PC2 correlates with the presence of corticotropin-like intermediate lobe peptide in human adrenocorticotropin-secreting tumors. J Clin Endocrinol Metab 1994; 79:1503-1506.

CHAPTER 3

The Mammalian Precursor Convertases: Paralogs of the Subtilisin/Kexin Family of Calcium-Dependent Serine Proteinases Nabil G. Seidah, Majambu Mbikay, Mieczyslaw Marcinkiewicz, Michel Chrétien

Introduction

O

ver the last 30 years,1-4 our understanding of the complex cellular processing of inactive secretory precursors into active polypeptides and proteins by limited proteolysis has greatly matured. It is now becoming clear that following removal of the signal peptide, precursor cleavage can occur either intracellularly, at the cell surface or within the extracellular milieu. The sites of cleavage are either composed of: 1. single or pairs of basic residues (K or R) within the general motif (R/K)-(X)n-(K/R)↓, where n = 0, 2, 4, or 6 (Table 3.1); 2. hydrophobic amino acids (e.g., Leu, Phe, Val or Met) (Table 3.2); or 3. small amino acid residues such as Ala or Thr (Table 3.2). The subdivision of precursors cleaved at basic residues into four types (Table 3.1), is based on the amino acids surrounding the cleavage site which could be critical for efficient processing. For example, cleavage of type I precursors within the motif R-X-(K/R)-R↓ is usually accomplished within the trans-Golgi network (TGN) by one or more resident enzymes, which often process precursors expressed in cells devoid of dense core secretory granules, the products(s) of which reach the cell surface by the constitutive secretory pathway.14 In contrast, cleavage of type II precursors at pairs of basic residues usually occurs within immature secretory granules and involves precursors whose products are stored in dense core granules and exit the cell via the regulated secretory pathway. Cleavage at monobasic residues (type III) and of type IV precursors can occur in either of the above secretory pathways. Some of the proteinases involved in intracellular endoproteolytic events resulting in cleavage at specific basic residues have recently been identified and molecularly characterized. They form a family of calcium-dependent serine proteinases of the subtilisin/kexintype, of which seven mammalian members (paralogs) are known so far (for recent reviews, see refs. 4, 15-18). The various names given to these “Precursor Convertases” (PCs) are presented in Table 3.3. For simplicity, throughout this chapter we will refer to the seven convertases as PC1, PC2, furin, PC4, PC5, PACE4, and PC7. In a combinatorial fashion, these enzymes determine the cell type and time at which biologically active products are derived from a given inactive precursor protein. Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

50


Table 3.1. Precursor classification based on basic (B) amino acid cleavage motifs Precursor Protein

Cleavage Site Sequence

Type I precursors [R-X-(K/R)-R]

P6 P5 P4 P3 P2 P1 [X- X- R- X- K/R- R Thr-His-Arg-Ser-Lys-Arg Gln-Val-Arg-Glu-Lys-Arg Pro-Ile-Arg-Arg-Lys-Arg Leu-Ala-Arg-Gly-Arg-Arg Asn-Ser-Arg-Lys-Lys-Arg Val-Gln-Arg-Glu-Lys-Arg Gln-Ser-Arg-Met-Arg-Arg Val-Glu-Arg-Val-Lys-Arg His-Gln-Arg-Ala-Arg-Arg Asn-Leu-Arg-Met-Lys-Arg Arg-Asn-Arg-Gln-Lys-Arg Glu-Arg-Arg-Lys-Arg-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X- X] Ser-Ser Leu-Asp Ser-Ile Ser-Leu Glu-Ile Ala-Val Ala-Ala Arg-Ala Ser-Val Asp-Thr Phe-Val Ser-Val

P6 P5 P4 P3 P2 P1 [X- X- X- X- K/R- R Pro-Val-Gly-Lys-Lys-Arg Pro-Pro-Lys-Asp-Lys-Arg Thr-Pro-Lys-Thr-Arg-Arg Gly-Ser-Leu-Gln-Lys-Arg Ser-Gln-Pro-Met-Lys-Arg Ala-Pro-Leu-Thr-Lys-Arg His-Val-Ile-Ser-Lys-Arg Ser-Cys-Lys-Leu-Lys-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X- X] Arg-Pro Tyr-Gly Glu-Ala Gly-Ile Leu-Thr His-Ser Ser-Thr Arg-Gly

ProβNGF Leptin proreceptor ProPDGF-A ProPDGF-B Integrin α6 HIV-1 gp160 Profertilin α proTACE proKUZ (mouse)—site 1 ————————site 2 proStromelysin-3 Pro7B2 Type II precursors [(K/R)-(K/R)] POMC (α−MSH/CLIP) (γ−LPH/β−END) Proinsulin (B/C chain) (C/A chain) ProRenin PACAP-RP Integrin α4 Profertilin β Type III precursors [Single R]

P8 P7 P6 P5 P4 P3 P2 P1 (B)- X- (B)- X- (B)- X- X R Arg-Gln-Phe-Lys-Val-Val-Thr-Arg Arg-Ala-Leu-Leu-Thr-Ala-Pro-Arg Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg Leu-Lys-Pro-Thr-Lys-Ala-Ala-Arg Ala-Thr-Pro-Ala-Lys-Ser-Glu-Arg Glu-Asp-Gly-His-His-Leu-Asp-Arg Asp-Leu-Arg-Trp-Trp-Glu-Leu-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X - X Ser-Gln Ser-Leu Ser-Ala Ser-Ile Asp-Val Asn-Ser His-Ala

P8 P7 P6 P5 P4 P3 P2 P1 (B)- X- (B)- X- (B)- X- (B)-R/K PACAP-RP Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg ProMullerian Inhibiting Substance Glu-Gly-Arg-Gly-Arg-Ala-Gly-Arg Proglucagon Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg ProIGF-II Glu-Ala-Phe-Arg-Glu-Ala-Lys-Arg ProPTPµ receptor Val-Glu-Glu-Glu-Arg-Pro-Arg-Arg

↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X -R/K Tyr-Lys Ser-Lys Asn-Arg His-Arg Thr-Lys

Prodynorphin (C-peptide) ProANF Prosomatostatin (SS-28) ProIGF-I ProIGF-II ProEGF (N-terminal) ProEGF (C-terminal) Type IV precursors [P2'= R/K]

The precursors listed in this table have been reported previously [4]. The motif recognized in each precursor type is emphasized. This list represents only a limited repertoire of the known PC substrates.

The Mammalian Precursor Convertases

51

As an alternative to cleavage at basic residues, some precursors are processed intracellularly at single or paired hydrophobic amino acids (e.g., Leu, Val, Ile, Phe and Met and combinations thereof), or following small amino acids, usually Ala or Thr (Table 3.2). Although so far the proteinases responsible for such processing are not known, efforts towards their identification are underway in a number of laboratories, especially since they could play major roles in the regulation of cholesterol and fatty acid metabolism7 and in Alzheimer disease.12

Subtilisin/Kexin-like Precursor Convertases (PCs): Structural and Cellular Considerations A schematic representation of the primary structure and proposed domains of the known 7 mammalian convertases and their membrane-bound isoforms, as compared to yeast kexin and bacterial subtilisin BPN’ is shown in Figure 3.1. The degree of sequence identities between each member within each of the pro-, catalytic and P-domains are compared in Tables 3.4-3.6. In all three domains, the best sequence identity is between PACE4 and PC5, varying between 67%, 75% and 61%, respectively. So far, it is apparent that while furin15 and PC732-36 are always first synthesized as type I membrane-bound enzymes, the isoforms PACE4-E28 and PC5-B31,37 are also membrane-associated (Fig. 3.1). Downstream of the signal peptide (Fig. 3.1), the subtilases contain a highly basic prosegment which, by analogy to bacterial subtilisins, is thought to act as an intramolecular chaperone guiding their folding within the endoplasmic reticulum (ER). Once cleaved by an autocatalytic mechanism, the pro-segment becomes a potent inhibitor regulating the intracellular site of activation of the enzyme (for reviews see ref. 38) until it is disposed of via a secondary cleavage(s) during cellular transit to the trans-Golgi Network (TGN).4,18,39 Overall, PC7 exhibits the least conserved pro-segment among the mammalian PCs (Table 3.4). Intramolecular autocatalytic zymogen cleavage of the pro-segment occurs in the neutral pH environment of the ER for furin,40 PC1,41-44 PC5,37 PACE428,45-47 and PC7.36 For these convertases, it is likely that the cleaved pro-segment remains bound as a complex to the enzyme until it reaches the TGN, where it is thought that a secondary event(s), e.g., a second cleavage within the pro-segment,39 causes the disassembly of the complex and the generation of the active convertase, which can then process other precursors in trans. In contrast, removal of the pro-segment of PC2 occurs within the acidic milieu of the TGN/ immature secretory granules (ISG)42,44 and is highly facilitated by the presence of 7B2,48,49 a PC2-specific binding protein.16,50-53 Thus, PC2 is unique among the convertases in that it is activated late along the secretory pathway and requires the presence of 7B2 for optimal activation. The catalytic domain of each convertase which contains the Asp, His and Ser of the catalytic triad and the oxyanion hole Asn (except for PC2, where it is an Asp) is the most conserved segment (Table 3.5), emphasizing its critical role in the recognition of single or paired basic residues within substrates. The conserved oxyanion hole Asp+ in PC2 seems to be important for its interaction with the precursor of 7B2.53 Alignment of the catalytic domain of all the PCs reveals that, with the exception of PC2, within the 350 amino acid catalytic domain 9 residues are conserved in all the mammalian PCs (Fig. 3.2). In PC2, these include 3 conservative and 6 nonconservative substitutions of which 2 are not conserved in all species and, hence, only 4 are considered to be significant changes. All these amino acid substitutions occur between the PC2's active site •D166 and the oxyanion hole +D . They include: 309 1. the oxyanion hole +D309 itself (as opposed to N); 2. two in the segment S81-S-N-D-P-Y86-P-Y-P-R, in which the underlined S81 and Y86 are unique, as they are N and D respectively in all other PCs; and finally 3. a Q134 replacing the usual G in all other PCs.

52


Table 3.2. Precursor classification based on hydrophobic (ϕ) and/or small (σ) amino acid cleavage motifs Precursor Protein

Cleavage Site Sequence P8 P7 P6 P5 P4 P3 P2 P1 P1' P2' ϕσ -X- Bϕσ ϕσ -X- Bϕσ ϕσ -X- ϕσ - ϕσ ↓ X- ϕσ Bϕσ

(r)proRelaxin (B-chain)

Ala-Ser-Val-Gly-Arg-Leu-Ala-Leu ↓ Ser-Gln

(h)SREBP-2 (h)SREBP-1a

Ser-Gly-Ser-Gly-Arg-Ser-Val-Leu ↓ Ser-Val His-Ser-Pro-Gly-Arg-Asn-Val-Leu ↓ Gly-Thr

(h) proCCK (CCK5)

Arg-Ile-Ser-Asp-Arg-Asp-Tyr-Met ↓ Gly-Trp

(r)α-Endorphin (r)γ-Endorphin

Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu ↓ Phe-Lys Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe ↓ Lys-Asn

(r)proAVP (CPP)

Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu ↓ Arg-Leu

(r)proRenin

Lys-Ser-Ser-Phe-Thr-Asn-Val-Thr ↓ Ser-Pro

(b)Chromogranin A (65↓66) (b)Chromogranin A (291↓292) (b)Chromogranin B (609↓610) (b)Chromogranin B (614↓615)

Leu-Leu-Lys-Glu-Leu-Gln-Asp-Leu ↓ Ala-Leu Met-Ala-Arg-Ala-Pro-Gln-Val-Leu ↓ Phe-Arg Glu-Leu-Glu-Asn-Leu-Ala-Ala-Met ↓ Asp-Leu Ala-Ala-Met-Asp-Leu-Glu-Leu-Gln ↓ Lys-Ile

(h)β-APP β-Secretase site β-Secretase site (Swedish) βε1-Secretase site βε2-Secretase site γ40-Secretase site γ42-Secretase site

Glu-Glu-Ile-Ser-Glu-Val-Lys-Met ↓ Asp-Ala Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu ↓ Asp-Ala Ile-Ser-Glu-Val-Lys-Met-Asp-Ala ↓ Glu-Phe Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr ↓ Glu-Val Gly-Leu-Met-Val-Gly-Gly-Val-Val ↓ Ile-Ala Met-Val-Gly-Gly-Val-Val-Ile-Ala ↓ Thr-Val

The precursors listed in this table include those of rat relaxin [5], human cholescytokin to produce CCK5 [6] and human sterol responsive element binding proteins 1a and 2 [7], all of which involve cleavage at either a hydrophobic (ϕ) or small (σ) residue and exhibit the presence of a basic (B) R or K at P4 and either a σ or ϕ amino acid at P2' or a B/ϕ/σ at either P4 and/or P8. Some of these cleavages could involve an enzyme(s) recognizing the motif (R/K)-X-X-ϕ↓ [7]. Other cleavages occur at either single or pairs of hydrophobic residues such as in the production of α- and γ-Endorphin [8], the above mentioned chromogranin A (9) and chromogranin B (10) sites, vasopressin C-terminal glycopeptide fragment CPP 1-19 [11] and processing of the human amyloid precursor protein (β-APP) processed at either the native or Swedish mutation of the β-secretase site or at the γ-secretase-40 site [12]. Some precursors are also cleaved at σ amino acids, e.g., in the γ-secretase-42 site and rat proRenin. Often in such precursors we note the presence of a ϕ or σ amino acid at either P4, P6 or P8. In addition, in some precursors we also observe the presence of a Gly at P5. The βε1- and βε2-secretase sites have been recently reported to increase with age in the brain of Azheimer and Down syndrome patients (13).


53

Table 3.3. Various names of Precursor Convertases (PCs) Adopted Convertase Name (Reference)

Alternative Name(s)(Reference)

Furin (19)

PACE (20) SPC1 (17)

PC1 (21,22)

PC3 (23) SPC3 (17)

PC2 (21,24)

SPC2 (17)

PC4 (25,26)

SPC4 (17)

PACE4 (27,28)

SPC5 (17)

PC5 (29)

PC6 (30,31) SPC6 (17)

PC7 (32)

LPC (33) PC8 (34) SPC7 (35)

Nomenclature of convertases with the original references shown in brackets.

Table 3.4. Sequence identities of the Pro-domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4

hPC5

hPC7

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin

35% 35% 47% 50% 41% 35% 25%

31% 43% 41% 35% 31% 29%

53% 45% 44% 36% 26%

60% 58% 34% 29%

67% 35% 26%

30% 29%

23%

Table 3.5. Sequence identities of the catalytic domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4 hPC5

hPC7

ykexin

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin subt. BPN’

55% 59% 64% 63% 60% 55% 50% 36%

56% 58% 54% 53% 51% 47% 35%

70% 62% 62% 53% 46% 34%

68% 67% 54% 49% 37%

75% 54% 48% 34%

48% 34%

34%

54% 48% 34%

54


Fig. 3.1. Schematic representation of the primary structures of the PCs as compared to those of Subtilisin BPN’ and yeast kexin. The various domains are emphasized, together with the active sites Asp, His and Ser and the oxyanion hole Asn (Asp for PC2).


55

Table 3.6. Sequence identities of the P-domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4

hPC5

hPC7

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin

43% 42% 44% 43% 39% 37% 41%

43% 47% 52% 52% 42% 32%

55% 43% 43% 38% 33%

45% 51% 38% 32%

61% 45% 33%

43% 38%

32%

We have already obtained the D/N mutant of the oxyanion hole of PC2. This substitution abrogates the binding of proPC2 to pro7B2 in the ER but not that of PC2/7B2 in the TGN.53 These data suggest that the unique oxyanion hole of proPC2 is important for its binding to pro7B2 in the ER but is not needed for the predicted second binding site of 7B2. Finally, based on homology modeling to subtilisin, suggestions have been made identifying the PC residues potentially critical for substrate recognition.54 Resolving the crystal structure of one or more PCs should shed more light on the intricate details of their structures and their ability to discriminate between various substrates. The P-domain has been shown to be critical for zymogen cleavage and for enzyme secretion of prokexin55 proPACE4,46 proPC2,56 for the substrate cleavage activity of furin,57,58 and for the sorting of PC2.59 The N-terminus of the P-domain starts at the end of the catalytic domain, delimiting the end of the homologous subtilisin sequence. Its C-terminal border has been defined as that amino acid close to an L-X-(L/F)-X-G sequence18 (Fig. 3.3), beyond which further deletions would irreversibly abolish the enzymatic activity of the PC.46,55,57 Interestingly, this functional definition coincides with the endpoint of similarity among the PCs.18,55 It is noted, however, that the P-domain of PC7 extends beyond the L-X(L/F)-X-G sequence.36 In addition to the above consensus sequence at the end of the P-domain of mammalian PCs, this segment exhibits the presence of 20 other identical amino acids, 10 of which are absolutely conserved among all PC orthologues18 (residues bold and underlined in Fig. 3.3). Among these, and with the exception of PC7, the RRGDL sequence is conserved in the other mammalian PCs, and its RG dipeptide is always present in all PC orthologues reported. In view of the RGD sequence found in the PCs and in fibronectin, it was originally suggested that this motif may be important for the interaction of the PCs with cell adhesion integrins.21 Interestingly, three RGD copies exist in Hydra PC1. The PCs which do not contain an RGD sequence include mammalian (human, rat and mouse) PC7, Drosophila furin 2, Lymnaea stagnalis PC2, Aplysia californica PC1 and PC2 and yeast XPR6 (reviewed in ref. 18). The conservation of this motif suggested a structural or functional role which may be common to most mammalian convertases. Binding of fibronectin to its integrin receptor was shown to be dependent on the RGD sequence and to be abrogated when it was replaced by RGE.60 Recently, we addressed the question of the functional relevance of the RRGDL motif within the model enzyme mouse PC1.61 The parameters examined included its importance for POMC cleavage, precursor processing (pro-segment removal and C-terminal cleavage), stability of the enzyme and intracellular trafficking. The data revealed that the RRGDL motif is critical for the stability of PC1 and for its zymogen activation in the ER, as well as for the elaboration of maximal PC1 activity releasing β-LPH from POMC. In addition, since the resulting RGD/E mutant enzyme does not enter secretory

YFNDPiWsnm YFNDPKWPsm eptDPKFPQQ vptDPwFskQ lFNDPmWnQQ nmNDPlFtkQ hFNDPKYPQQ ---DP-----

NYDsyASYDv NYDalASCDv NYDPgASFDv NYDPlASYDF NYDPEASYDF NYnsDASYDF NYsPEgSYDL NY----S-D+ MLDGD.VTDV MLDGD.VTDm MLDGE.VTDa MLDGa.ITDI MLDGi.VTDa MLDqpfmTDI vLDGp.lTDs -LD----TD-

10 ...qaRsdSl dYdlsRaqSt ......dVyq .......slV .svprdsaln gYrdineIdI ........SI ---------70 IERNHPDLAp IERtHPDLmq IEkNHPDLAg IEkdHPDLwa lEWNHtDiya IDYlHPDLAy VEhtvqDiAp ------D--130 AYNAKIGGIR AFNAKIGGVR AYNArIGGVR AFNArIGGVR AYNsKVGGIR AYNsKVaGIR AYgsrIaGIR A------G-R

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus



150 VEAkSLgirP VEAkSvsYNP VEArSLgLNP VEAqSLsLqP IEAsSigFNP IEAsSishmP mEAvaFnkhy -EA-------

+ + 90 NgNDyDPsPR NgNDlDPmPR NDqDPDPqPR NDyDPDPqPR NDNDhDPfPR ssNDPyPyPR NsNDPDPmPh ---D--P-P-

30 WYMHCaDkNs WYMHCsDNth WYL.....sg WYM.....Nk WYLqdTrmta WYLfnTgqad WhL....NNr ----------

nyIDIYSASW QHVhIYSASW nHIhIYSASW QHIhIYSASW gHVDIYSASW QlIDIYSASW QinDIYScSW ----IYS-SW

• YDasNENKHG YDasNENKHG YtqmNDNrHG YtpndENrHG YDptNENKHG YtddwfNsHG pDeeNgNhHG ----N---HG

Rcrs.EMNVq pcqs.DMNIe vtqr.DLNVk eieq.DLNIl slPkLDLhVi gtPgLDLNVa RsPgrDiNVt W---------

+ 170 GPDDDGKTVD GPDDDGKTVD GPEDDGKTVD GPEDDGrTVD GPnDDGKTVE GPtDnGKTVD GPDDDGKTVD GP-D-G-TV-

110 TRCAGEVAAs TRCAGEVAAt TRCAGEVAAv TRCAGEVsAt TRCAGEIAmq TRCAGEVsAa TRCAGEIAAv TRCAGE----

50 aAWkRGYTGK gAWkRGYTGK eAWaqGFTGr kvWnqGLTGr pvWqkGiTGK eAWelGYTGK gvWeRnvTGr --W----TG-

GPGRLakQAF GPapLtrQAF GPaRLaeeAF GPGlLtqeAF GPGRLaqkAF GPreLtlQAM GPhqLgkaAL GP--L---A-

+ ANNSYCiVGI ANNShCtVGI ANNgvCGVGV ANNgFCGaGV ANNhkCGVGV AsNniCGVGV pNNSFCaVGV --N--C--G-

• nVVVTILDDG nIVVTILDDG GIVVsILDDG GVVVsILDDG GVVITVLDDG GVtIgIMDDG GVtVvVvDDG -------DDG


kTSRPAhLkA RTSRaghLNA qTSkPAhLNA RaSRPAqLqA WTSeydpLan lTSkrnqLhd FT..atqyed ----------

310 TWRDvQHLlV TWRDvQHvIV TWRDMQHLVV TWRDLQHLVV TWRDMQHLVV TWRDMQHLtV TWRDvQHiIV TWRD-QH--V

GhKVSHLYGF GFKVSHLYGF GrKVSHsYGY GrqVSHhYGY GLmVnsrFGF GLefnHLFGY GFshSHqhGF G-------G-

+ 350 GLvDAeALVl GLMDAeAMVm GLLDAGAMVa GLLDAGlLVd GLLnAkALVd GvLDAGAMVk GLLnAwrLVn G---A---V-

eA.. eAE. LA.. LA.. LADp MA.. aA.. -A--

ALALEANnqL ALALEANPfL ALtLEANknL ALALEANPlL ALALEANPnL ALALEANvdL ALmLqvrPcL AL-L-----L

hkPWYlEECa kkPWYlEECS nvPWYsEaCS RvPWYsEaCa lsPWYaEkCS RtaLYdEsCS RmPFYaEECa

Fig. 3.2. Alignment of the catalytic subunits of the 7 PCs. (•) = catalytic triad and the oxyanion hole. (+) = conserved residues, except for PC2 which is underlined.

330 s..DWKvNGA N..DWKTNaA N..DWaTNGv e..DWriNGv N.pgWKkNGA evhqWrrNGv hraDWlTNeA ----W--N--

290 vSAPMvAGII ASAPMAAGII ASAPLAAGII ASAPLAAGmI ASAPLAAGIf AaAPeAAGVf AaAPLAAGmI --AP--AG--


+ • .CTDGHTGTS .CTDnHTGTS .CTEsHTGTS .CTDkHTGTS .CTEtHTGTS .CTlrHsGTS gCTEGHTGTS -CT--H-GTS

afyErk..IV EsyDkk..II nqnEkq..IV vvtDpq..IV Dytnqr..It rkrnpeagVa Dkmlr..sIV ----------

250 STLATTYSSG STLATTYSSG STLATTYSSG STFtTTFSSG STLATsYSSG STLAsTFSnG SmLAvTFSgG S------S-G


270 TTDLRQr... TTDLRQr... TTDLRQk... TTDLhhq... saDLhnd... TTDLygn... TTDwdlqkgt --D-------

190 +/• 210 + 230 EyGIKKGRQG LGSIFVWASG NGGRegDhCs CDGYTNSIYT ISVSStTEnG EnGVrmGRrG LGSVFVWASG NGGRskDhCs CDGYTNSIYT ISISStaEsG frGVsqGRgG LGSIFVWASG NGGRehDsCN CDGYTNSIYT lSISSaTqfG rrGVtKGRQG LGtlFIWASG NGGlhyDnCN CDGYTNSIhT lSVgStTrqG EyGVKqGRQG kGSIFVWASG NGGRqgDyCd CDGYTdSICT ISISSasqqG adGVnKGRgG kGSIYVWASG dGG.syDdCN CDGYasSmWT ISInSaindG qhGVmaGRQG FGSIFVvASG NGGqhnDnCN YDGYaNSIYT VtIgavdEeG -G-GR-G -G--ASG -GG-D-C- -DGY-S-T ----G --Y-E-C-


The Mammalian Precursor Convertases 57

ysRRGDLhVt atRRGDLnIn hPRRGDLqIh hPRRGDLaIy ynRRGDLaIh ysRRGDLeIf hPRRGsLelk --RRG-L---

vGtWTLkVtD RGtWTLEl.g eGEWTLEVqD aGDWvLEVyD sGEWvLEIEn qGlWTLglEn RGvYrLvIrD -G---L----

51 EHVQfeaTIe EHVQavITVn EHVvVRIsIS EHVvVRITIt EHVQaRlTlS EHVQVqlslS EHVaVtVsIt EHV------101 MsVHtWGEnp MTtHtWGEDA MTVHCWGEkA MTIHCWGErA MTtHsWdEDp MstHYWdEDp sTVrCWGErA -----W-E--

rPC1 rPC2 rPACE4 rPC5 rFurin rPC4 rPC7 Consensus


Fig. 3.3. Alignment of the P-domain of rat convertases.

eCIIkdnnfE hCVggsvq.n mCVatadK.r vCVestdr.q KCIIeIla.E KCtIrVvh.t sYVspmlK.E ----------

1 RtWrnVPekk kdWkTVPerf RkWTaVPsQh ekWTTVPqQh qnWTTVapQr RvWlptkpQk kiWTsVPyla -W--------


msgrMq...N fvgsap...q ipsQvRnpek tpsQLRnfkt ts....eanN kg....yyyN vgd...eplq ----------

LTSaaGTstv MTSPmGTkSi LiSPsGTkSq LTSPsGTRSq LiSPmGTRSt LTSPmGTRSt LfcPsGmmSl -----G----

PrAlkangEV PekIPPtgkl PrsIPvvqvl iKtIrPnsaV PKdIgkrlEV PtpIlPrmlV nKAVPrsphs ----------

eGrivnWkLI kGlLKEWTLm qGkLKEWsLI pGkLKEWsLV yGtLtkFTLV tGtLyycTLl vGiLqqWqLt -G------L-

LLAeReR.Dt LLsrRPRdDd LLAkRl.LDF LLAnRl.FDh LLAaRP.hDY LvAiRP.LDi igApRs.MDs ----R---D-

iveipTrACE vlTLqTnACE RtTalTnACa RsiykasgCs RKT.VTaclg pKn.VTvcCD levLwnvsrt ----------

144 LhGT LhGT LYGT LYGT LYGT LYGT LYGs L-G-

100 SPnGFkNWDF SkvGFdkWpF SnEGFtNWEF SmEGFkNWEF SaDGFNdWaF SgqGYNNWiF dPnGFNdWtF ---G---W-F

50 gqEN.aInsL gkEN.FVrYL DhsdqrVvYL DnpNhhVnYL Epnh..IsrL gsrrrLIrsL DlEmsglktL ---------L



59

granules, it does not undergo C-terminal truncation into its 66 kDa form and accordingly exits the cell via the constitutive secretory pathway.61 Future work should define whether these results obtained for PC1 are applicable to other members of the PC family which undergo zymogen cleavage in the ER, such as furin, PACE4, PC4 and PC5, and whether the integrity of the RRGDL sequence is also critical for the formation of the C-terminally truncated, granule-associated, 65 kDa PC537 and/or 76 kDa furin.62 In addition, it will be informative to define whether the variant RRGSL motif found in PC7 also plays a similar role for this convertase.

Ontogeny, Tissue Expression and Subcellular Localization Based on their tissue distribution and intracellular localization, the mammalian subtilisin/kexin-like serine proteinases could be subdivided into four classes4 where: 1. furin and the recently discovered PC7 process precursors which reach the cell surface via the constitutive secretory pathway; 2. PC1, PC2 and the isoform PC5-A process precursors whose products are stored in secretory granules; 3. PACE4 and PC5, which are expressed in both endocrine and nonendocrine cells, conceivably process precursors in both the constitutive and regulated secretory pathways; and 4. PC4 is predominantly synthesized in testicular germ cells. The class I convertases furin63,64 and PC736 are localized within the TGN and could cycle to the cell surface and enter endosomal compartments.63 Class II convertases traverse the TGN but are ultimately localized within mature secretory granules.37,65 PC5 could be found in the TGN and/or in granules.37 However, its isoform PC5-B mostly resides in the TGN and cycles to cell surface processing precursors within the constitutive secretory pathway.37 The intracellular localizations of PC4 and PACE4 are not yet known with certainty. Defining the cellular colocalization of each PC with its cognate substrate(s) is necessary in order to ascribe a role for these enzymes in particular precursor processing events.65-71 In addition, dramatic developmental changes in the tissue expression of the PCs have been reported.4,71-74 In the adult, numerous publications dealt with the cell and tissue distribution of the PCs.75-82 They demonstrate a unique distribution of each PC,4,16,17,32 for example in the central nervous system (CNS),70,75-77 pituitary,66,71,77 peripheral nervous system (PNS),78 and some peripheral organs including the heart,79 as well as the thyroid,80 adrenals,29,81 gut and gonads.82 The study of the coregulation of PCs and their cognate substrate(s)17,66,70,71,83-87 also provided hints on their physiological role(s) in vivo. The expression of PCs has been observed in the development of the cerebral cortex, neuromuscular junctions, pituitary gland, pancreatic islets, as well as in the development of cardiovasculature, ossification and chondrification centers. The early expression of PCs suggests their participation in the plasticity of tissue development. However, little is known about the role of specific PCs during embryonic development. For example, it is likely that the growth, differentiation and patterning of embryonic tissue which depends on secretion of growth factors will require proteolytic maturation. Some already published data on the expression of PCs in fetal mice and rats suggest that they are involved in processing of many morphogenetic factors.4,35,73,74,82,87 It is, however, not known which critical steps of organogenesis depend upon PCs, whether they be cell differentiation, cell proliferation or both. Furin is already expressed in both endoderm and mesoderm in the primitive streak stage on embryonic day 7 (e7), while in embryonic ectoderm, from which the nervous system is derived, its mRNA transcripts are undetectable. The overall expression pattern outside the presumptive nervous system is maintained until e10. Higher furin expression is observed in the cardiovasculature and liver primordia, while cephalic regions only express

60


low to medium levels of transcripts.73 In the rat, furin is transiently expressed in Rathke’s pouch region from e13 to e18, suggesting a role in development of the future hypophysis.73 The nervous system remains negative for furin until e18 in the rat73 or e16 in the mouse.82 Maximal expression of furin appears postnatally, but is much lower in comparison to that found in some peripheral tissues (Fig. 3.4), including the lacrimal gland, Bowman’s glands in olfactory turbinate tissues, the liver and Bruner’s glands in the gut.82 Finally, appreciable hybridization levels can be seen in chondrification centers, including chondrocytes and their immediate precursors. Based on comparative expression studies, a role of furin, PACE4, PC5 and PC7 in processing of bone morphogenetic proteins has been suggested.35,74,87 In Figure 3.4, we present some original data to compare the ontogeny of furin and PC7 in whole mouse embryos, and in Figure 3.5 we also present evidence that their tissue mRNAs are sometimes coordinately regulated during development. In situ hybridization ontogeny studies using whole body sections provided a clear demonstration for the presence of furin and PC7 transcripts in each and every tissue. These include skin, muscles, bones and tooth, central and peripheral nervous system, endocrine and exocrine tissues, gut and reproductive organs (Fig. 3.4). However, they exhibit a mosaic expression revealing unique and developmentally regulated local concentrations for each convertase. A representative example is the cardiovascular apparatus in mouse embryo on day 10 (e10) in which a relatively high furin hybridization signal is detected (H in Fig. 3.4a), and the signal remains elevated on e18 (Fig. 3.4b), and then declines on postnatal day 16 (p16; Fig. 3.4c). In comparison, the cardiovascular levels of PC7 mRNA are also detectable on e10 and e18, and postnatally they do not decline as dramatically as for furin. On the other hand, on e18 we note the high expression levels overall of both furin (Fig. 3.4b) and PC7 (Fig. 3.4e), which postnatally (p16) exhibit a dramatic decrease (Fig 3.4c and f). These data suggest that both convertases are functionally implicated in the development of striatal muscle (B) within a narrow developmental window, occurring late in uterine life. Interestingly, this period is critical for the formation of neuromuscular junctions, and hence may require the activity of furin and/ or PC7 either directly or as part of an enzymatic cascade (see ADAM family below). Thus, a picture of furin and PC7 distribution cannot be sealed in a rigid frame of ubiquity, but has to be envisaged with notions that admit plasticity, heterogeneity and developmental regulation of expression patterns. Aside from areas which exhibit a transient increase in PCs’ mRNA concentration, one can also observe that in some tissues (e.g., in the stomach’s pyloric glands) both furin and PC7 transcripts show elevated levels on p16 (Fig. 3.4c and f). This result indicates that, in the adult, PC7 and furin may also be relevant to normal tissue physiology. In order to further probe the developmental changes that occur from birth up to adulthood, in Figure 3.5, Northern blots compare the relative levels of PC7 and furin in selected mouse tissues, revealing cell-specific trends in mRNA expression levels. Thus, in the thymus, brain and male submaxillary gland, mRNA levels of PC7 and/or furin decrease from p1 to p42 (young adults), suggesting that they are developmentally downregulated in these tissues. However, the levels of these PCs do not appreciably change in liver, duodenum and in the cardiac section of the stomach especially for PC7. In contrast, within this postnatal period, dramatic increases in PC7 and furin mRNA levels are observed within the glandular portion of the stomach (pyloric region) and in lacrimal glands. It is interesting that the observed dramatic rise in PC mRNAs occurs in tissues potentially exposed to external pathogens and which specialize in neutralizing the toxic effects of the hostile environment. Thus, the predicted involvement of PCs in host defense mechanisms deserve further studies. More confined expression patterns have been found for PC1 and PC2 mRNAs in embryos, indicating specialization within limited structures. They are already evident by midgestation (e10 and e11) in pancreatic primordia.72 Expression in pituitary primordia and in


61

Fig. 3.4. In situ hybridization histochemistry (ISH) showing furin (a-c) and PC7 (d-f) mRNA expression sites at the anatomical level in mouse embryos on day 10 (e10) and 18 (e18) and on postnatal day 16 (p16), using antisense (as) (a-f) and, to exemplify the extent of nonspecific hybridization, control sense (ss) riboprobes (g-i). A hybridization signal was detected in all tissues studied, exhibiting some heterogeneity in terms of its local concentration. Whereas most tissues were labeled with furin and PC7 on e10, high signal was observed in embryonic cardiovasculature with furin only (H in a). On e18 (b) furin mRNA is detectable in all tissues, with a low signal in brain and spinal cord (Br) and high level hybridization in visceral tissues, striated muscles (Mu) and mandibular bone rudiments (M). The PC7 hybridization pattern on e18 (e) resembles that of furin, with the exception of thymus (Th) which was strongly labeled. ISH on p16 reveals PCspecific patterns of furin (c) and PC7 (f) mRNA distribution. High concentrations of furin mRNA were found in the olfactory neuroepithelium within olfactory turbinates (OT), submaxillary gland (SM), liver (L) stomach pyloric region (Stp), and kidney (k), but not cardiac region (Stc) nor kidney (K). PC7 mRNA was in general less abundant on p16 and, with the exception of the thymus (Th), its distribution resembled that of furin. Magnification x 3.5; bar in (e) = 1cm.

some neuronal structures including the cortical plate has been observed 1 or 2 days later. During the later periods of embryonic development (e16-e19), the expression of PC1 and PC2 reaches appreciable levels both in intensity and extent in the future brain, spinal cord and cranial ganglia, including the trigeminal ganglion (TriG) and spinal ganglia.78 PC1 expression seems to increase maximally by adulthood, while higher levels of PC2 expression are found postnatally in the brain78 and pituitary gland, showing transient presence in corticotrophs.71 Interestingly, PC2 expression coincides with short-time span production of des-acetyl-α-MSH in the same corticotrophs, suggesting that at a high level of this particular convertase, ACTH1-39 processing into ACTH1-17 and CLIP takes place.71 Thus, analysis of temporal expression patterns provides insights into physiologically-relevant functions of PCs. Transient production of des-acetyl-α-MSH in neonatal corticotrophs is an example.

62


Fig. 3.5. Northern blot analysis showing the levels of (A) furin and (B) PC7 mRNA during postnatal developmental stages in mice on postnatal days 1, 16 and 42. The selected tissues are: thymus, brain, male submaxillary gland (S/MAX), liver, duodenum (DUOD), stomach cardiac (ST C) and stomach pyloric (ST P) regions, as well as lachrymal gland (LACR GL). 5 µg of total RNA were loaded in each lane. The X-ray film was exposed for 3 days. Northern blots were obtained with cRNA probes, as described.32,70,76 The observed sizes of the mRNAs of furin and PC7 are 4.4 and 3.9 kb, respectively. The migration position of the 28S ribosomal RNA is indicated.

PC5 expression has been noted in mouse fetal and postnatal ontogeny. A strong PC5 hybridization signal is detected in developing intestine tissue. Only a few telencephalic and mesencephalic centers in the spinal cord synthesize PC5. Its expression appears to be associated with connective tissue around the cardiovascular apparatus and ossification centers35,74,82 and with the exception of ribs, in regions of developing bone.87 These correlative data suggest an important role of PC5 in processing of factors involved in bone morphogenesis, such as the TGFβ-like bone morphogenic proteins BMP-2 and BMP-435,87 and the lefty and nodal proteins which are involved in establishing left-right asymmetry in the mouse.88 In a very recent study, PC5 was found to be expressed both during implantation and embryogenesis.87 Thus, strong PC5 mRNA expression was detected in maternal decidua during implantation, overlapping the region where TGFβ family members and the inhibitor TIMP-3 are expressed.87 This is the first example of the possible role of a PC in implantation and hence, a participation in the uterine-embryo dialogue that requires endocrine-mediated interactions between the embryo and the uterus for implantation to succeed. In summary, PCs demonstrate a remarkable temporal and spatial specificity of expression patterns. Based on this, it seems that they are available in various proportions and combinations in different loci. This raises questions about their redundancy vs. their specialization. Since the PC family actually counts 7 paralogs (Fig. 3.1) with some exhibiting additional isoforms produced by alternative splicing (PACE4 and PC5), the distribution picture remains open to future exploration. Essential steps for understanding PC functions in embryogenesis would involve their modulation in living embryos, with subsequent observation of changes in cell differentiation and organogenesis.

Structure, Loci, and Evolution of PC Genes The intron-exon organization of the genes for furin, PC1, PC2 and PC4 has been determined.89-92 It shows a remarkable conservation of exon order and size in the catalytic and


63

P domains, an additional indication of a common origin of these genes. The Asp, His and Ser of the catalytic triad are each contained within a distinct exon, in line with the hypothesis that exons define functional domains. The Asn of the oxyanion hole is also contained within an exon. In the human PC2 gene, however, the homologous exon is interrupted; and the Asp of the oxyanion hole is located in the 3' portion of the split exon.90 The number and the size of exons flanking the conserved domains are more variable. They probably carry domains that determine properties unique to each enzyme, including its subcellular location, its cleavage site preference and its half life.93 For furin, PC1 and PC4, the use of alternate promoters, splicing sites or polyadenylation signals during transcription generates optional exons found only in some of their mRNA isoforms.91,92,94 The differences in size among PC genes largely result from variation of intron lengths. In this regard, the PC4 gene is noticeable by its short size (about 7 kb)92 and the PC2 gene by its large size (about 130 kb).90 The conservation of the catalytic and P domains and the variability around these domains suggests that PC genes evolved from a common ancestral gene through various mechanisms including duplications, translocations, insertions or deletions. Except for furin and PACE4, which are closely linked, all the other PCs gene are dispersed on various chromosomes (Table 3.7) (reviewed in ref. 95). The synteny between mouse and human for all the chromosomal regions carrying PC loci suggests that their multiplication and divergence occurred before the branching apart of human and murine evolutionary lines about 80 million years ago.

Antisense Transgene Inhibition Cellular Functions of PCs The multiplicity of PC genes suggests both redundancy and differentiation of functions among their products. Redundancy serves the survival of a biological system while differentiation affords it complexity. The challenge in such a case is in the delimiting of the degree of overlap and distinctiveness. Co-expression of substrates and convertases in transfected cells provided invaluable insights into the substrate and cleavage site preferences of many convertases. Some of these relationships have been confirmed in cells expressing transgene-directed antisense RNA that blocks endogenous expression of a convertase. Using this approach, Bloomquist et al84 showed that PC1 deficiency induced by antisense RNA in AtT20 cells caused POMC to accumulate and its conversion to ACTH to diminish, supporting a critical role of PC1 in the production of this corticotrophic hormone.96,97 Similarly, transgene-directed antisense RNA has been used to demonstrate the converting action on several other precursors in various cell types: furin on proparathyroid hormone-related peptide (proPTH-RP) in monkey kidney COS-7 cells;98 PC2 on POMC in rat somatomammotrope GH3;99 PC1 and PC2 on proglucagon in mouse glucagonoma aTC1-6 cells;100,101 PC2 on proneuropeptide Y (proNPY) in cultured sympathetic neurons;102 PC2 on proneurotensin (proNT) in rat medullo-thyroid carcinoma rMTC 6-23 cells;103 and PC1 on procholecystokinin (proCCK) in rat insulinoma Rin5F cells.104 In human colon carcinoma LoVo cells, spontaneous mutations have affected both alleles of the furin gene, making them deficient in furin activity.105,106 These cells are unable to process receptors for insulin-like growth factor (IGF-R) and hepatocyte growth factor (HGF-R),105,106 while they still can convert HIV gp160,107 probably through the action of coresident PACE4 and PC7.16,32 A dependence of IGF-R processing on furin may explain the anti-proliferative effect of antisense transgene or oligonucleotide on mouse gastric mucus GDM6 cells108 and on the well differentiated pancreatic beta cell line MIN6.109

20p (c)

15q (c)

15q (c, s)

11q (c, s)

PCSK2

PCSK3 PCSK4

PCSK5 PCSK6

PCSK7

PC2

Furin

PC5

PACE4

PC7

Pcsk7

Pcsk5 Pcsk6

Pcsk3 Pcsk4

Pcsk2

Pcsk1

Locus Symbol

9 (l)

7 (l)

19 (l)

10 (l)

7 (c, l)

2 (c, l)

13 (c, l)

Chr (c, s, l)

Mouse (m)

nd

nd

nd

7 (m)

12 (h)

> 130 (h)

35 (h), 41 (m)

Size (kb)

nd

nd

nd

15 (m)

16 (h)

12 (h)

14 (h) 15 (m)

No of Exons

nd

nd

nd

Reduced male fertility, preimplantation embryonic lethality (m)

Embryonic dysmorphisms and lethality (m)

Slow growth rate, hyperplasia of pancreatic alpha and delta cells (m)

Obesity, hypogonadotropic hypogonadism, hypercortisolism (h)

Gene Structure (Species) Morbid Phenotype (Species)

PC7. References on chromosomal localizations were reviewed by Mbikay et al (95). Localization was either by cytogenetic (c) methods (including in situ hybridization), by analysis of somatic (s) cell hybrids or by linkage (l) analysis in human (h) or mouse (m).

aReferences on PC gene structure and on morbid phenotypes of PC mutations are presented in the text. This has not been determined (nd) for PC5, PACE4 and

11q (c, s)

19 (s)

5q (c)

PCSK1

PC1

PC4

Chr (c, s, l)

Human (h)

Chromosomal Localization

Locus Symbol

PC

Table 3.7. PC Genes: chromososomal localization, structure, and phenotypic consequences of their inactivationa



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Implication of PCs in Disease States: The Model of Atherosclerosis The endovascular cell proliferation causing atherosclerosis is due in part to the activation of growth factors. Among them, many are produced as inactive precursors which are cleavable by one or more PCs (Table 3.1), e.g., platelet-derived-growth factors (PDGFs), epidermal-growth-factor (EGF), insulin-like growth factors (IGFs) and their receptors and transforming growth factors (TGFs). These molecules require activation by limited proteolysis at the motif (K/R)-Xn-R (where n = 0, 2, 4, 6). Based on the fact that high levels of PC5 transcripts were detected in cells lining rat coronary vessels,79 we explored the possibility that PC5 may play an important role in human arterial restenosis.110 Thus, by in situ hybridization histochemistry the PC expression in normal and atherosclerotic human coronaries revealed that occluded vessels exhibit a marked increase in the level of PC5 mRNA within smooth muscle cells, whereas coronaries without occlusions were PC5 negative. Accordingly, incubation of rabbit smooth muscle cells with a 17-mer PC5-specific antisense oligonucleotide caused a dose-dependent inhibition of cellular proliferation with a maximal effect of 81.6% ± 1.6% at 10 µM, a dose causing a significant reduction in the protein level of PC5.110 The results of this in vitro assay were further extended using an in vivo model of induced injury of rabbit carotid arteries. In this model, the selected PC5-derived 17-mer antisense phosphorothioate oligonucleotide caused a 50% inhibition of neointimal hyperplasia when compared to sense or random oligonucleotides.110 These data suggest that PC5 (and possibly other PCs) could play an important role in the development of arterial restenosis following injury to blood vessels.

Heritable Deficiency of PC in Human and Mouse Oftentimes in contemporary biology, efforts to understand the physiological function of a specific molecule gains significant momentum with the discovery of spontaneous mutant organisms or the production of induced ones. When a female patient with history of massive childhood obesity, hypogonadotropic hypogonadism and secondary hypocortisolism was also found to have large amounts of circulating proinsulin and des-64,65 proinsulin, a possible defect in PC1 was immediately suspected.111 This presumption has been recently confirmed: The patient is a compound PC1 heterozygote, carrying a missense mutation on one allele and a splicing mutation on the other allele.112 The product of the first allele carries a Gly→Arg483 mutation which leads to proPC1 retention in the ER. The second mutation on the second allele affects the donor site of intron 5 and causes skipping of exon 5 during transcription, resulting in the loss of 26 amino acids, a frameshift and creation of a premature stop codon within the catalytic domain, thus leading to an inactive PC1.112 The clinical picture of this patient clearly implicates PC1 in the intricate network of signaling pathways that control body mass and gonadal development, most likely through its processing/activating action on prohormones (proinsulin and POMC) and proneuropeptides. With a mouse model of PC1 deficiency, it should be possible to determine which proneuropetides fail to be appropriately processed and to assess their implication in the pathology by replacement therapy. The PC2 locus was recently inactivated in mouse by homologous recombination.113 Besides a small decrease in growth, the mouse is otherwise normal. However, pancreatic islets in these mice are deficient in their processing of precursors to glucagon, insulin and somatostatin. This deficiency is associated with a secondary hyperplasia of α and δ cells. The mild phenotype resulting from PC2 inactivation is so in contrast with its relative abundance in most neuronal cells, and some endocrine cells, that one is attempted to ascribe to this enzyme a backup role for PC1, its coresident homolog in many of these cells. These results imply that a PC1-null mouse may present a viable, albeit more morbid, phenotype and that PC1/PC2 double-nulls may not be viable.

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Targeted inactivation of the PC4 locus in mouse results in a significant decrease in male fertility, consistent with its restricted expression in spermatogenic cells.114 Spermatogenesis is apparently normal in this mouse. In vitro, its spermatozoa are less efficient at fertilizing ova, and zygotes derived from them fail to develop to the blastocyst stage, suggesting that PC4 activates proproteins that are necessary for fertilization as well as early embryonic development. Whether the PC4 gene is expressed at these early stages remains to be determined. Furin, whose transcripts become detectable on embryonic day e7, is apparently expressed very early during development.73,74 It certainly plays a critical role in embryogenesis since its targeted inactivation leads to dysmorphic embryos that die on e11.5.115 The observed dysmorphisms lend support to the experimental data derived from cells transfected with inducible furin sense or antisense transgenes, which show that furin level influences the equilibrium between cell proliferation and cell differentiation.108,109 In conclusion, the availability of these PC mutant mice enriches our understanding of their biological functions and provides useful models of human pathologies, ultimately leading to the identification of their physiological substrates.

Inhibitors of PCs In view of the potential clinical and pharmacological role of the convertases,93 it was of interest to produce specific PC inhibitors. The proposed strategies involved the development of either peptide-based PC inhibitors,116-119 or protein-based inhibitors.120,121 So far, the in vitro peptide-based approach has not succeeded in effectively inhibiting the PCs intracellularly, and more work in this direction is needed in order to improve the cellular permeability of the designed inhibitors. In humans, the 394 amino acid α1-antitrypsin (α1-AT) is the physiological inhibitor of neutrophil elastase.122 A naturally occurring mutation, known as α1-AT-Pittsburgh (α1-PIT), at the α1-AT reactive site AIPM358 into AIPR358, changed the specificity of this serpin from an inhibitor of elastase into an inhibitor of thrombin.123 Consistent with the critical importance of Arg at the P1 and P4 positions of substrates recognized by furin,124,125 a second mutation in α1-PIT giving the sequence RIPR358 was engineered by Anderson et al, resulting in a potent inhibitor of this convertase.120 This new variant, called α1-AT-Portland (α1-PDX), was shown to inhibit 50% of the furin-catalyzed in vitro cleavage of a small fluorogenic substrate at 0.5 nM concentrations.120 Furthermore, Decroly et al demonstrated that, in vitro, α1-PDX is an inhibitor of all tested PCs.68 However, Vollenweider et al126 demonstrated that in AtT20 cells α1-PDX only partially inhibits the endogenous processing of gp160 or exogenous processing by furin, PACE4 and PC5-B. This observation was recently rationalized by the fact that α1-PDX acts primarily within the constitutive secretory pathway.127 Biosynthetic and immunocytochemical analyses of AtT20 cells stably transfected with α1-PDX cells demonstrated that this 64 kDa serpin is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as an inactive 56 kDa product resulting from cleavage of the active 64 kDa form at the engineered RIPR358 site.127 Furthermore, expression of α1-PDX resulted in modified contents of mature secretory granules with increased levels of partially processed products, suggesting a delayed processing. Accordingly it became apparent that α1-PDX may not inhibit the processing of all precursors to a similar extent and that processing inhibition occurs primarily within the constitutive secretory pathway. Therefore, α1-PDX is a very useful lead protein to inhibit processing of precursors including endogenous growth factors and imported viral surface glycoproteins in constitutive cells, and may exhibit a limited toxicity to cells in vivo. Finally, further variations in the structure of this serpin may lead to a more specific inhibitor which may better discriminate between the convertases.


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Swapping of the pro-domains of PCs demonstrated that the prosegment of PC1 can replace that of PACE4128 and that the furin prosegment can replace that of either PC1 or to a lesser extent that of PC2.128 However, the prodomains of PC1 and PC2 are not interchangeable and the prodomain of PC2 cannot replace that of furin.129 These results further emphasize the uniqueness of PC2, which is the only convertase undergoing pro-segment removal late along the secretory pathway (TGN/immature granules), which has an Asp instead of the usual Asn at the site of the oxyanion hole, and which requires the participation of a specific binding protein 7B2 for its efficient zymogen activation. Nevertheless, these domain-swap data suggested that some of the pro-segments are interchangeable and hence could act as inhibitors of more than one PC. Accordingly, in an effort to design specific protein-based inhibitors which would selectively inhibit the various members of the PC family, the potency and selectivity of their pro-segments were tested in vitro. Thus, a recent report demonstrated that the prosegment of furin exhibits in vitro an inhibitory activity on furin with a K0.5 of 14 nM when used as a fusion protein to glutathione S-transferase.130 We have also recently shown that the pro-segment of PC7 itself when purified from bacterial cultures is a very potent slow binding inhibitor of PC7 in vitro, exhibiting a marked preferential inhibition of PC7 over furin (M. Zhong, J. S. Munzer, N.G. Seidah, unpublished data). Additionally, Lazure et al demonstrated that the pro-segment of PC1 is also a very potent inhibitor of this enzyme in vitro131 and Basak et al132 showed that synthetic peptides derived from the prosegments of proprotein convertase PC1 and furin are potent inhibitors of both proteinases. A recently identified peptide-based inhibitor which shows promise in terms of selectivity and potency is the C-terminal 31 amino acid of the PC2-specific binding protein 7B2,51,52,133,134 called CT-peptide.132 This peptide is highly selective for PC2 and requires the presence of the Lys-Lys bond in the CT-peptide of 7B2 for maximal activity.135 Another very recently identified inhibitory polypeptide is the C-terminal segment of PC1, which is a potent inhibitor of this enzyme, dampening its activity on certain substrates, e.g., proRenin, until the enzyme/substrate complex reaches immature secretory granules.136 In conclusion, future efforts to understand the selectivity embedded within the prosegment, C-terminal sequence of certain PCs and of 7B2's CT-peptide may shed some light on the structures needed to achieve both potent and selective PC inhibition.

Enzymatic Cascades: ADAM Family and PCs In studies involving the definition of the cleavage specificity of the PCs, human aspartyl protease proRenin, responsible for the processing of angiotensinogen into angiotensin I, was shown to be processed quite efficiently by either PC141 or PC5.137 This was the first example of a cascade phenomenon where a PC would activate an enzyme which in turn would process another enzyme precursor. The second example came from the work on the human metalloproteinase Stromelysin-3 (Table 3.1), which is effectively processed by either furin or PACE4.138,139 In a similar vein, in vitro furin can activate the type I membraneassociated matrix metalloproteinase MT1-MMP at the Arg-Arg-Lys-Arg111 ↓ Tyr112-Ala site,140 though the authors report that another enzyme found in CHO-cells can process MT1-MMP intracellularly at the alternative Arg-Arg-Lys-Arg-Tyr112 ↓ Ala113-Ile site,140 a motif similar to that recognized in SREBPs (Table 3.2). Recently, a new family of type I membrane-bound metalloendopeptidases was identified. The members of this so called “ADAM” family contain both a disintegrin and metalloprotease domain. This is an emerging gene family, which in the mouse genome consists of at least 20 members (for review see ref. 141). As prototype paralogs of this family we can mention the fertilins α (ADAM-1) and β (ADAM-2), which are proteins at the surface of sperm implicated in sperm-egg binding during fertilization via an interaction with an


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integrin α6β1 receptor.142 Our results in PC4-null mice suggested that the processing of fertilins, and/or Cyritestin (ADAM-3) could be affected in these mice (ref. 88 and M. Mbikay, unpublished results) and thus provide other examples of enzymatic cascades, whereby a PC would activate an ADAM protease, which in turn would cleave other substrates. Two other examples emerged recently suggesting the generality of this mechanism. One of them involves tumor necrosis factor-alpha-converting enzyme (TACE), which processes membranebound proTNFα to TNFα at the cell surface during an inflammatory response.143,144 In this example, proTACE is first processed intracellularly into active TACE at the type I precursor sequence Arg-Val-Lys-Arg214 ↓ Arg-Ala143 (Table 3.1). The other example involves the likely furin activation of the novel ADAM protease KUZ (Table 3.1), needed for the downstream processing of the Notch protein.145 The latter encodes an ≈300 kDa transmembrane protein146 that acts as a receptor in a cell-cell signaling mechanism controlling cell fate decisions throughout development.146,147 Finally, since proteolytic cleavage at pairs of basic residues found at interdomain boundaries of ADAM proteinases regulates some of their functions, the PCs are likely to exert a key regulatory role by processing various ADAM precursors.

Conclusions The recent identification of a family of serine proteinases of the subtilisin/kexin-type and proof of their function(s) as intracellular processing enzymes recognizing the general motif Arg-(X)n-Arg↓ where n = 0, 2, 4 or 6, resolved a longstanding quest for some of the precursor convertases. Other processing sites, including those occurring at single or pairs of hydrophobic and/or small amino acids are yet to be identified. Furthermore, the generation of PC1-, PC2-, PC4- and furin-null animals suggested that while a limited redundancy in the in vivo activities of these enzymes is possible, convertases expressed early during development (e.g., furin) are indispensable and their silencing inevitably leads to a lethal phenotype. Structure-function and cellular localization studies performed on the PCs exposed the functional complexity of their various domains and the likely cooperativity between some of them. What is emerging are complex, well orchestrated, genetic events, mRNA regulation and protein-protein interactions that regulate the temporal expression, tissue and intracellular localization, and the fine substrate specificity of processing events. Additionally, the involvement of new convertases, distinct from the PCs (see chapters 2, 5, and 7), further emphasize the complexity of regulatory steps needed for high fidelity and efficient precursor processing. Medical applications of this new, but still incomplete and fragmented, knowledge are now beginning to take shape, and it is hoped that in the future these will lead to novel rational therapeutical approaches in a number of pathologies, including cancer, neurological disorders, proliferative diseases and opportunistic pathogenic infections.

Acknowledgments This work was supported by grants from Medical Research Council of Canada (GR 11474), NeuroScience Network and Protein Engineering Network of Centres of Excellence (PENCE). We thank Jon Scott Munzer for Figure 3.1.

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99. Friedman TC, Cool DR, Jayasvasti V, Louie D, Loh YP. Processing of pro-opiomelanocortin in GH3 cells: Inhibition by prohormone convertase 2 (PC2) antisense mRNA. Mol Cell Endocrinol 1996; 116:89-96. 100. Rouillé Y, Westermark G, Martin SK, Steiner DF. Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 Cells. Proc Natl Acad Sci USA 1994; 91:3242-3246. 101. Rothenberg ME, Eilertson CD, Klein K, Mackin RB, Noe BD. Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: An antisense study. Mol Endocrinol 1996; 10:331-341. 102. Paquet L, Massie B, Mains RE. Proneuropeptide Y processing in large dense-core vesicles: Manipulation of prohormone convertase expression in sympathetic neurons using adenoviruses. J Neurosci 1996; 16:964-973. 103. Rovère C, Barbero P, Kitabgi P. Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin. J Biol Chem 1996; 271:11368-11375. 104. Yoon JY, Beinfeld MC. Prohormone convertase 1 is necessary for the formation of cholecystokinin 8 in Rin5f and STC-1 cells. J Biol Chem 1997; 272:9450-9456. 105. Takahashi S, Kasai K, Hatsuzawa K, Kitamura N, Misumi Y, Ikehara Y, Murakami K, Nakayama K. A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochem Biophys Res Commun 1993; 195:1019-1026. 106. Takahashi S, Nakagawa T, Kasai K, Banno T, Duguay SJ, Van de Ven WJM, Murakami K, Nakayama K. A second mutant allele of furin in the processing-incompetent cell line, lovo. evidence for involvement of the homo b domain in autocatalytic activation. J Biol Chem 1995; 270:26565-26569. 107. Ohnishi Y, Shioda T, Nakayama K, Iwata S, Gotoh B, Hamaguchi M, Nagai Y. A furindefective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1. J Virol 1994; 68:4075-4079. 108. Konda Y, Yokota H, Kayo T, Horiuchi T, Sugiyama N, Tanaka S, Takata K, Takeuchi T. Proprotein-processing endoprotease furin controls the growth and differentiation of gastric surface mucous cells. J Clin Invest 1997; 99:1842-1851. 109. Kayo T, Sawada Y, Suzuki Y, Suda M, Tanaka S, Konda Y, Miyazaki J, Takeuchi T. Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic beta cell line MIN6. J Biol Chem 1996; 271:10731-10737. 110. Chrétien M, Fleser A, Day R, Martel R, Leclerc G, Seidah NG. Role of the pro-protein convertases (PCs) in arterial restenosis: a novel therapeutical approach. J Invest Med 1997; 45(3):197A-293A. 111. O’Rahilly S, Gray H, Humphreys PJ, Krook A, Polonsky KS, White A, Gibson S, Taylor K, Carr C. Brief report: Impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function. New Engl J Med 1995; 333:1386-1390. 112. Jackson RS, Creemers JWM, Ohagi S, Raffin-Sanson M-L, Sanders L, Montague CT, Hutton JC, O’Rahilly S. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 1997; 16:303-306. 113. Furuta M, Yano H, Zhou A, Rouillé Y, Holst JJ, Carroll, Ravazzola M, Orci L, Furuta H, Steiner DF. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 1997; 94:6646-6651. 114. Mbikay M, Tadros H, Ishida N, Lerner CP, de Lamirande E, Chen A, El-Alfy M, Clermont Y, Seidah NG, Chrétien M, Gagnon, Simpson EM. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 1997; 94:6842-6846. 115. Roebroek AJM, Pauli I, van Leuven F, Van de Ven WJM. Targeted inactivation of the FUR gene in mice. Keystone Symposium: Molecular and Cellular Biology. Processing of Peptide Hormones, Neurotransmitters, Growth Factors and Viral Proteins. Taos, New Mexico. March 3-9, 1997; Abstract #218. 116. Jean F, Boudreault A, Basak A, Seidah NG, Lazure C. Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor. J Biol Chem 1995; 270:19225-19231.


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117. Decroly E, Vandenbranden M, Cogniaux J, Ruysschaert JM, Howard SC, Jacob G, Marshall G, Kompelli A, Basak A, Jean F, Lazure C, Benjannet S, Chrétien M, Day R, Seidah NG. The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). J Biol Chem 1994; 14:12240-12247. 118. Hallenberger S, Bosch V, Angliker H, Shaw E, Klenk H-D, Garten W. Inhibition of furinmediated cleavage activation of HIV-1 glycoprotein gp160. Nature 1992; 360:358-361. 119. Jean F, Basak A, DiMaio J, Seidah NG, Lazure C. An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor. Biochem J 1995; 307:689-695. 120. Anderson ED, Thomas L, Hayflick JS, Thomas G. Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant. J Biol Chem 1993; 268:24887-24891. 121. Lu WY, Zhang WL, Molloy SS, Thomas G, Ryan K, Chiang YW, Anderson S, Laskowski M. Arg(15)-Lys(17)-Arg(18) turkey ovomucoid 3rd domain inhibits human furin. J Biol Chem 1993; 268:14583-14585. 122. Kurachi K, Chandra T, Friezner Degen SJ, White TT, Marchioro TL, Woo SLC, Davie EW. Cloning and sequence of cDNA coding for alpha 1-antitrypsin. Proc Natl Acad Sci USA 1981; 78:6826-6830. 123. Owen MC, Brennan SO, Lewis JH, Carrell RW. Mutation of antitrypsin to antithrombin alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder. N Engl J Med 1983; 309:694-698. 124. Hatsuzawa K, Murakami K, Nakayama K. Molecular and enzymatic properties of furin, a Kex2-cleavage at Arg-X-Lys/Arg-Arg sites. J Biochem Tokyo 1992; 111:296-301. 125. Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, Brake AJ, Barr PJ, Thomas G. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-b-NGF in vivo. J Cell Biol 1990; 111:2851-2859. 126. Vollenweider F, Benjannet S, Decroly E, Savaria D, Lazure C, Thomas G, Chrétien M, Seidah NG. Comparative cellular processing of the human immunodeficiency virus (HIV1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases. Biochem J 1996; 314:521-532. 127. Benjannet S, Savaria D, Laslop A, Chrétien M, Marcinkiewicz M, Seidah NG. α1-antitrypsin-Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J Biol Chem 1997; 272:26210-26218. 128. Zhou A, Paquet L, Mains RE. Structural elements that direct specific processing of different mammalian subtilisin-like prohormone convertases. J Biol Chem 1995; 270:21509-21516. 129. Rehemtulla A, Dorner AJ, Kaufman RJ. Regulation of PACE propeptide-processing activity—requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc Natl Acad Sci USA 1992; 89:8235-8239. 130. Anderson ED, Vanslyke JK, Thulin CD, Jean F, Thomas G. Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J 1997; 16:1508-1518. 131. Lazure C, Boudreault A, Gauthier D, Seidah NG, Chrétien M, Basak A. The use of recombinant baculovirus expressed mPC1 and mPC2 and synthetic peptides to probe the role of their respective propeptide. Keystone Symposia: Molecular & Cellular Biology. Processing of Peptide Hormones, Neurotransmitters, Growth Factors and Viral Proteins. Taos, New Mexico. March 3-9, 1997; Abstract #111. 132. Basak A, Gauthier D, Seidah NG, Lazure C. Synthetic peptides derived from the prosegments of proprotein convertase PC1 and furin are potent inhibitors of both proteases. Fifteenth American Peptide Symposium, Nashville, TN. June 14-19, 1997. 133. Martens GJM. Cloning and sequence analysis of human pituitary cDNA encoding a novel polypeptide 7B2. FEBS Lett 1988; 234:160-164. 134. Mbikay M, Grant SGN, Sirois F, Tadros H, Skowronski J, Lazure C, Seidah NG, Hanahan D, Chrétien M. cDNA sequence of neuroendocrine protein 7B2 is expressed in beta cell tumors of transgenic mice. Int J Pept Protein Res 1989; 33:39-45.

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135. Zhu X, Rouillé Y, Lamango NS, Steiner DF, Lindberg I. Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: A potential mechanism for its inactivation. Proc Natl Acad Sci USA 1996; 93:4919-4924. 136. Jutras I, Seidah NG, Reudelhuber TL, Brechler V. Two activation states of the prohormone convertase PC1 in the secretory pathway. J Biol Chem 1997; 272:15184-15188. 137. Mercure C, Jutras I, Day R, Seidah NG, Reudelhuber TL. Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex. Hypertension 1996; 28:840-846. 138. Santavicca M, Noel A, Stoll I, Angliker H, Stoll I, Segain J-P, Anglard P, Chrétien M, Seidah NG, Basset P. Characterization of structural determinants and molecular mechanisms involved in pro-stromelysin-3 activation by 4-aminophenylmercuric acetate and furintype convertases. Biochem J 1996; 315:953-958. 139. Pei DQ, Weiss SJ. Furin-dependent intracellular activcation of the human Stromelysin-3 zymogen. Nature 1995; 375:244-247. 140. Sato H, Kinoshita T, Takino T, Nakayama K, Seiki M. Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinase (TIMP) 2. FEBS Lett 1996; 393:101-104. 141. Wolfsberg TG, Primakoff P, Myles DG, White JM. ADAM, a novel family of membrane proteins containing a disintegrin and metalloprotease domain: Multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 1995; 131:275-278. 142. Blobel CP, Wolfberg TG, Turck CW, Myles DG, Primakoff P, White JM. A potential fusion peptide and an integrin ligand domain in a protein active sperm-egg fusion. Nature 1992; 356:248-252. 143. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson M F, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumor necrosis factor-α from cells. Nature 1997; 385:729-733. 144. Moss ML, Jin SLC, Milla ME, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, Becherer JD. Cloning of a disintegrin metalloproteinase that processes precursor tumor-necrosis factor-α. Nature 1997; 385:733-736. 145. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah HG, Israel A. The Notch1 receptor is constitutively cleaved by a furin-like convertase. Proc Natl Acad Sci, USA 1998; 95:8108-8112. 146. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during drosophila and vertebrate neurogenesis. Cell 1997; 90:271-280. 147. Blaumueller CM, Qi H, Zagouras P, Artavanis-Tsakonas S. Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell 1997; 90:278-291.

CHAPTER 4

The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5 Margery C. Beinfeld

Introduction

T

he cloning of the cDNAs of most of the major peptide prohormones revealed that the active moieties are almost always flanked by single or paired basic residues. The recognition that the processing of these prohormones probably takes place in acidifying secretory granules in the presence of significant calcium concentrations has defined the location and some of the biochemical requirements of potential prohormone processing enzymes. They should be trypsin-like enzymes which have the ability to be sorted into regulated secretory vesicles, which are activated by calcium and which operate at moderately acidic pH (5.5-6.5). The discovery of the subtilisin family of enzymes, with the correct anatomical and subcellular distribution and precisely these biochemical properties, has provided a number of likely candidates for the endoproteolytic cleavage of a number of proproteins, prohormones and propeptides in both the regulated and constitutive secretory pathway. This chapter focuses on the three members of this family (PC1, PC2 and PC5) whose distribution in neuroendocrine tissues and endocrine tumor cells and their ability to process a wide range of precursors makes them most likely to participate in the processing of a number of prohormones and proneuropeptides in the regulated secretory pathway.

The Discovery of the Subtilisin Family of Prohormone Convertases Discovery of the prohormone convertase (PC) family of enzymes which now numbers 7 members represented a major advance in the prohormone processing field.1,2 These mammalian enzymes include PC1 (also known as PC3),3,4 PC2,5 PC4,6 PACE 4,7 PC5 (also known as PC6),8,9 PC7/8,10 and furin (also known as SPC1 or PACE, paired basic amino acid cleaving enzyme).11,12 This confusing nomenclature has arisen from the near simultaneous discovery of the same enzymes by different groups. PC5 also has multiple splice variants (PC5B), further complicating the nomenclature. The relationship between these enzymes is depicted schematically in Figure 4.1. Discovery of the Kex2 yeast enzyme which processes the alpha mating factor and killer toxin precursor inspired the search for similar enzymes which could process mammalian precursors. PC25 was cloned by PCR based on its similarity to the catalytic domain of kex2. A database search with the PC2 sequence uncovered the partial sequence of a related human protein fur, found in the c-fes/fps protooncogene. Cloning of this protein, which was later called furin, provided another member of the family.11,12 The other family members PC1, PC4, PC5, PC7/8 and PACE4 were subsequently cloned based on their highly homologous Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Fig. 4.1. Schematic representation of the structure of the subtilisin family of prohormone convertases. The catalytic domain of subtilisin, containing active side D (asp), H (His), N (Asn) and S (Ser) residues is strongly conserved in all family members. P is the P domain, AH is the amphipathic alpha helical domain, TM is the transmembrane spanning domain. PC5 and PC5B are alternately spliced proteins produced from the same gene. The arrow shows where the protein sequences diverge.

catalytic domains. PC5 and PC5B are alternate splice variants of the same gene, as shown in Figure 4.1. PC5B is much larger, has a transmembrane spanning domain and cycles to the cell surface. PC1, PC2, PC4 and PC5 are sorted and activated in the regulated secretory pathway and are candidates for processing prohormones whose secretion is regulated. PC4 is found only in testis and may be involved in prohormone processing in this tissue. The others like furin, PACE4, PC5B and PC7/8 have a transmembrane spanning domain, cycle to the cell surface and are involved in the processing of proteins and precursors in the constitutive pathway.13 These calcium-activated serine proteases which work at mildly acidic pH all have a signal sequence, an amino terminal propeptide of about 80-90 amino acids which is removed at a cleavage site with the motif RXK/RR. They all have a highly conserved catalytic domain with active site aspartic, histidine and serine residues which is very similar to sub-

The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5

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tilisin from Bacillus subtilis. They also have a conserved P or Homo B domain of about 150 residues with an integrin recognition motif RDG.

Distribution of PC1, PC2 and PC5 PC1 and PC2 mRNA are widely distributed in neural and endocrine tissues (as well as tumor cells derived from these tissues) including pituitary, adrenal, pancreas, islet cells, brain and intestine.3,4 PC2 is more abundant than PC1 in most tissues. AtT20 cells are the most abundant source of PC1 mRNA. PC1 and PC2 immunoreactive cells have a distinct distribution in the gastrointestinal tract.14 PC1 is widely distributed throughout the gut and is colocalized with a number of G.I. peptides. PC2 is more abundant in the gastric pylorus and proximal duodenum where it is colocalized with gastrin, CCK and somatostatin. In the stomach, the PCs are more abundant in the mucosa than in the muscular layer. Both PC1 and PC2 are present in the pancreas, but PC2 is much more abundant in pancreatic β cells where it is colocalized with glucagon, somatostatin and pancreatic polypeptide, while PC1 is colocalized with insulin in islet cells.15 PC1 and PC2 are both present in superior cervical ganglia.16 PC1 is present in the ovary. PC1 and PC2 have been visualized in bovine chromaffin granules and their release is stimulated by carbamoylcholine chloride. 17 PC2 has been colocalized with proenkephalin in human adrenal medulla and in many human pheochromocytomas.18 PC1 and PC2 have been found in rat neutrophils and macrophages.19 PC1, PC2 have been visualized by in situ hybridization in the intracardiac para-aortic ganglia of the rat heart.20 PC5 is expressed in brain while both PC5 and PC5B are expressed in intestine. The alternate splice variant PC5B is much larger, has a transmembrane spanning domain, cycles to the cell surface like furin,21 and is probably involved in protein processing in the constitutive pathway.22,23 PC5 is fairly widely distributed, but it is much more abundant in intestine than either PC1 or PC2, as well as being more abundant in brain than PC1. PC5 has also been localized in glucagon-containing dense core secretory granules in the pancreas.23 PC5 has been found in the endothelial cells lining the coronary vessels and the valve leaflets of the rat heart.20 PC5 is known to be expressed in PC12, NB-1 neuroblastoma cells and adrenocortical Y-1 cells, but it is not expressed in RIN5F or AtT20 cells.22

Biosynthesis and Activation of PC1, PC2 and PC5 Detailed examination of the life cycle, intracellular localization, catalytic activity and tissue distribution of these PCs has yielded a gold mine of information. During their biosynthesis, they all undergo autoactivation with the removal of the propeptide. ProPC1 (88 kDa) is converted into 83 kDa, and an 84 kDa sulfated and glycosylated form is secreted.24 The carboxyl terminal of PC1 is not required for maturation although the active site mutant (Ser to Ala mutation) is not able to mature itself.25 PC1 activation is thought to occur in the endoplasmic reticulum with a pH of 7-8 with no added calcium, while PC2 is activated much more slowly than PC1. It is thought to occur in the trans Golgi network with a pH of about 5.5 to 6 and a millimolar calcium concentration.26 This difference in the time course of activation may explain why PC1 frequently cleaves prohormones before PC2. PC1 and PC2 are both glycosylated, and inhibition of PC2 glycosylation by tunicamycin causes intracellular degradation of PC2.24 PC2 is converted from 76 to 64/66 kDa with a half time of about 140 min. Its release is stimulated by glucose in islet cells. PC1 and insulin biosynthesis are also stimulated at the translational level by glucose.27 Production of active PC2 requires an additional protein called 7B2.28,29 It serves as a chaperone, helping PC2 to reach its correct cellular destination. The amino terminal 21 kDa

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protein is responsible for this chaperone action and enhances transport of PC2, while the 31 amino acid peptide is an inhibitor of PC2 activity which is active at a nanomolar concentration. Activation of PC2 in the trans Golgi network is accompanied by the cleavage of this inhibitory domain of 7B2 by PC2.30 The PC enzymes have been reported to be sulfated in vivo but whether this sulfate is on tyrosine or sugar resides is not clear. Much less is known about PC5. When it is expressed in AtT20 cells, it is processed to 117 kDa and 65 kDa forms which are secreted into the media, and their secretion is stimulated by 8Br-cAMP.23 The major form found in rat brain tissue extracts is the 65 kDa form.31 PC5B is expressed as a 210 KDa protein intracellularly and a shorter form of 170 kDa is secreted, although its secretion is not stimulated by 8Br-cAMP, suggesting that it is secreted constitutively.

Regulation of PC Expression Overexpression of CCK mRNA in AtT20 cells does not alter PC1 expression in these cells,32 although pharmacological elevation of cAMP levels and treatment with phorbol esters increased CCK, PC1 and PC2 mRNA levels in rat thyroid medullary carcinoma WE cells and in human neuroblastoma cells, suggesting that the expression of both genes is probably regulated by protein kinase A and C.33 The PC1 gene has multiple transcriptional start sites and its 5' promoter confers both basal and hormone-regulated activity.34 The PC1 and PC2 promoters contain AP-1, SP-1 and cAMP response elements. In rat pituitary, the expression of PC1, PC2, POMC, the amidating enzyme and carboxypeptidase H are all decreased by dopamine agonists.35,36 Birch et al37 have shown that both PC1 and PC2 are present in the supraoptic and paraventricular nuclei of the hypothalamus (PVN), and that expression of both of them is increased by chronic osmotic stimulus. CCK synthesis in this region is increased by the same stimulus.38 PC1 expression in PVN is regulated by glucocorticoids39 and PC1 expression is transiently increased in pilocarpine-induced and kindled seizures.40 In AtT20 cells ACTH secretion is also regulated by ICER (inducible cAMP early repressor), which appears to work through transcriptional control of the PC1 gene rather than expression of the POMC gene.41

Experimental Systems Used to Study Processing Much of the experimental work on the temporal order of cleavages and elucidation of the enzymes responsible for these cleavages initially relied on COS-7 or other fibroblastic cells or neural and endocrine tumor cells in culture. More recently the production of recombinant prohormones and recombinant processing enzymes has allowed processing reactions to be studied in vitro under controlled conditions. The production of processing enzyme mutant mice has added yet another important model. None of these techniques are ideal and utilization of several of them may be required to completely understand the processing of specific prohormones. A wide variety of neural and endocrine tumor cells have been used: neuroblastomas, pituitary (AtT20 and GH3), pancreatic (RIN5F), intestinal (STC-1), adrenal (PC12), and thyroid (WE) cells which normally express peptide mRNAs and some of the processing enzymes and secrete processed peptides. Secretion of these processed peptides is stimulated in response to secretagogues like potassium or cAMP.32,42-44 If these cells do not express the peptide mRNA of interest, it can be stably transfected. Specific processing enzymes can also be coexpressed in these cells with the prohormone with standard techniques, or by infection with vaccinia45 or adenovirus46 vectors. As a first approximation, these endocrine cells appear to be a good model of peptide processing. Some of these endocrine cells normally


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express several PC enzymes, so knowing which of them is responsible for specific cleavages is sometimes difficult. COS-7, L cells and other fibroblastic cells have been used to transiently express peptide prohormones (like proCCK) with individual processing enzymes. When expressed in fibroblastic cells in the absence of specific processing enzymes, the intact prohormones (like proCCK) are generally secreted into the media. When PC1 is transiently coexpressed with proCCK in these cells, the CCK prohormone is processed at appropriate sites and these products are secreted into the media. This makes another system to study the ability of individual processing enzymes to cleave specific prohormones. Many of these cells may also express furin and other related enzymes, which may complicate the analysis because they may also have some ability to cleave expressed prohormones. PC enzyme mRNAs have also been successfully expressed in Xenopus oocytes,26 where they are activated as in mammalian cells. The production of recombinant prohormones and processing enzymes in quantity in bacteria,47 mammalian cells29,48 and insect cells with baculovirus vectors49 has permitted several studies of prohormone processing in vitro.29,48,50-53 These experiments, which take place under more defined conditions, have allowed a more precise determination of the properties of these enzymes with their physiological substrates as well as with synthetic fluorogenic and chromogenic substrates. The Km for most of these enzymes with synthetic substrates is in the 100-200 micromolar range.29,48,52 This is not surprising, as prohormones are known to be considerably concentrated in the secretory granules and this may not be far from their physiological concentration. These studies have provided many new insights, but in some cases there have been discrepancies between cleavages observed in transfection experiments in endocrine cells and in vitro incubations. These differences can perhaps be attributed to the difficulty in reproducing the milieu of the condensing secretory vesicles in vitro.

Enzymatic Activity of PC1, PC2, and PC5 These enzymes cleave mainly at dibasic pairs such as Lys-Arg, Arg-Lys, and Arg-Arg, while some monobasic sites (mainly Arg) are also cleaved.52,54 PC1 and PC2 are widely distributed in neural and endocrine tissues and cell lines and have been shown to be good candidates for the processing of POMC (proopiomelanocortin),45,55,56 insulin,57 glucagon,58 CCK,52,59,60 gastrin,61 dynorphin,54 enkephalin,62 TRH,63 neurotensin,64 somatostatin,65,66 NPY,16 chromogranin A,67 secretogranin II,68 and procorticotropin releasing hormone.69 The newly discovered PC5 is a good candidate for prohormone processing in the brain and intestine.8,9,22 Little is known about the activity of PC5. When studied with renin substrates, PC5 appears to resemble PC1 in terms of its catalytic activity, but with a stricter requirement for paired basic over single basic sites.

Antisense PC1 and PC2 Strategies to Study Proneuropeptide Processing In order to address which of these enzymes is responsible for proneuropeptide cleavage in specific endocrine cells, an antisense strategy has been used because there are no specific, nontoxic inhibitors of these enzymes. This strategy, in which partial PC1 or PC2 cDNAs consisting of the proregions are expressed stably in endocrine cells in the antisense orientation, has provided evidence that PC1 and/or PC2 is involved in POMC, 70 proCCK,59,60,71 proenkephalin,72 neurotensin64 and glucagon processing.58 This is a technique limited to cells that can be transfected or infected. It is a successful strategy in general, but complete inhibition of PC expression with antisense methods has been difficult to achieve.

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Possible toxic effects of overexpression of antisense mRNAs on the expression of peptide or other processing enzyme mRNAs may also limit this technique. Although complete inhibition of PC expression was not always achieved with this technique, there was a large enough decrease in enzyme protein expression to see alteration of prohormone processing. In the case of CCK, inhibition of PC1 in STC-1 and RIN 5F cells caused a selective depletion of CCK-8 while sparing CCK-22, while inhibition of PC2 depleted CCK-22, sparing CCK-8. Antisense experiments have more recently used an inducible promoter system73 and adenovirus vectors to express the PC antisense mRNA.16

Endoproteases in CCK Processing, a Case in Point The question of which of these many enzymes are responsible for individual proCCK cleavages in tissues is difficult to answer. Although detailed studies of the colocalization of CCK with these enzymes have not been performed, PC1, PC2, and PC5 are found in CCKergic cell types and neuronal populations in both the brain and intestine. PC1, PC2 and PC5 are colocalized with oxytocin in the supraoptic and paraventricular nuclei of the hypothalamus. The oxytocin cells are known to also contain CCK,74 and thus these enzymes have a distribution which is consistent with a role in CCK processing.39,75 The colocalization of CCK with these three enzymes appears to hold throughout the rat CNS. AtT20 cells which express PC1 but neither PC2 nor PC5 when transfected with the CCK cDNA can generate CCK-8 in the absence of PC2 and PC5. PC1 by itself has the ability to cleave proCCK at the Arg-Asp and Arg Arg-Ser bonds flanking CCK-8 to generate CCK-8 Gly Arg Arg in L cells. This peptide is thought to be the immediate precursor of CCK-8 amide and accumulates in carboxypeptidase E deficient fat/fat mice.76 COS cells or L cells transfected with PC1 and proCCK can also cleave proCCK to liberate the propeptide (between the signal peptide cleavage site and the amino terminal of CCK-58), a peptide that is found in rat brain77 and endocrine cells.78 Antisense studies also support a role for PC1 and PC2 in proCCK processing. Inhibition of PC1 protein expression by expression of antisense PC1 mRNA in STC-1 and RIN 5F cells caused a selective depletion of CCK-8 while sparing CCK-22. Inhibition of PC2 protein expression by a similar strategy depleted CCK-22, sparing CCK-8. These results further support a role for PC1 in production of CCK-8 and suggest that PC2 activity may be responsible for the production of larger forms of amidated CCK (such as CCK-58, -33, and -22) found in gastrointestinal tissues. Recent work in progress has shown that other cell lines lacking PC1 (which have PC5 with or without PC2) can also process proCCK to CCK-8. This suggests that either PC5 can substitute for PC1 or that there is redundancy so that all three enzymes can produce CCK-8 Gly Arg Arg by themselves.

Processing Enzyme Knockouts and Mutations The most promising approach to understanding the physiological relevance of these enzymes is the production of mice in which these enzymes are specifically deleted. PC279 and furin knockout mice have been produced and efforts are underway to make PC1 knockout mice. PC2 mice are viable and fertile and have defective processing of insulin, somatostatin, glucagon, dynorphin, and enkephalin. Furin knockout mice are embryonic lethals. PC4 knockout mice are viable but the homozygous male mice have reduced fertility, and eggs fertilized in vitro by homozygous male sperm had reduced viability.80 A human patient with defective PC1 enzyme has been described.81 The affected individual was heteroallelic, one allele produced an abnormal Gly to Arg483 substitution while the other allele had a mutation producing a stop codon. The Gly to Arg substituted protein when expressed in CHO cells was retained in the ER, so no active PC1 enzyme was pro-


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duced. The affected individual had severe early-onset obesity, impaired glucose tolerance, secreted excess proinsulin and pro opiomelanocortin. She displayed hypogonadotrophic hypogonadism (but was not sterile) and hypocortisolism. All of her three children inherited one of the Gly to Arg substituted alleles but were clinically unaffected. The ability to use human monocyte-derived macrophage RNA for diagnostic analysis should greatly simplify genetic screening for other human patients with prohormone convertase deficiencies.82

Future Challenges Knowledge of the actual enzymes responsible for prohormone cleavages in specific tissues lags behind progress in other areas of the field due to the technical difficulty of working with intact tissues and whole animals. It is clear that the subtilisin-like PC enzymes are major players, but that there may be additional enzymes which are not in this family which are also required for specific cleavages. Progress in our ability to develop specific, bioavailable inhibitors for these enzymes as well as to develop conditional, tissue-specific knockouts will allow us to more directly address the question of which enzymes are physiologically relevant. Detailed analysis of the recently produced knockout mice should provide much insight into the physiological role of these enzymes. Detailed investigation of the regulation of these processing enzymes and the complex process of their biosynthetic activation is likely to provide many answers. The possibility that tissue-specific differences in their activation and the presence of tissue specific inhibitory substances which play a role cannot be excluded. The most interesting questions are still unanswered, although work is in progress to address them. What structural features of the prohormone determine where it will be processed and how it will be recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? What is the physiological and clinical significance of this regulation? In summary, the last five years have seen enormous progress but many of the most interesting questions remain to be answered.

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47. Hook VYH, Schiller MR, Azaryan AV. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70kDa aspartic proteinase show preference among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 1996; 328:107-14. 48. Zhou Y, Lindberg I. Purification and characterization of the prohormone convertase PC1(PC3). J Biol Chem 1993; 268:5615-23. 49. Wang W, Yum L, Beinfeld MC. Expression and purification of rat pro CCK in bacteria and from media from insect cells infected with recombinant baculovirus. Peptides 1997; 18:1295-1299. 50. Hook VYH, Hegerle D, Affolter HU. Cleavage of recominant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules. Biochem Biophys Res Commun 1990; 167:722-30. 51. Andreasson KI, Tam WWH, Feurst TO et al. Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by a POMC-converting enzyme. FEBS Lett 1989; 248:43-7. 52. Wang W, Beinfeld MC. Cleavage of CCK-33 by recombinant PC 2 in vitro. Biochem Biophys Res Commun 1997; 231:149-52. 53. Rufaut NW, Brennan SO, Hakes DJ et al. Purification and characterization of the candidate prohormone processing enzyme SPC3 produced in a mouse L cell line. J Biol Chem 1993; 268:20291-8. 54. Dupuy A, Lindberg I, Zhou Y et al. Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. FEBS Lett 1994; 337:60-5. 55. Benjannet S, Rondeau N, Day R et al. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-8. 56. Zhou A, Bloomquist BT, Mains RE. The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 1993; 268:1763-9. 57. Smeekens SP, Montag AG, Thomas G et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC3, and PC3. Proc Natl Acad Sci USA 1992; 89:8822-6. 58. Rouille Y, Westermark G, Martin SK et al. Proglucagon is processed to glucagon by prohormone convertase PC 2 in α TC1-6 cells. Proc Natl Acad Sci USA 1994; 91:3242-6. 59. Yoon JY, Beinfeld MC. Prohormone convertase 1 (PC 1) is necessary for the formation of CCK-8 in Rin5F and STC-1 cells. J Biol Chem 1997; 272:9450-6. 60. Yoon JY, Beinfeld MC. Prohormone convertase 2 (PC2) in necessary for the formation of CCK-22 but not CCK-8 in RIN5F and STC-1 cells. Endo 1997; 138:3620-3. 61. Dickinson CJ, Sawada M, Guo YJ et al. Specficity of prohormone convertase endoproteolysis of progastrin in AtT-20 cells. J Clin Invest 1995; 96:1425-31. 62. Breslin MB, Lindberg I, Benjannet S et al. Differential processing of proenkephalin by prohormone convertase I (3) and 2 and furin. J Biol Chem 1993; 268:27084-93. 63. Nillni EA, Friedman TC, Todd RB et al. Pro-thyrotropin-releasing hormone processing by recombinant PC1. J Neurochem 1995; 65:2462-72. 64. Rovere C, Barbero P, Kitabgi P. Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin. J Biol Chem 1996; 271:11368-75. 65. Galanopoulou AS, Kent G, Rabbani SN et al. Heterologous processing of prosomatostatin in constitutive and regulated secretory pathways. Putative role of the endoproteases furin, PC 1 and PC 2. J Biol Chem 1993; 268:6041-9. 66. Galanopoulou AS, Seidah NG, Patel YC. Direct role of furin in mammalian prosomatostain processing. Biochem J 1995; 309:33-40. 67. Arden SD, Rutherford NG, Guest PC et al. The post-translational processing of chromogranin A in the pancreatic islet: Involvement of the eukaryote subtilisin PC2. Biochem J 1994; 298:521-8.


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68. Hoflehner J, Eder U, Laslop A et al. Processing of secretogranin II by prohormone convertases: Importance of PC1 in generation of secretoneurin. FEBS Lett 1995; 360:294-8. 69. Perone MJ, Ahmed I, Linton EA et al. Procorticotrophin releasing hormone is endoproteolytically processed by the prohormone convertase PC2 but not PC1 with stably transfected CHO-K1 cells. Biochemical Society Transactions 1996; 24:497S 70. Bloomquist BT, Eipper BA, Mains RE. Pro-hormone converting enzymes: Regulation and evaluation of function using antisense RNA. Mol Endo 1991; 5:2014-24. 71. Yoon JY, Beinfeld MC. Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK-8 biosynthesis. Reg Peptides 1995; 59:221-7. 72. Johanning K, Mathis JP, Lindberg I. Role of PC2 in proenkephalin processing: Antisense and overexpression studies. J Neurochem 1996; 66:898-907. 73. Eskeland NL, Zhou A, Dinh TQ et al. Chromoganin A processing and secretion: Specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway. J Clin Invest 1996; 98:148-56. 74. Vanderhaeghen JJ, Lotstra F, Vandesande F et al. Co-existence of cholecystokinin and oxytocin-neurophysin in some magnocellular hypothalamophypophyseal neurons. Cell Tis Res 1981; 221:227-31. 75. Shafer MKH, Day R, Cullinan WE et al. Gene expression of prohormone and proprotein convertases in the rat CNS: A comparative in situ hybridization analysis. J Neurosci 1993; 13:1258-79. 76. Cain BM, Wang W, Beinfeld MC. Cholecystokinin (CCK) levels are severely decreased in the forebrains of Cpefat/Cpefat mice: A regional difference in the involvement of carboxypeptidase E (CPE) in pro CCK processing. Endo 1997; 138:4034-7. 77. Beinfeld MC. Cholecystokinin (CCK) gene-related peptides: Distribution and characterization of immunoreactive pre-pro CCK, pro-CCK and an amino terminal pro-CCK fragment in rat brain. BrainRes 1985; 344:351-5. 78. Cao GH, Beinfeld MC. Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells. Peptides 1992; 13:1087-90. 79. Furuta M, Yano H, Zhou A et al. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 1997; 94:6646-51. 80. Mbikay M, Tadros H, Ishida N et al. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 1997; 94:6842-6. 81. Jackson RS, Creemers JWM, Ohagi S et al. Obesity and impared prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nature Genetics 1997; 16:303-6. 82. LaMendola J, Martin SK, Steiner DF. Expression of PC3, carboxypeptidase E and enkephalin in human monocyte-derived macrophages as a tool for genetic studies. FEBS Lett 1997; 404:19-22.

CHAPTER 5

‘Prohormone Thiol Protease’ (PTP), a Novel Cysteine Protease for Proenkephalin and Prohormone Processing Vivian Y.H. Hook, Yuan-Hsu Kang, Martin Schiller, Nikolaos Tezapsidis, Jane M. Johnston and Ada Azaryan

Introduction

P

roduction of peptide hormones and neurotransmitters requires several steps which involves transcription of the pro-hormone gene, translation of the corresponding mRNA, packaging of the prohormone into secretory vesicles, processing by proteolytic mechanisms, storage of mature neuropeptides in secretory vesicles, and regulated secretion of bioactive peptides. Among these steps, posttranslational processing is required for converting the inactive protein precursor into biologically active neuropeptides. Clearly, limited proteolysis is critical for generating neuropeptides.1-3 Endoproteases and exoproteases are required for prohormone processing, which occurs in the regulated secretory pathway of neuroendocrine cells. These potent neuropeptides are stored and secreted from secretory vesicles. The released peptide hormones and neurotransmitters mediate cell-cell communication in neuroendocrine systems.

Features of Prohormone Processing Several proteolytic steps are involved in prohormone processing (Fig. 5.1). The preprohormone is first synthesized from its mRNA at the rough endoplasmic reticulum (RER). The signal peptide of the preprohormone is then removed at the RER by signal peptidase. The resultant prohormone is routed through the Golgi apparatus and packaged into newly formed secretory vesicles, the primary site of prohormone processing.2 Endoproteolytic processing occurs at characteristic paired basic residues—Lys-Arg, ArgArg, Lys-Lys, or Arg-Lys—that flank the NH2- and COOH-termini of the active peptide within its precursor (Fig. 5.1). In addition, processing occasionally occurs at the monobasic arginine residues of some prohormones.4 Endoproteolytic processing at the paired basic residues may occur at three possible sites: 1. at the COOH-terminal side of the dibasic residues; 2. between the dibasic residues; or 3. at the NH2-terminal side of the paired basic residues. Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Fig. 5.1. Proteolysis in Prohormone Processing. The illustrated model neuropeptide precursor contains one copy of the active peptide. The NH2-terminal signal sequence is first removed by signal peptidase at the rough endoplasmic reticulum. Upon packaging of the prohormone into secretory vesicles, endoproteolytic processing occurs at paired basic residues—Lys-Arg (shown in this figure), Arg-Arg, Lys-Lys, or Arg-Lys—at one of three cleavage sites. Endoproteolytic cleavage at paired basic residues may occur at the positions indicated by #1, #2, or #3. Processing by carboxypeptidase and aminopeptidase exoproteolytic processing enzymes is then required to remove basic residues from COOH- and NH2-termini of peptide intermediates, respectively, to complete the series of proteolytic steps needed to produce active neuropeptides.

Resultant peptide intermediates then require removal of the basic residues at their COOH- and NH2-termini by carboxypeptidase and aminopeptidase enzymes,3,5 respectively, to complete the series of proteolytic steps required to generate active neuropeptides.

Evidence for Neurosecretory Vesicles as a Major Site of Prohormone Processing Two important experimental approaches have provided evidence that prohormone processing occurs primarily within secretory vesicles of neuroendocrine tissues. In one approach, the defined neuroanatomical features of the rat hypothalamo-neurohypophyseal system was advantageously utilized to demonstrate that provasopressin and prooxytocin processing occurs during axonal transport of secretory vesicles from neuronal cell bodies of the hypothalamus to nerve terminals of posterior pituitary.6 In a second approach, studies of proinsulin processing by immunoelectron microscopy of proinsulin and insulin, with pH-sensitive indicators, demonstrated that proinsulin processing occurs during maturation and acidification of secretory vesicles.7,8

Prohormone Thiol Protease (PTP)

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Fig. 5.2. The hypothalamo-neurohypophyseal system: Prohormone processing during axonal transport of secretory vesicles. Hypothalamic neuronal cell bodies located in the SON (supraoptic nucleus) and PVN (paraventricular nucleus) send axons through the ME (median eminence) and pituitary stalk, which terminate in the posterior pituitary. Secretory vesicles are axonally transported from cell bodies to nerve terminals.

The hypothalamo-neurohypophyseal system consists of neuronal cell bodies of the SON (supraoptic nucleus) and PVN (paraventricular nucleus) in the hypothalamus, which project axons through the median eminence to the posterior pituitary, known as the hypothalamo-neurohypophyseal system (HNS, Fig. 5.2). Microdissection of these regions allows analyses of radiolabeled 35S-cysteine forms of vasopressin and oxytocin in hypothalamic cell bodies, axons of the median eminence, and nerve terminals in the posterior pituitary. Analyses of 35S-(Cys)-provasopressin and 35S-(Cys)-prooxytocin processing in the hypothalamo-neurohypophyseal system indicated the presence of precursor forms of vasopressin and oxytocin in neuronal cell bodies of the hypothalamus, partially processed peptide forms in axons of the median eminence, and fully processed vasopressin and oxytocin peptides in nerve terminals of the posterior pituitary. These results demonstrate processing of vasopressin and oxytocin precursors during axonal transport of vesicles to nerve terminals. Elegant studies of proinsulin processing have been achieved by immunoelectron microscopic detection of proinsulin and insulin (with specific antibodies to proinsulin and insulin), combined with measurement of internal vesicle pH with the pH-sensitive reagent DAMP (3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine). These studies demonstrated that proinsulin processing occurs within the maturing secretory vesicle while it undergoes internal acidification.7,8 Significantly, proinsulin was present in immature secretory vesicles with an internal pH near 6.0. However, insulin was present in mature vesicles with a more acidic internal pH near 5.0. These results demonstrate that proinsulin processing takes place during vesicle maturation and acidification, suggesting that the processing enzymes within the vesicle become active as the internal vesicular pH becomes acidified to allow conversion of proinsulin to insulin.

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Chromaffin Granules as a Model Neurosecretory Vesicle System for Identification of Proenkephalin and Prohormone Processing Enzymes Evidence for prohormone processing occurring within secretory vesicles6-8 indicates that relevant processing enzymes should be present within isolated secretory vesicles. The bovine adrenal medulla provides an abundant source of homogeneous neurosecretory vesicles9,10 that contain high levels of the opioid peptide (Met)enkephalin (Tyr-Gly-GlyPhe-Met) (Fig. 5.3),11-13 as well as enkephalin-related peptides derived from proenkephalin.14,15 Proenkephalin-derived intermediates (10-25 kDa) and peptides are well-defined in chromaffin granules.12,13,16-18 The chromaffin granule, therefore, serves as an excellent model secretory vesicle system to investigate proenkephalin processing proteases. In vitro and cellular studies of relevant proenkephalin cleaving activity in chromaffin granules have resulted in identification and characterization of relevant proteases that are responsible for converting proenkephalin to enkephalin peptide products. In addition to (Met)enkephalin, chromaffin granules contain a number of neuropeptides including neuropeptide Y (NPY),19 somatostatin,20 vasoactive intestinal polypeptide (VIP),21 and galanin.22 These neuropeptides are also synthesized as precursors that require proteolytic processing at paired basic residues to generate the smaller active peptides. Knowledge gained from studies of proenkephalin processing enzymes can be extended in future investigations to determine the proteases that are involved in processing other proneuropeptides.

The Novel ‘Prohormone Thiol Protease’ (PTP): A Major Proenkephalin Processing Enzyme in Chromaffin Granules Relevant prohormone processing enzymes should meet the following criteria to be considered as serious candidate proteases for authentic prohormone processing.1-3 The processing enzyme should: 1. be colocalized in secretory vesicles with peptide products; 2. demonstrate activity at the acidic pH range of pH 5.5-6.0 that corresponds to the intravesicular pH in vivo; 3. convert precursor substrate to products that are present in vivo; 4. undergo full characterization for comparison to other processing enzymes and proteases; and 5. be coordinately regulated with cellular stimulation of neuropeptide levels. Finally, inhibition of the processing enzyme should reduce cellular levels of the neuropeptide. This chapter describes how the novel cysteine protease known as ‘prohormone thiol protease’ (PTP) meets these criteria, to indicate PTP as an important proenkephalin and prohormone processing enzyme.

Recombinant Enkephalin Precursor as Substrate for Processing Enzymes Identification and characterization of relevant proenkephalin and prohormone processing enzymes requires use of full-length precursor substrates, since the prohormone processing enzyme(s) may require the conformation of the precursor protein for proper recognition and specificity of processing. For this reason, recombinant 35S-(Met)enkephalin precursor was generated from the corresponding rat preproenkephalin cDNA14 by in vitro transcription and translation.23-25 The resultant 35S-(Met)-preproenkephalin (35S-(Met)-PPE) was utilized as a model substrate for detection of candidate proenkephalin processing proteases. 35S-(Met)-PPE was generated from the rat preproenkephalin (PPE) cDNA subcloned downstream of the SP6 promoter of the pSP65 transcription vector. Efficient in vitro transcription with SP6 RNA polymerase routinely generates approximately 20 µg RNA from


93 Fig. 5.3. Immunoelectron microscopy of (Met)enkephalin in chromaffin granules. Sections of isolated chromaffin granules were incubated with anti-(Met)enkephalin (rabbit) serum in PBS (phosphate-buffered saline), and incubated with anti-rabbit IgG labeled with 5 nm gold. Electron dense 5 nM gold particles are visualized directly over the granules.

one µg plasmid DNA.24 Subsequent in vitro translation with wheat germ extract produces up to 20 million cpm 35S-(Met)enkephalin precursor from 4-5 µg RNA. Overall, one µg plasmid DNA can yield 1 x 108 cpm 35S-(Met)enkephalin precursor of high radiospecific activity.23,24 This approach of in vitro transcription and translation of cloned prohormone cDNAs allows efficient production of 35S-(Met)-precursors for studies of in vitro processing.

Purification and Biochemical Characterization of PTP Purification of 35S-(Met)enkephalin precursor cleaving activity from bovine adrenal medullary chromaffin granules results in the isolation of the novel ‘prohormone thiol protease’ (PTP) as the major proenkephalin processing activity.23,25 The majority, 80-90%, of enkephalin precursor cleaving activity from these granules is present in the soluble component of granules.22 Purification by Concanavalin A-Sepharose, Sephacryl S200 gel filtration, chromatofocusing, and thiopropyl-Sepharose results in purification of 33 kDa PTP. PTP is a glycoprotein with a pI of 6.0 and pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0.23 The thiol dependence of PTP is indicated by its stimulation by DTT (dithiothreitol), as well as by its inhibition by the cysteine protease reagents p-hydroxymercuribenzoate, mercuric chloride, cystatin C, E-64c, and E-64d. Importantly, peptide microsequencing of PTP indicates that it possesses a unique NH2-terminal primary sequence that bears no homology to any other known proteases.26 PTP has been characterized with recombinant proenkephalin (PE) in in vitro processing studies.27 In vitro concentrations of PE near estimated in vivo levels29 at 10–5 to 10–4 M were achieved by high level expression of PE in E. coli.27,28 PTP converted purified recombinant PE to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by SDS-PAGE gels and peptide microsequencing. Also, products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOHterminus of (Met)enkephalin-Arg6-Gly7-Leu8. Thus, PTP generates PE products in vitro (Fig. 5.4) that resemble those in adrenal medulla in vivo. These results indicate that PTP possesses appropriate cleavage sites for paired basic residues, and generates relevant PE-related peptide products. Kinetic studies with recombinant proenkephalin showed that PTP possesses a Km(app) of 18.6 µM PE and Vmax(app) of 1.98 mmol/hr-mg.27 PTP’s affinity for PE is compatible with the estimated concentration of PE within the secretory vesicle of approximately 10–4 M,29 indicating that PTP would be functional in vivo.

Cleavage Site Specificity at Dibasic and Monobasic Residue Sites PTP cleavage site specificity has been assessed by examining PTP cleavage of the enkephalin-containing peptides (Fig. 5.5) known as peptide F and BAM-22P that are generated in vivo from proenkephalin.23,30 PTP cleaves peptide F at Lys-Lys and Lys-Arg paired basic residue sites, with cleavage occurring between the two basic residues and at the

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Fig. 5.4. PTP in vitro processing of proenkephalin. PE products generated by PTP are illustrated below the schematic structure of PE. Bars with apparent molecular sizes indicate products that were identified by SDS-PAGE, peptide microsequencing, and reactivity to PE-18 monoclonal antibody.

Fig. 5.5. PTP cleavage of enkephalin-containing peptides. PTP cleavage sites within the enkephalin-containing neuropeptides known as peptide F and BAM-22P are shown by the arrows. The active opioid peptide (Met)enkephalin, YGGFM, is underlined. Amino acids representing paired basic or monobasic cleavage sites are shown as bold letters. Results indicate that PTP generates the final peptide (Met)enkephalin from the proenkephalin-derived intermediates known as peptide F and BAM-22P.

NH2-terminal side of the dibasic residues. PTP also cleaves the Arg-Arg site within BAM-22P at the NH2-terminal side of the dibasic residues. Interestingly, PTP cleaves at a monobasic arginine site within BAM-22P. Cleavage at this monobasic site in vivo is predicted as a necessary processing step in the formation of metorphamide, a bioactive opioid peptide.31 PTP thus possesses appropriate cleavage specificity for paired basic residues and monobasic arginine residues that are required for proenkephalin processing. Importantly, PTP processing at these basic residue sites generates the final product (Met)enkephalin.23,30 PTP’s cleavage sites at paired basic residues differs from the subtilisin-like PC1/3 and PC2 proteases (PC = prohormone convertase)32-38 and the pituitary aspartyl protease (known as ‘POMC converting enzyme,’ or PCE).39,40 PTP cleaves enkephalin-containing peptides at the NH2-terminal side of the dibasic residues or between the dibasic residues, as well as on the NH2-terminal side of monobasic arginine residues.23,30 In contrast, PC1/3, PC2, and PCE cleave at the COOH-terminal side of the dibasic residues, and between the dibasic residues.32-38


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Table 5.1. Comparison of PTP, PC1/3, and PC2 hydrolysis of peptide-MCA substrates, in the absence and presence of aminopeptidase M Peptide-MCA Substrate

-APM

+APM

-APM

+APM

-APM

+APM

PTP Activity (nmol AMC/h/mg)

PC1/3 Activity (nmol AMC/h/mg)

PC2 Activity (nmol AMC/h/mg)

Peptide-MCA Substrate

-APM

+APM

-APM

+APM

-APM

+APM

Dibasic substrates: Z-Arg-Arg-MCA Boc-Gln-Arg-Arg-MCA Boc-Gly-Arg-Arg-MCA Z-Arg-Val-Arg-Arg-MCA Boc-Gly-Lys-Arg-MCA Boc-Glu-Lys-Lys-MCA

16,000 15,000 7,000 19,000 21,000 7,000

49,000 118,000 42,000 50,000 73,000 26,000

ND 125 116 351 208 32

ND 192 118 345 300 51

ND 345 336 762 515 81

ND 364 360 747 552 104

Monobasic substrates: Z-Phe-Arg-MCA 1,176,000 1,915,000 Bz-Arg-MCA 7,000 24,000 Boc-Gln-Gly-Arg-MCA 6,000 84,000 Bz-Val-Leu-Lys-MCA 695,000 885,000 Ac-Lys-MCA 0 3,000

ND ND 2 ND ND

ND ND 29 ND ND

ND ND 6 ND ND

ND ND 36 ND ND

PTP, PC1/3, and PC2 were purified from bovine chromaffin granules, as previously described.23,38 Proteolytic activities with peptide-MCA substrates for each enzyme was determined under the established optimum buffer conditions for each enzyme. ND = not determined.

Further analysis of PTP cleavage sites utilized fluorogenic peptide-MCA substrates containing dibasic and monobasic residues.41,42 PTP cleavage of these peptide substrates indicates cleavage at the COOH-terminal side of dibasic and monobasic residues (Table 5.1). However, it is noted that if cleavage occurs at the NH2-terminal side of basic residues, resultant peptide-MCA products are not detected since only free AMC (not peptide-MCA products) is fluorometrically detected. Therefore, to assess cleavage between dibasic residues, or at their NH2-terminal side, aminopeptidase M (APM) was utilized after PTP hydrolysis to convert peptide-MCA products to free AMC that can be detected fluorometrically. Assays with APM showed several-fold higher activity compared to PTP alone, indicating that PTP preferentially cleaves at the NH2-terminal side of basic residues (Table 5.1). PTP’s preference for cleavage at NH2-terminal sides of basic residues compared to cleavage at the COOH-terminal sides, is illustrated in a bar graph plot (Fig. 5.6). In contrast, PC1/3 and PC2 cleave the dibasic peptide-MCA substrates primarily at the COOH-terminal side, since addition of APM makes little difference in their proteolytic activity (Table 5.1). In contrast to PTP, PC1/3 and PC2 possess much lower activity with monobasic peptide substrates. These results demonstrate PTP’s high specific activity for cleaving both dibasic and monobasic processing sites. A striking difference between PTP and the PC enzymes is the high PTP activity detected with these model substrates. PTP specific activity is 100-500 times greater (average range) than the specific activity measured for PC1/3 and PC2 (Table 5.1). The high activity of PTP, compared to PC1/3 and PC2, is also evident with several prohormone substrates, discussed in the next section.


96

Proteolytic Activity (umol AMC/hr/mg)

a

Dibasic Peptides

Proteolytic Activity (umol AMC/hr/mg)

Peptide-MCA substrate

Monobasic Peptides NH2-Terminal Side COOH-Terminal Side

Peptide-MCA substrate Fig. 5.6. PTP cleavage of peptide-MCA substrates at NH2- and COOH-terminal sides of basic residues in peptide-MCA substrates. Cleavage at NH2-terminal (filled bars) and COOH-terminal (hatched bars) sides of basic residues are represented by PTP activity with aminopeptidase M (APM) minus activity without APM, and PTP activity without APM, respectively. (a) Dibasic peptides: Dibasic peptides numbered 1-6 are Z-Arg-ArgMCA, Boc-Gln-Arg-Arg-MCA, Boc-Gly-Arg-Arg-MCA, Z-Arg-Val-Arg-Arg-MCA, BocGly-Lys-Arg-MCA, and Boc-Glu-Lys-Lys-MCA, respectively (indicated in Table 5.1). (b) Monobasic peptides: Monobasic peptides numbered 1-5 are Z-Phe-Arg-MCA, Bz-ArgMCA, Boc-Gln-Gly-Arg-MCA, Bz-Val-Leu-Lys-MCA, and Ac-Lys-MCA, respectively (indicated in Table 5.1).


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Fig. 5.7. Recombinant prohormones generated with the pET3c expression vector. Proenkephalin, proneuropeptide Y, and proopiomelanocortin (POMC) structures are illustrated. Proenkephalin contains four copies of (Met)enkephalin (M), one copy of (Leu)enkephalin (L), and the enkephalin-related octapeptide (O) and heptapeptide (H). ProNPY contains NPY- and COOHterminal peptide (CTP) sequences. POMC contains ACTH and β-endorphin-related bioactive peptides.

Selectivity for Prohormone Substrates Although prohormones contain similar dibasic or monobasic processing sites, the unique primary structures of prohormones predict that each prohormone possesses a unique conformation that may be preferentially recognized by particular processing enzyme(s). To test the hypothesis that PTP and other processing enzymes may possess selectivity for different prohormone substrates, recombinant proenkephalin (PE), proneuropeptide Y (proNPY), and proopiomelanocortin (POMC) (Fig. 5.7) were expressed in E. coli, and the relative rates of in vitro processing of these precursors were measured (Fig. 5.8).27,28,43,44 PTP efficiently processed PE and proNPY at rates of 383 and 420 µmol/hr/mg enzyme, respectively; however, PTP was much less effective in cleaving POMC. Comparison of four candidate processing enzymes showed that PTP processed PE at rates that were 1,665 and 383,000 times greater than that by the 70 kDa aspartyl protease (also known as ‘POMCconverting enzyme’ or PCE) or PC (PC1/3 and PC2) enzymes, respectively. PTP also processed proNPY 35,000 and 84,000 times more efficiently than the 70 kDa aspartyl protease or the PC enzymes, respectively. In contrast, the 70 kDa aspartyl protease was most effective in POMC processing,43,45,46 and the PC enzymes were also very good;43 however, PTP showed little cleavage of POMC. Furthermore, PTP does not cleave proinsulin (Hook and Gorman, unpublished observations). These results indicate that PTP possesses a high degree of selectivity for particular prohormone substrates.

Immunohistochemistry and Immunoelectron Microscopy of PTP: Localization to Secretory Vesicles To develop antisera for immunolocalization studies of PTP, antisera were generated against a synthetic peptide that corresponds to the NH2-terminus of purified PTP that was determined by peptide microsequencing. The determined NH2-terminal peptide sequence of PTP is a novel sequence,26 as assessed by comparison with sequences in the protein database. The anti-PTP serum detects 33 kDa PTP26 in chromaffin granules, as well as a band of 55 kDa that may represent a zymogen form of PTP. The antibody immunoprecipitates

98


Fig. 5.8. Comparison of PTP, 70 kDa apartyl protease, and PC enzymes in rates of processing recombinant proenkephalin, proNPY, and POMC. The relative rates of processing proenkephalin, proNPY, and POMC—panels (a), (b) and (c), respectively—by PTP, 70 kDa aspartyl protease (PCE, indicated as AP), PC1/3, and PC2 were determined by incubating recombinant prohormones with purified processing enzymes, and the rates of prohormone cleavages were quantitated by SDS-PAGE gels and densitometric scanning of prohormone bands. The specific activity of prohormone processing was calculated as µmol prohormone cleaved per hour per milligram enzyme protein (µmol/h/mg enzyme).


99

Fig. 5.9. Chromaffin cell localization of PTP by immunoelectron microscopy. Chromaffin granules were prepared for immunoelectron microscopy, as described in Figure 5.3. Sections of isolated chromaffin granules were incubated with anti-PTP (rabbit) serum in PBS (phosphate-buffered saline), and incubated with anti-rabbit IgG labeled with 5 nm gold. Electron dense 5 nM gold particles are visualized directly over the granules. The average diameter of the chromaffin granule is approximately 0.1 to 0.2 µM.53

purified PTP activity, indicating that the determined NH2-terminal sequence corresponds to PTP. Immunofluorescence immunocytochemistry indicates punctate, perinuclear PTP staining of chromaffin cells in primary culture (Hook et al, manuscript in preparation). This discrete staining pattern is consistent with a secretory vesicle localization of PTP. The subcellular localization was further examined by immunoelectron microscopy of PTP in isolated chromaffin granules (Fig. 5.9). Immunogold labeling shows the presence of PTP within the chromaffin granules. These results provide definitive evidence for the secretory vesicle localization of PTP.

(Met)enkephalin and PTP are Coordinately Regulated by cAMP in Chromaffin Cells Indication of PTP as the primary proenkephalin processing activity from in vitro studies suggests that PTP may be involved in the regulation of (Met)enkephalin biosynthesis. Forskolin, a stimulator of adenylate cyclase, raises intracellular cAMP, and leads to a 2-fold elevation of (Met)enkephalin levels in chromaffin cells.26,47 During this stimulation, enkephalin precursor cleaving activity from forskolin-treated chromaffin cells was elevated 2-fold in chromaffin granules. Importantly, the elevated processing activity was immunoprecipitated by anti-PTP antibodies, and was inhibited by the cysteine protease inhibitor E64c that is a potent inhibitor of PTP. These results indicate cAMP stimulation of PTP and (Met)enkephalin in chromaffin cells. Moreover, a role for PTP in cellular PE processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by incubation of cells with Ep453,26 which is converted intracellularly to E-64c that potently inhibits PTP.41


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Table 5.2. Properties of ‘prohormone thiol protease’ (PTP) – Secretory vesicle localization – 33 kDa glycoprotein, with pI of 6.0. – Cysteine protease, as determined by protease inhibitors. – pH optimum of 5.5, consistent with intravesicular pH. – Cleaves dibasic and monobasic sites: Dibasic: Lys-Arg, Lys-Lys, Arg-Arg Monobasic: Arg – High specific activity compared to other processing enzymes. – Distinct selectivity for prohormone substrates.

These results demonstrate that cAMP-stimulation of PTP is involved in regulating cellular production of (Met)enkephalin.

Participation of PC1/3 and PC2 Subtilisin-Like Proteases, and 70 kDa Aspartyl Protease (PCE) in Proenkephalin Processing in Chromaffin Granules It is noted that while PTP represents the major proenkephalin processing enzyme in chromaffin granules, lesser activities were identified as the subtilisin-like PC1/3 and PC2 proteases,38 as well as the 70 kDa aspartyl protease45,46 (also known as ‘POMC converting enzyme’ or PCE). The presence of the subtilisin and aspartyl processing proteases in chromaffin granules indicates that PC1/3, PC2 and PCE may also be involved in certain steps of proenkephalin processing. Studies described in this chapter have compared proteases that generate the first cleavage(s) in proenkephalin and other prohormones. Based on differences in primary and therefore, tertiary, structures of prohormones and proteolytic products, processing of intermediates may involve similar or different proteases. It will be important in future studies to determine the order of proteases that are responsible for proteolytic steps of the prohormone processing pathway.

Conclusions Results of these studies indicate the ‘prohormone thiol protease’ (PTP) as a distinct protease among candidate prohormone processing enzymes (Table 5.2). Determination of PTP’s NH2-terminal peptide sequence indicates it as a unique cysteine protease. PTP is a 33 kDa glycoprotein localized to secretory vesicles. It displays a pH optimum of 5.5 that is consistent with the intravesicular pH.1-3 PTP preferentially cleaves dibasic residue sites between the two residues, and at the NH2-terminal side of the paired basic residues; PTP also cleaves at the NH2-terminal side of monobasic arginine residues. PTP possesses extremely high specific activity compared to PC1/3 and PC2 when assayed with dibasic and monobasic peptide-MCA substrates (Table 5.1). Significantly, PTP possesses a high degree of selectivity for proenkephalin and certain prohormone substrates. These properties suggest PTP as an important proenkephalin processing enzyme that possesses selectivity for particular prohormones. It is of interest to note that the primary proteolytic activities within secretory vesicles in vivo from different tissues parallel each protease’s preferences for prohormone substrates (Table 5.3). Proenkephalin processing in chromaffin granules is achieved primarily by PTP, and PTP shows high activity with PE as substrate. POMC processing in pituitary secretory vesicles is primarily accomplished by PCE,39,40 and PCE (which presumably is represented by the 70 kDa aspartyl protease) shows highest activity with POMC (compared to PE and


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Table 5.3. Primary processing proteases identified in isolated neuroendocrine secretory vesicles

Secretory Vesicles

Endogenous Prohormone

Primary Protease for Processing the Endogenous Prohormone

Bovine adrenal medulla chromaffin granules

Proenkephalin

‘Prohormone Thiol Protease’(PTP)*

Pituitary intermediate lobe secretory vesicles

POMC

‘POMC Converting Enzyme’ (PCE)+

Pancreatic insulin vesicles

Proinsulin

PC1/3 and PC2# (PC = prohormone convertase)

* PTP studies23,27,28,30,43,44;+ PCE studies39,40; # PC1/3 and PC2 studies1-3,48,49

proNPY) as substrate. Furthermore, pancreatic insulin secretory vesicles contain PC1/3 and PC2 as primary proinsulin processing proteases.48,49 These observations suggest that distinct, specific processing proteases may be involved in converting different prohormones into active neuropeptides. These studies of PTP, combined with other studies in the field of prohormone processing, indicate that at least three mechanistic groups of proteases are involved in prohormone processing: 1. the unique cysteine protease PTP; 2. the subtilisin-like PC1/3 and PC2 enzymes; and 3. the 70 kDa aspartyl protease known as ‘POMC converting enzyme’ (PCE). It will be important in future studies to determine the specific roles of multiple processing proteases in the conversion of different precursors into active peptide hormones and neurotransmitters.

Acknowledgments This work was supported by grants from the National Institute of Drug Abuse (NIH), National Institute of Neurological Disease and Stroke (NIH), and the National Science Foundation.

References 1. Docherty K, Steiner DF. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol 1982; 44:625-638 2. Gainer H, Russel JT, Loh YP. The enzymology and intracellular organization of peptide precursor processing: The secretory vesicle hypothesis. Neuroendocrinology 1985; 40:171-184 3. Hook VYH, Azaryan AV, Hwang SR, Tezapsidis N. Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J 1994; 8:1269-1278. 4. Devi L. Consensus sequences for processing of peptide precursors at monobasic sites. FEBS Lett 1991; 280:189-194. 5. Fricker LD. Peptide processing exopeptidases: Amino and carboxypeptidases involved with peptide biosynthesis. In: Fricker LD, ed. Peptide Biosynthesis and Processing. Boca Raton: CRC Press, 1991:199-229. 6. Gainer H, Sarne Y, Brownstein MG. Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides. J Cell Biol 1977; 73:366-381.

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7. Orci L, Ravazzola M, Storch MG, Anderson RGW, Vassalli JD, Perrelet A. Proteolytic maturation of insulin is a post-golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987; 49:865-868. 8. Orci L, Halban P, Perrelet A, Amherdt M, Ravazzola M, Anderson RGW. pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle. J Cell Biol 1994; 126:1149-1156. 9. Carmichael SW, Winkler H. The adrenal chromaffin cell. Sci Am 1985; 253:40-49. 10. Hook VYH, Eiden LE. Two peptidases that convert 125I-Lys-Arg-(Met)enkephalin and 125I(Met)enkephalin-Arg6, respectively, to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules. FEBS Lett 1984; 171:212-218. 11. Schultzberg M, Hokfelt T, Lundberg JM, Terenius L, Elfvin LG, Elde R. Enkephalin-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medullary gland cells. Acta Physiol Scand 1978; 103:475-477. 12. Udenfriend S, Kilpatrick DL. Biochemistry of the enkephalins and enkephalin-containing peptides. Arch Biochem Biophys 1983; 221:309-323. 13. Liston DR, Vanderhaeghen JJ, Rossier J. Presence in brain of synenkephalin, proenkephalinimmunoreactive protein which does not contain enkephalin. Nature 1983; 302:62-65. 14. Yoshikawa K, Williams C, Sabol SL. Rat brain preproenkephalin mRNA, cDNA cloning, primary structure, and distribution in the central nervous system. J Biol Chem 1984; 259:14301-14308. 15. Gubler U, Seeburg P, Hoffman BJ, Gage LP, Udenfriend S. Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides. Nature 1982; 295:206-208. 16. Spruce BA, Jackson S, Lowry PJ, Lane DP, Glover D. Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. J Biol Chem 1988; 263:19788-19795. 17. Birch NP, Christie DL. Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum. J Biol Chem 1986; 261:12213-12221. 18. Hook VYH, Liston D. Distribution of enkephalin containing peptide within bovine chromaffin granules. Neuropeptides 1987; 9:263-268. 19. Carmichael WS, Stoddard SL, O’Connor DT, Yaksh TL, Tyce GM. The secretion of catecholamines, chromogranin A and neuropeptide Y from the adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: Different anatomic routes based on size. Neuroscience 1990; 34:433-440. 20. Lundberg JM, Hamberger B, Schultzberg M, Hokfelt T, Granberg PO, Efendie S, Terenius L, Goldstein M, Luft R. Enkephalin-and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma. Proc Natl Acad Sci USA 1979; 76:4079-4083. 21. Holzward MA. The distribution of vasoactive intestinal peptide in the rat adrenal cortex and medulla. J Auton Nerv Syst 1984; 11:269-283. 22. Rokaeus A, Brownstein MG. Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc Natl Acad Sci USA 1986; 83:6287-6291. 23. Krieger TK, Hook VYH. Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor. J Biol Chem 1991; 266:8376-8383. 24. Hook VYH, Schiller MR, Nguyen C, Yasothronsrikul S. Production of a radiolabeled neuropeptide precursors by in vitro transcription and translation. Peptide Res 1996; 9:183-187. 25. Hook VYH, Hegerle D, Affolter HU. Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine adrenomedullary chromaffin granules. Biochem Biophys Res Commun 1990; 167:722-730. 26. Tezapsidis N, Noctor S, Kannan R, Krieger TK, Mende-Mueller L, Hook VYH. Stimulation of ‘prohormone thiol protease’ (PTP) and (Met)enkephalin for forskolin: Blockade of elevated (Met)enkephalin by cysteine protease inhibitor of PTP. J Biol Chem. 1995; 270:13285-13290. 27. Schiller MR, Mende-Mueller L, Miller KW, Hook VYH. ‘Prohormone thiol protease’ (PTP) processing of recombinant proenkephalin. Biochemistry 1995; 34:7988-7995.


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28. Hook VYH, Moran K, Kannan R, Kohn A, Lively MO, Azaryan A, Schiller M, Miller K. High-level expression of the prohormone proenkephalin, proneuropeptide Y, proopiomelanocortin, and protachykinin for in vitro prohormone processing. Prot Express Purif 1997; 10:80-88. 29. Ungar A, Phillips JH. Regulation of the adrenal medulla. Physiol Rev 1983l 63:787-843. 30. Kreiger TK, Mende-Mueller L, Hook VYH. Prohormone thiol protease and enkephalin precursor processing: Cleavage at dibasic and monobasic sites. J Neurochem 1992; 59:26-31. 31. Weber E, Esch FE, Bohlen P, Paterson S, Corbett AD, McKnight AT, Kosterlitz HW, Barchas JD, Evans CJ. Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain. Proc Natl Acad Sci USA 1982; 80:7362-7366. 32. Benjannet D, Rondeau N, Day R, Chretien M, Seidah NG. PC1 and PC2 are proprotein convertases capable of cleaving propiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-3568. 33. Thomas L, Leduc R, Thorne B, Smeekens SP, Steiner DF, Thomas G. KEX2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-5301. 34. Sheenan KIJ, Smeekens SP, Steiner DF, Docherty R. Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes. FEBS Lett 1991; 284:277-280. 35. Zhou Y, Lindberg I. Purification and characterization of the prohormone convertase PC1 (PC3). J Biol Chem 1993; 268:5615-5623. 36. Friedman TC, Loh YP, Birch NP. In vitro processing of proopiomelanocortin by recombinant PC1 (PC3). Endocrinology 1994; 135:854-862. 37. Nillni EA, Friedman TC, Todd RB, Birch NP, Loh YP, Jackson IM. Pro-thyrotropin-releasing hormone processing by recombinant PC1. J Neurochem 1995; 65:2462-2472. 38. Azaryan AV, Krieger TK, Hook VYH. Characteristics of the candidate prohormone processing proteases, PC2 and PC1/3, from bovine adrenal medulla chromaffin granules. J Biol Chem 1995; 270:8201-8208. 39. Loh YP, Parish DC, Tuteja R. Purification and characterization of paired basic residuespecific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J Biol Chem 1985; 260:7194-9205. 40. Loh YP, Beinfeld MC, Birch NP. Proteolytic processing of prohormone and pro-neuropeptides. In: Loh YP, ed. Mechanisms of Intracellular Trafficking and Processing of Proproteins. CRC Press 1993:179-224 41. Azaryan AV, Hook VYH. Unique cleavage site specificity of ‘prohormone thiol protease’ related to proenkephalin processing. FEBS Lett 1994; 341:197-202. 42. Azaryan AV, Hook VYH. Distinct properties of ‘prohormone thiol protease’ (PTP) compared to cathepsins B, L, and H: Evidence for PTP as a novel cysteine protease. Arch Biochem Biophys 1994; 314:170-177. 43. Hook VYH, Schiller MR, Azaryan AV. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70 kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 1996; 328:107-1147. 44. Schiller MR, Kohn AB, Mende-Mueller L, Miller K, Hook VYH. Expression of recombinant pro-neuropeptide Y, proopiomelanocortin, and proenkephalin: Relative processing by ‘prohormone thiol protease’ (PTP). FEBS Lett 1996; 382:6-10. 45. Azaryan AV, Schiller M, Mende-Mueller L, Hook VYH. Characteristics of the chromaffin granules aspartic proteinase involved in proenkephalin processing. Neurochem 1995; 65:1771-179. 46. Azaryan AV, Schiller MR, Hook VYH. Chromaffin granule aspartic proteinase processes recombinant proopiomelanocortin (POMC). Biochem Biophys Res Comm 1995; 215:937-944.

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47. Hook VYH, Eiden LE, Pruss RM. Selective regulation of carboxypeptidase peptide hormone processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells. J Biol Chem 1985; 260:5991-5997. 48. Bennet DL, Bailyes EM, Nielson E, Guest PC, Rutherford NG, Arden SD, Hutton JC. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem 1992; 267:15229-14236. 49. Bailyes EM, Shennan KI, Seal AJ, Smeekens SP, Steiner DF, Hutton JC, Docherty K. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochem J 1992; 285:391-394. 50. Studier FW, Rosenberg AH, Dunn JJ, Dubenforff JW. Use of the T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990; 185:60-89. 51. Kang, YJ, Carl M, Watson LP, Yaffe L. Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and anti-Leu-7 antibodies. J Immunol Methods 1985; 84:177-196. 52. Kang YH, Dwivedi RS, Lee CH. Ultrastructural and immunocytochemical study of the uptake and distribution of bacterial lipopolysaccharide in human monocytes. J Leuko Biol 1990; 48:316-332. 53. Darchen F, Senyshyn J, Brondyk WH, Taatjes DJ, Holz RW, Henry JP, Denizot JP, Macara IG. The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells. J Cell Sci 1995; 108:1639-1649.

CHAPTER 6

Regulation of Prohormone Conversion by Coordinated Control of Processing Endopeptidase Biosynthesis with that of the Prohormone Substrate Terence P. Herbert, Cristina Alarcon, Robert H. Skelly, L. Cornelius Bollheimer, George T. Schuppin and Christopher J. Rhodes

I

t has been established for many years that maintenance of intracellular store levels for mature polypeptide hormones in neuroendocrine cells can be regulated at the level of both gene expression and mRNA translation. Recent evidence has unveiled a specific coordinated control of the appropriate prohormone convertase(s) in parallel to their particular prohormone substrate, both at the level of transcription and translation. In general, translational regulation applies to the short-term (4 hours) there is additional transcriptional regulation. The specific parallel regulation of prohormone biosynthesis with that of the appropriate prohormone processing endopeptidases implicates unique common control mechanisms. For gene transcription this coordinated control likely resides in common ciselements in these genes and trans-acting factors peculiar to a given neuroendocrine cell type. For specific translational regulation, it is unlikely that this coordinated control is via initiation or elongation factor activity regulation, since this would affect protein synthesis translation in general. Thus, the focus has been on potential common regulatory elements in the untranslated regions of prohormone and proprotein convertase mRNAs.

Introduction Polypeptide hormones are often synthesized as larger inactive precursor prohormones which are post translationally processed into biologically active hormones by limited proteolysis.1,2 One feature of prohormone biosynthesis is the rapid changes in the demand for a particular hormone to exogenous stimuli (e.g., the increased demand for insulin in response to a rise in blood glucose concentration3,4). To ensure that optimal intracellular storage levels of a peptide hormone are maintained for regulated secretion, increases in demand for hormone secretion are often met by a corresponding increase in prohormone gene expression and/or biosynthesis. Such an increase in prohormone biosynthesis in turn leads to an increased demand in the specific proteolytic processing enzymes to assure efficient Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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processing of prohormone to biologically active hormone. A means by which this increased demand is met is the specific coordinate regulation of gene expression and/or biosynthesis of proteolytic processing enzymes with that of their prohormone substrate. This chapter focuses on the current knowledge for this latter aspect of the mechanism behind regulated polypeptide hormone production.

Coordinated Regulation of Prohormone and Processing Enzyme mRNA Levels There have been several reports of coordinate gene expression of a prohormone with its specific proprotein-processing endopeptidases. The most extensively studied of these is that of preproinsulin and proopiomelanocortin (POMC).

The Preproinsulin Gene Expression Example The pancreatic β cell is the site of production, storage and regulated secretion of insulin. Preproinsulin gene expression and proinsulin biosynthesis in β cells is regulated by many factors, including certain amino acids and some nutrients,4-10 but glucose is the most physiologically relevant.5,6,11 Proinsulin conversion to insulin is catalyzed by endopeptidic cleavage of human proinsulin at the carboxyl-terminal side of the dibasic amino acid sequences Arg31-Arg32 and Lys64-Arg65.1,12 PC1/PC3 cleaves at the Arg-Arg site, and PC2 has a strong preference for the Lys-Arg site.12-14 Once an endopeptidic cleavage has been made the newly exposed basic amino acids are removed by the exopeptidase carboxypeptidase-H (CP-H). Long term exposure of pancreatic β cells to glucose (>6 h) induces an increase in preproinsulin transcription,15-17 which in turn results in an increase in preproinsulin mRNA levels in the pancreatic beta cell.18,19 The specific levels of the proinsulin converting endopeptidases PC2 and PC1/3 have been shown to be coordinately regulated in parallel to preproinsulin mRNA levels in response to long term exposure to glucose (>12 h) in insulinproducing βTC3 cells.17 Interestingly, mRNA levels of the proinsulin processing exopeptidase CP-H did not appear to be affected by glucose in βTC3 cells. In an in vivo study, coordinate pancreatic islet expression of preproinsulin mRNA with that of its processing enzymes PC2, PC1/3 and CPH has been examined.20 Pancreatic islets were isolated from hyperglycemic rats infused with 50% (w/v) glucose over a 48 hour period. The hyperglycemia specifically increased levels of preproinsulin and PC1/3 mRNAs about 3-fold. However the expression of PC2 and CPH mRNA was unaffected by the treatment. The apparent discrepancy in the coordinate expression of PC2 with preproinsulin observed in βTC3 cells with that observed in pancreatic islets is likely explained by the heterogeneity of the islet cell population. The islet is a heterogeneous population of cells made up of α, β, δ and PP cells.21 PC2 is more abundantly expressed in α, PP and δ cells,22 and although glucose induces a specific induction of PC2 expression in β cells, it is masked by the expression of PC2 in islet non-β cells which are not responsive to glucose. The specific glucose-induced coordinate increase in mRNA levels for preproinsulin, PC1/3 and PC2 in the pancreatic β cell is likely a reflection of two factors: 1. the rate of gene transcription; and/or 2. the rate of mRNA decay. Nuclear run-on experiments were used to measure the rate of transcription of PC2, PC3 and preproinsulin genes in βTC3 cells in response to glucose.17 The transcription rate of preproinsulin, PC2 and PC1/3 genes increased approximately 2-fold in response to a prior 1 h exposure to glucose,17 implicating glucose regulation mediated at the transcriptional level. No glucose regulation of CP-H transcription in β cells has been observed. It should be noted, however, that although glucose can induce preproinsulin, PC2 and PC1/3 gene transcription within 1 hour,16,17,23 an increase in mRNA levels in the β cell cytoplasm is

Regulation of Prohormone Conversion by Control of Endopeptidase Biosynthesis

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not reflected until >6 hours glucose exposure.15-17,24,25 This is due to pre-mRNA processing intron excision occurring in the nucleus prior to mRNA export, which does not appear to be regulated in pancreatic β cells.19,26 The human preproinsulin gene contains 3 exons and 2 intervening sequences,19,26 the mouse PC1/3 gene 15 exons/14 introns,27,28 the human PC2 gene 12 exons/11 introns,29 and rat CP-H 9 exons/8 introns.30 Cytosolic levels of mRNAs can also be altered by changes in their half-life. In pancreatic β cells, the half-life of preproinsulin mRNA is unusually long (>48 h),17,31 and has been shown to be slightly increased by very long term glucose exposure (>24 h).31 In contrast to preproinsulin mRNA, half-lives of PC2, PC1/3 and CP-H mRNAs in β cells (between 4-6 hours) are more characteristic of mRNAs in general, with no apparent glucose regulation of their stability.17 Thus, coordinate regulation of preproinsulin, PC1/3 and PC2 mRNA levels is primarily mediated at the gene transcription level.17 Nonetheless, it should be noted that glucose-induced preproinsulin gene transcription (and that of PC1/3 and PC217), minimally requires more than 6 hours exposure to elevated glucose levels.18,19 However, under normal circumstances in vivo, circulating glucose concentrations are tightly controlled by insulin secretion.12,32 Thus, glucose regulated preproinsulin, PC1/3 and PC2 gene transcription is really only applicable to unusual circumstances of prolonged hyperglycemia,33,34 or refeeding after starvation35 and/or hypoglycemia.23,36 The preproinsulin, PC1/3 and PC2 gene promoter sequences (see below) all contain a cAMP response element (CRE). However, elevation of cAMP in pancreatic β cells does not appear to induce specific transcription of preproinsulin, PC1/3 and PC2 genes, in spite of a marked cAMP induction of c-jun and c-fos genes in the same β cells and of phosphorylation activation of the CRE-binding protein transcription factor.16,17,25

The Proopiomelanocortin (POMC) Gene Expression Example The prohormone POMC is expressed mostly in the neuro intermediate lobe, as well as in ~5% of anterior pituitary cells.37 In the anterior lobe of the pituitary POMC is primarily processed into adrenocorticotropin (ACTH) and β-lipotrophin (LPH). In the neuro intermediate lobe of the pituitary POMC is processed more extensively to β-endorphin, corticotrophin-like intermediate lobe peptide (CLIP), melanocyte stimulating hormone (MSH), and LPH.37 The prohormone processing endopeptidases believed to be primarily responsible for the processing of POMC are PC2 and PC1/3.38-40 Coexpression of PC1/3 and POMC results only in the production of ACTH and β-LPH, whereas coexpression of both PC1/3 and PC2 results in the production of ACTH, LPH and β-endorphin in cells which contain only a constitutive pathway; in cells containing a regulated secretory pathway ACTH is further cleaved to MSH and CLIP.38,39 Complementary to these observations, levels of PC2 mRNA are higher than PC1/3 in the intermediate lobe of the pituitary, but, conversely, in the anterior pituitary lobe PC1/3 mRNA levels are higher than PC2 mRNA levels.41 In the intermediate lobe, POMC gene transcription can be regulated by the neurotransmitter dopamine via the D2 dopamine receptor.42 Bromocryptine, a dopamine agonist, decreases POMC mRNA, whereas haloperidol, a dopamine receptor agonist, increases POMC mRNA.42 It has been found that PC2 and PC1/3 mRNA levels coordinately increase in parallel to POMC mRNA in response to haloperidol.41,43,44 Concordantly, in response to bromocryptine, PC2 and PC1/3 mRNAs fell in parallel to that of POMC.41,43,44 Further, in intermediate pituitary cells, mRNA levels for CP-H and peptidylglycine alpha-amidating monooxygenase (PAM) also decrease in parallel to POMC after bromocryptine treatment and increase after haloperidol treatment.44 In pituitary cells coordinated regulation of CP-H mRNA levels, in parallel to the POMC mRNA levels, contrasts with that seen in pancreatic β cells where CP-H mRNA levels remain constant when preproinsulin mRNA levels increase.20 In contrast to intermediate pituitary cells, haloperitol treatment of anterior pituitary cells

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markedly increases PC2 mRNA levels, whereas PC1/3 mRNA levels do not change.41 Thus, regulation of PC1/3 gene expression differs in anterior versus intermediate pituitary cells. In pituitary cells, the dopamine D2 receptor signal transduction pathway involves heterotrimeric G protein coupled interaction with adenylate cyclase, which in turn alters intracellular cAMP levels in response to bromocriptine or haloperidol.42 This could subsequently result in phosphorylation activation of the CRE-binding protein and increased gene transcription of responsive genes that contain a cAMP-response element (CRE),45 including POMC, PC1/3 and PC2,27-29,42,46 and/or induction of transcription factors such as c-Fos and c-Jun17,45 which in turn induce transcription of other genes via binding activation to an AP-1 site in the promoter.47 It has been shown that under certain circumstances (e.g., regulation by CRH) that induction of POMC gene transcription is dependent on c-Fos/c-Jun expression.48 A similar c-Jun/c-Fos requirement might also be necessary for PC1/3 and PC2 gene expression in intermediate pituitary cells. It should be noted, however, that in intermediate lobe pituitary cells, unlike pancreatic β cells,17 elevated cAMP can induce POMC, PC1/3 and PC2 gene transcription, likely via CRE.41,43,44,49,50 However, in anterior pituitary cells haloperitol does not induce PC1/3 mRNA levels in spite of increased POMC and PC2 gene expression, and therefore different transcriptional regulation applies in different pituitary cell types. Nonetheless, the pattern in pituitary cells for the majority of the time is that effects on POMC expression are generally accompanied by a parallel increase/decrease in PC1/3 and PC2 gene expression. Other circumstances which have previously been shown to affect POMC gene expression, such as hypothyriodism or dexamethazone treatment, also affect PC1/3 and PC2 gene expression.41 Thus, in pituitary cells, dopamine, thyroid hormones and corticosteroids are implicated in regulation of PC1/3 and PC2 gene expression in parallel to that of POMC.

Processing Enzyme and Prohormone Substrate Promoter Regions Regulatory elements within the 5'-promoter regions of both the prohormone and its conversion enzyme genes may be shared, which in turn confers the ability for the transcription of these genes to be coordinately regulated in response to an appropriate stimulus in a given cell type. As outlined in Table 6.1, some common regulatory elements are found in both prohormone substrates (proinsulin and POMC are used as examples in this instance) and the relevant processing enzyme gene promoter sequences. However, a good deal of care should be taken in interpreting the significance of coincidental expression of transcription factor coding elements, and the interpretations must be experimentally tested before making any formative conclusions. Some transcription factors, such as SP-1, are ubiquitous19 and unlikely to be involved in specific regulation of gene expression. Certain other transcription factors, such as Pan-1 (a helix-loop-helix binding protein), form heterodimers with a variety of other transcription factors in a cell-specific manner, that in turn affects its DNA-binding and transcription of a given gene.19 In the example of the preproinsulin gene promoter in pancreatic β cells, Pan-1 is able to form heterodimers with the transcription factors Meso-1,51 Mash-1,52 NeuroD/Beta253 and also inhibitory transcription factors Id1 and Id3.19 However, it is not clear under what circumstances a particular Pan-1-containing transcription factor heterodimer complex will bind the preproinsulin gene promoter region and what will then be the consequences of that DNA binding for regulation of preproinsulin gene transcription. Furthermore, some transcription factors are apparently more critical than others. The homeodomain transcription factor pdx-1 (also known as IPF-1, STF-1, IUF-1, Idx-1) appears to be very important for not only preproinsulin gene transcription but also endocrine pancreatic development.54-57 Homozygous pdx-1 knockout transgenic mice do not develop a pancreas and die soon after birth.58 pdx-1 is also

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Regulation of Prohormone Conversion by Control of Endopeptidase Biosynthesis

Table 6.1. Some consensus regulatory elements present in known promoter regions of processing enzyme and prohormone genes Gene

Consensus Regulatory Elements Present

Ref.

Human Preproinsulin Human POMC Murine PC1/3 Human PC2 Rat CPH Human 7B2

SP-1, CRE, Pan-1, NRE, TATA Box, CCAAT Box, GAGA Box SP-1, CRE, Pan-1, AP-1, TRS, TATA Box, GAGA Box, SP-1, CRE, Pan-1, AP-1, ICS, GHF-1, GAGA Box SP-1, CRE, AP-2, TRS, Pan-1 SP-1, AP-2, NF-1, Pan-1 CRE, AP-1, Pan-1, TRS, HSE, Pit-1/GHF-1

19 46 27 29 30 93

The 5'-promoter primary sequences for human preproinsulin, human POMC, murine PC1/3, human PC2, rat CP-H and human 7B2 (a specific chaperone for PC294) have been cloned and contain some putative regulatory elements. Abbreviations are: SP-1, consensus binding site for the ubiquitous transcription factor SP-1; CRE, cAMP response element; Pan-1, consensus binding site for the helixloop-helix transcription factor Pan-1; NRE, negative regulatory element; AP-1, consensus binding site for the AP-1 transcription factor complex containing c-fos; AP-2, consensus binding site for protein kinase-C sensitive transfactor complexes; TRS, thermal stress response consensus sequence; ICS, interferon consensus sequence; HSE, heat shock consensus sequence; Pit-1/GHF-1, consensus binding site for POU proteins; NF-1, consensus binding site for the transcription factor NF-1.

important for somatostatin gene transcription,56 indicative that certain transcription factors have multiple functions dependent on the cell type. To date the 5'-promoter region of the preproinsulin gene has been shown to bind at least twelve different transcription factors to different elements in that region.18,19 This, in turn, suggests that the combination of transcription factors required to drive expression of a prohormone gene are likely to be cell specific. Nonetheless, it remains to be shown under what regulatory circumstances certain factors associate to the preproinsulin gene promoter, and what combination of transcription factors is required to drive preproinsulin gene transcription in pancreatic β cells. In general, similar circumstances will also be appropriate for the regulated gene expression of other prohormones and processing enzymes in other endocrine cell types. However, although some transcription factors might be common, the final composition of factors required will ultimately be specific for a given prohormone and peculiar to the cell where that the prohormone is expressed. It should also be noted that presence of a primary sequence encoding for a transcription factor binding in a promoter region of a gene does not necessarily mean that it is functional, and could be dependent on whether that element is functional in a particular cell type. For example, a CRE is present in the preproinsulin, PC1/3 and PC2 gene promoter sequences, but cAMP does not appear to regulate expression of these genes in pancreatic β cells.17 In contrast to pancreatic β cells, in intermediate pituitary cells cAMP does appear to regulate gene expression of POMC, PC1/3 and PC2;41 in anterior pituitary cells only PC2 mRNA, and not that of PC1/3 is increased by cAMP mediated pathways.41 Therefore, as well as the combination of transcription factors required to drive expression of a given prohormone/processing enzyme being specific to an endocrine cell-type, another important consideration is that transcription factors are responsive to an appropriate signal transduction pathway in that cell.

110


Coordinated Translational Regulation of Specific Prohormone and Processing Enzyme Biosynthesis Intracellular content of polypeptide hormones in endocrine cells in dense core secretory granules is continually maintained at optimal levels so that a hormone is readily available for rapid regulated release in response to an extracellular stimulus. The secreted hormone lost from the cell in response to the stimulus is rapidly replaced by a corresponding specific stimulation of prohormone biosynthesis. It is becoming apparent that this specific regulation of prohormone biosynthesis is mediated at the level of translation. Such an increase in the biosynthesis of a prohormone places an obvious increased demand on the proteolytic processing of that precursor; however this is provided for by a coordinated regulation of the appropriate proprotein convertase biosynthesis in parallel to the prohormone substrate. This is especially appropriate to the regulation of proinsulin biosynthesis in pancreatic β cells, which will be more extensively described here. It should be noted that the general mechanism for proinsulin biosynthesis translation could well apply to prohormones in other endocrine cell types; for example, there is recent evidence that the biosynthesis of POMC is also translationally regulated.44

Translational Regulation of Proinsulin Biosynthesis Proinsulin biosynthesis is specifically regulated in a pancreatic β cell by a variety of nutrients, hormones, pharmaceutical and physiological factors; however the most physiologically relevant of these is glucose.59 Short term (

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