Essentials of Apoptosis

Second Edition

Xiao-Ming Yin l Zheng DongEditors

Essentials of Apoptosis

A Guide for Basic and Clinical Research

Second Edition

1 3

Editors

Xiao-Ming YinDepartment of PathologyUniversity of Pittsburgh3500 Terrace StreetPittsburgh PA 15261Scaife Hall, 7th FloorUSAxmyin@pitt.edu

Zheng DongDepartment of Graduate StudiesMedical College of Georgia1459 Laney Walker Blvd.Augusta GA 30912USAzdong@mcg.edu

ISBN 978-1-60327-380-0 e-ISBN 978-1-60327-381-7DOI 10.1007/978-1-60327-381-7

Library of Congress Control Number: 2009921154

# Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009All rights reserved. This workmay not be translated or copied in whole or in part without the writtenpermission of the publisher (Humana Press, c/o Springer ScienceþBusinessMedia, LLC, 233 SpringStreet, New York, NY 10013, USA), except for brief excerpts in connection with reviews orscholarly analysis. Use in connection with any form of information storage and retrieval,electronic adaptation, computer software, or by similar or dissimilar methodology now known orhereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if theyare not identified as such, is not to be taken as an expression of opinion as to whether or not they aresubject to proprietary rights.

Printed on acid-free paper

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Preface to Second Edition

The first edition of this book was published in 2003. It was intended to providethe basic and essential information of apoptosis for those who were new to thefield and who wanted to apply the knowledge to their own research. The booktherefore focused on the concepts, the basic molecular architecture, and thepathophysiological significance of apoptosis. Since the first edition, there havebeen tremendous new developments in the field of apoptosis and cell death ingeneral. The concept of various types of cell death has been further developed.The studies in both basic and clinical disciplines have been greatly expanded.In particular, notable progress has been made in extending the work into thetherapeutic arena. As a result, the field hasmatured considerably and developedextensive cross-talk with works in other fields.

We strive to incorporate and reflect these new developments in the secondedition of this book. Our goal is to provide readers with the most updatedand advanced knowledge in the field, while maintaining the fundamentalinformation as presented in the first edition. To this end, the book has beensignificant expanded, not only in the page and chapter numbers but moreimportantly in the depth of coverage. Most of the chapters have been revisedand/or rewritten, and 15 new chapters have been added to give rise to a total of31 chapters. In Part I, in addition to the discussion of the major apoptosismolecules, the activation and regulatory pathways, and the clearance ofapoptotic cells, we also present important issues that examine apoptosis frommore integrated points of view. Thus, the roles of reactive oxygen species,metabolism, and transcription control in apoptosis activation and regulationare explored. A new systems biology approach to studying apoptosis is alsointroduced. In Part II, we discuss apoptosis and cell death in four modelsystems, including plant, yeast, C. elegans, and Drosophila, which together havecontributed greatly to our current understanding of cell death. In Part III, we focuson mammalian cell death under various pathophysiological situations in all majorsystems, including the hematopoietic and immune system, the brain, the heart, theliver, the lung, and the kidney. Two integrated chapters discussing cell death innormal development and in cancer biology have also been included. Furthermore,a separate Part IV has been added to discuss the alternative mechanisms andpathways of cell death. Finally, Part V discusses the technical aspect of apoptosis

v

research. This new edition should be valuable for both novice and seasonedinvestigators as a comprehensive reference as well as a practical guide.

As broad as the content to which they have contributed, our more than80 contributors come from across the world, representing institutes from15 countries and regions. We would like to acknowledge the hard work by allthe authors, who are recognized experts and leaders in the field of apoptosisresearch. Without their dedicated contributions, this book would not have beenpossible. We are also especially grateful to our families for their wholeheartedand enduring support, which makes the edition of this book very rewardingand enjoyable. Finally, we wish to dedicate this edition to the memory ofDr. Stanley J. Korsmeyer (1950–2005), a beloved mentor, colleague, andfriend. Dr. Korsmeyer was a pioneer and a visionary in the field of apoptosis.His seminal works on the Bcl-2 family proteins, on the mitochondria andendoplasmic reticulum pathways of apoptosis activation and regulation, andon the pathophysiological significance of apoptosis in embryonic development,in immune response, and in cancer biology and cancer therapy tremendouslychanged and advanced the field. He will be remembered by all of us.

Pittsburgh, PA Xiao-Ming YinAugusta, GA Zheng Dong

vi Preface to Second Edition

Preface to First Edition

Life and death are topics that no one takes lightly. In the cell, death by apoptosisis just as fundamental as proliferation for the maintenance of normal tissuehomeostasis. Too much or too little apoptosis can lead to developmentalabnormality, degenerative diseases, or cancers. Although apoptosis, orprogrammed cell death (PCD), has been recognized for more than 100 years,its significance and its molecular mechanisms were not revealed until recently.

We have witnessed rapid progress in apoptosis research in the last decade.Apoptosis can now be defined not only by morphology, but also by molecularand biochemical mechanisms. As a result, there has been an information explosionin the field. On one hand, this has dramatically expanded our understanding of therole of apoptosis in both biology and medicine; on the other hand, it has made thestudy of apoptosis quite complicated, and sometimes confusing. One oftenwonders whether findings from other laboratories can be generalized orwheather the methods used can be made applicable to other systems.

Studies of apoptosis are unusual in that the common focus on a basic processthat is driven by specific sets of biochemical machinery is studied in an array ofvery diverse research areas. Investigators from different fields have documentedtheir views of apoptosis in numerous review articles. These reviews, published invarious scientific journals, are aimed at either summarizing the latest findings orproviding brief introductions to apoptosis. However, essential informationabout apoptosis, such as its mechanisms and pathophysiological roles, has yetto be presented in a systematic and concise way. This has posed a great hurdle tomany investigators who want to enter this field or to apply the knowledge totheir own research, and are not sure where and how to begin.

Essentials of Apoptosis: A Guide for Basic and Clinical Research serves as astarting point for those investigators who are relatively new to apoptosisresearch. Therefore, instead of describing detailed findings in one specificfield, we present the concepts, the molecular architecture (the molecules andthe pathways), and the pathophysiological significance of apoptosis.Controversial results are presented only if they are related to the essentialprocess. In addition, standard biochemical and cellular approaches toapoptosis research are described as a guideline for bench work. Essentials ofApoptosis: A Guide for Basic and Clinical Research is intended to provide

vii

readers with the basics of apoptosis in order to stimulate their interests and toprepare them for the commencement of apoptosis-related research in theirchosen areas. We hope that Essentials of Apoptosis: A Guide for Basic andClinical Research will prove useful reading for all those interested in apoptosisresearch.

Pittsburgh, PA Xiao-Ming Yin, MD, PhDAugusta, GA Zheng Dong, PhD

viii Preface to First Edition

Contents

Part I Molecules and Pathways of Apoptosis

1 Caspases: Activation, Regulation, and Function . . . . . . . . . . . . . . . . . 3Stefan J. Riedl and Fiona L. Scott

2 The Bcl-2 Family Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Wen-Xing Ding and Xiao-Ming Yin

3 The Mammalian IAPs: Multifaceted Inhibitors of Apoptosis . . . . . . . 63Eric C. LaCasse, Herman H. Cheung,Allison M. Hunter, Stephanie Plenchette,Douglas J. Mahoney, and Robert G. Korneluk

4 Structural Biology of Programmed Cell Death. . . . . . . . . . . . . . . . . . 95Yigong Shi

5 The Death Receptor Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Maria Eugenia Guicciardi and Gregory J. Gores

6 The Mitochondrial Pathway: Focus on Shape Changes . . . . . . . . . . . 151Silvia Campello and Luca Scorrano

7 The Endoplasmic Reticulum Pathway . . . . . . . . . . . . . . . . . . . . . . . . 177Michael W. Harr and Clark W. Distelhorst

8 Reactive Oxygen Species in Cell Fate Decisions . . . . . . . . . . . . . . . . 199Han-Ming Shen and Shazib Pervaiz

9 The Integration of Metabolism and Cell Death . . . . . . . . . . . . . . . . . 223Jonathan L. Coloff, Yuxing Zhao, and Jeffrey C. Rathmell

ix

10 Transcriptional Regulation of Apoptosis . . . . . . . . . . . . . . . . . . . . . . 239Crissy Dudgeon, Wei Qiu, Quanhong Sun, Lin Zhang,and Jian Yu

11 Clearance of Apoptotic Cells – Mechanisms

and Consequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261Carylyn J. Marek and Lars-Peter Erwig

12 Systems Biology Approaches to the Study of Apoptosis . . . . . . . . . . . 283Heinrich Huber, Eric Bullinger, and Markus Rehm

Part II Apoptosis in Model Organisms

13 Programmed Cell Death in Plants: Apoptotic but Not Quite . . . . . . . 301Naohide Watanabe and Eric Lam

14 Tracing the Roots of Death: Apoptosis

in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Didac Carmona-Gutierrez and Frank Madeo

15 Programmed Cell Death in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . 355Monica Darland-Ransom, Yi-Chun Wu, and Ding Xue

16 Cell Death in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375Dianne C. Purves, Jessica P. Monserrate,and Carrie Baker Brachmann

Part III Apoptosis in Mammalian Physiology and Pathogenesis

17 Cell Death: Defining and Misshaping Mammalian Embryos . . . . . . . 409Zahra Zakeri and Richard A. Lockshin

18 Matters of Life and Death in the Immune System . . . . . . . . . . . . . . . 423Christopher P. Dillon and Douglas R. Green

19 Cell Death in the Hematopoietic System . . . . . . . . . . . . . . . . . . . . . . 443Emma C. Josefsson and Benjamin T. Kile

20 Cell Death in Acute Neuronal Injury . . . . . . . . . . . . . . . . . . . . . . . . . 461R. Anne Stetler, Armando P. Signore, and Jun Chen

21 Apoptosis in Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . 479Qiuli Liang and Jianhua Zhang

x Contents

22 Apoptosis in Cardiovascular Pathogenesis . . . . . . . . . . . . . . . . . . . . . 505Hamid el Azzouzi, Meriem Bourajjaj,Paula A. da Costa Martins, and Leon J. De Windt

23 Apoptosis in Lung Injury and Disease . . . . . . . . . . . . . . . . . . . . . . . . 523Stefan W. Ryter, Hong Pyo Kim, and Augustine M. K. Choi

24 Apoptosis in Liver Injury and Liver Diseases . . . . . . . . . . . . . . . . . . . 547Yosuke Osawa, Ekihiro Seki, and David A. Brenner

25 Apoptosis in Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565Navjotsingh Pabla, Qingqing Wei, and Zheng Dong

26 Apoptosis in Cancer Biology and Cancer Therapeutics . . . . . . . . . . . 581Simone Fulda

Part IV Alternative Cell Death Mechanisms and Pathways

27 Necrosis: Molecular Mechanisms and Physiological Roles. . . . . . . . . 599Linde Duprez, Nele Vanlangenakker, Nele Festjens,Franky Van Herreweghe, Tom Vanden Berghe,and Peter Vandenabeele

28 Caspase-Independent Mitotic Death. . . . . . . . . . . . . . . . . . . . . . . . . . 635Katsumi Kitagawa

29 Lysosomal Proteases in Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . 647Nathalie Andrieu-Abadie

30 Autophagy and Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671Wentao Gao, Jeong-Han Kang, Yong Liao, Min Li,and Xiao-Ming Yin

Part V Approaches to the Study of Apoptosis

31 Analysis of Apoptosis: Basic Principles and Protocols . . . . . . . . . . . . 691Man Jiang, Craig Brooks, Guie Dong, Xiaoning Li,Hong-Min Ni, Xiao-Ming Yin, and Zheng Dong

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713

Contents xi

Contributors

Nathalie Andrieu-Abadie INSERM U858, 12 MR-Institut de Medecine

Moleculaire de Rangueil BP84225, 31432 Toulouse cedex 4, France,

nathalie.andrieu@inserm.fr

Meriem Bourajjaj Hubrecht Institute and Interuniversity Cardiology Institute

Netherlands, Royal Netherlands Academy of Sciences, Utrecht, The

Netherlands

Carrie Baker Brachmann Department of Developmental and Cell Biology,

School of Biological Sciences, University of California, Irvine, CA 92697-2300,

USA, cbrachma@uci.edu

David A. Brenner Department of Medicine, University of California,

San Diego School of Medicine, La Jolla, CA 92093, USA

Craig Brooks Department of Cellular Biology and Anatomy, Medical

College of Georgia and Charlie Norwood VA Medical Center, Augusta, GA

30912, USA

Eric Bullinger Industrial Control Centre, Department of Electronic and

Electrical Engineering, University of Strathclyde, Glasgow, Scotland, UK;

Hamilton Institute, National University of Ireland, Maynooth, Ireland

Silvia Campello Department of Cell Physiology and Metabolism, University

of Geneva, 1Rue Michel Servet, 1201, Geneva 4-CH, Switzerland

Didac Carmona-Gutierrez Institute for Molecular Biosciences, Karl-Franzens

University, Humboldtstrabe 50, 8010 Graz, Austria

Jun Chen Department of Neurology, University of Pittsburgh School

ofMedicine, Pittsburgh, PA 15237, USA; National Key Laboratory ofMedical

Neurobiology, Shanghai Medical School, Fudan University, Shanghai, China

Herman H. Cheung Apoptosis Research Centre, Children’s Hospital

of Eastern Ontario, Ottawa, Ontario K1H 8L1; Department of Biochemistry,

Microbiology, and Immunology, University of Ottawa, Ottawa K1H 8M5,

Canada

xiii

Augustine M.K. Choi Pulmonary and Critical Care Medicine, Brigham and

Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA

Jonathan L. Coloff Department of Pharmacology and Cancer Biology, Sarah

W. Stedman Nutrition and Metabolism Center, Duke University Medical

Center, Durham, NC 27710, USA

Paula A. da Costa Martins Hubrecht Institute and Interuniversity Cardiology

Institute Netherlands, Royal Netherlands Academy of Sciences, Utrecht,

The Netherlands

Monica Darland-Ransom Department of Molecular, Cellular and

Developmental Biology, University of Colorado, Boulder, CO 80309-0347,

USA

Leon J. De Windt Department of Medical Physiology, Division Heart

and Lungs, University Medical Center Utrecht, Utrecht, The Netherlands,

l.j.dewindt@umcutrecht.nl

Christopher P. Dillon Department of Immunology, St. Jude Children’s

Research Hospital, Memphis, TN 38105-2794, USA

Wen-xing Ding Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15236, USA

Clark W. Distelhorst Department of Medicine, Case Western Reserve

University, WRB 3-133, Cleveland, OH 44106-7285, USA, cwd@case.edu

Guie Dong Department of Cellular Biology and Anatomy, Medical College

of Georgia and Charlie Norwood VA Medical Center, Augusta, GA

30912, USA

Zheng Dong Department of Cellular Biology and Anatomy, Medical College

of Georgia and Charlie Norwood VA Medical Center, Augusta, GA 30912,

USA, zdong@mail.mcg.edu

Crissy Dudgeon Department of Pharmacology and Pathology, University

of Pittsburgh Cancer Institute andUniversity of Pittsburgh School ofMedicine,

Pittsburgh, PA 15213, USA

Linde Duprez Molecular Signaling and Cell Death Unit, Department for

Molecular Biomedical Research, VIB, 9052 Ghent; Department of Molecular

Biology, Ghent University, 9052 Ghent, Belgium

Hamid el Azzouzi Hubrecht Institute and Interuniversity Cardiology Institute

Netherlands, Royal Netherlands Academy of Sciences, Utrecht, The

Netherlands

Lars-Peter Erwig Department of Medicine and Therapeutics, Institute

of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK,

l.p.erwig@abdn.ac.uk

xiv Contributors

Nele Festjens Unit for Molecular Glycobiology, Department for

Molecular Biomedical Research, VIB, 9052 Ghent; Department

of Biochemistry, Physiology and Microbiology, Laboratory for Protein

Biochemistry and Biomolecular Engineering, Ghent University, 9052

Ghent, Belgium

Simone Fulda University Children’s Hospital, Eythstr. 24, D-89075 Ulm,

Germany, simone.fulda@uniklinik-ulm.de

Wentao Gao Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15236, USA

Gregory J. Gores Division of Gastroenterology and Hepatology, Mayo

Medical School, Clinic, and Foundation, Rochester, MN 55905, USA,

gores.gregory@mayo.edu

Douglas R. Green Department of Immunology, St. Jude Children’s Research

Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA,

douglas.green@stjude.org

Maria Eugenia Guicciardi Division of Gastroenterology and Hepatology,

Mayo Medical School, Clinic, and Foundation, Rochester, MN 55905, USA

Michael W. Harr Department of Medicine, Case Western Reserve University,

Cleveland, OH 44106-7285, USA

Heinrich Huber Systems BiologyGroup, Department of Physiology &Medical

Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street,

Dublin 2, Ireland

Allison M. Hunter Apoptosis Research Centre, Children’s Hospital of Eastern

Ontario, Ontario K1H 8L1; Department of Biochemistry, Microbiology,

and Immunology, University of Ottawa, Ottawa K1H 8M5, Canada

Man Jiang Department of Cellular Biology and Anatomy, Medical

College of Georgia and Charlie Norwood VA Medical Center, Augusta,

GA 30912, USA

Emma C. Josefsson Division of Molecular Medicine, The Walter & Eliza Hall

Institute ofMedical Research, 1GRoyal Parade, Parkville, VIC 3050, Australia

Jeong-Han Kang Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15236, USA

Benjamin T. Kile Division of Molecular Medicine, The Walter and Eliza Hall

Institute ofMedical Research, Parkville, VIC 3052, Australia, kile@wehi.edu.au

Hong Pyo Kim Division of Pulmonary, Allergy and Critical Care Medicine,

Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15231;

Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital,

Harvard Medical School, Boston, MA 02115, USA

Contributors xv

Katsumi Kitagawa Department of Molecular Pharmacology, St. Jude

Children’s Research Hospital, Memphis, TN 38105-2794, USA,

katsumi.kitagawa@stjude.org

Robert G. Korneluk Apoptosis Research Centre, Children’s Hospital

of Eastern Ontario, Ottawa, Ontario K1H 8L1; Department of Biochemistry,

Microbiology, and Immunology, University of Ottawa, Ottawa K1H 8M5,

Canada, bob@arc.cheo.ca

Eric C. LaCasse Apoptosis Research Centre, Children’s Hospital of Eastern

Ontario, Ottawa, Ontario K1H 8L1; Department of Biochemistry,

Microbiology, and Immunology, University of Ottawa, Ottawa K1H 8M5,

Canada, bob@arc.cheo.ca

Eric Lam Department of Plant Biology and Pathology, Biotechnology Center

for Agriculture and the Environment, Rutgers University, Foran Hall, 59

Dudley Road, New Brunswick, NJ 08901, USA, lam@aesop.rutgers.edu

Min Li Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15236, USA

Xiaoning Li Department of Cellular Biology andAnatomy,Medical College of

Georgia and Charlie, Norwood VAMedical Center, Augusta, GA 30912, USA

Qiuli Liang Department of Pathology, University of Alabama, Birmingham,

AL 35294, USA

Yong Liao Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15236, USA

Richard A. Lockshin Department of Biological Sciences, St. John’s University,

Queens, NY 11439, USA, lockshin@stjohns.edu

Frank Madeo Institute for Molecular Biosciences, Karl-Franzens University,

Humboldtstrabe 50, 8010 Graz, Austria, frank.madeo@uni-graz.at

Douglas J. Mahoney Apoptosis Research Centre, Children’s Hospital of Eastern

Ontario, Ottawa, Ontario K1H 8L1; Department of Biochemistry, Microbiology,

and Immunology, University of Ottawa, Ottawa K1H 8M5, Canada.

Carylyn J. Marek Department of Medicine and Therapeutics, Institute

of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK

Jessica P.Monserrate Department of Developmental and Cell Biology, School

of Biological Sciences, University of California, Irvine, CA 92697-2300, USA

Hong-Min Ni Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15213, USA

Yosuke Osawa Department of Gastroenterology, Gifu University Graduate

School of Medicine, Yanagido, Gifu 501-1194, Japan

xvi Contributors

Navjotsingh Pabla Department of Cellular Biology and Anatomy, Medical

College of Georgia and Charlie Norwood VA Medical Center, Augusta, GA

30912, USA

Shazib Pervaiz ROS and Cancer Biology Program, Department of Physiology,

Yong Loo Lin School of Medicine; NUS Graduate School for Integrative

Sciences and Engineering, National University of Singapore; Cancer and Stem

Cell Biology Program, Duke-NUS Graduate Medical School; Singapore-MIT

Alliance, Singapore 117597, Singapore, phssp@nus.edu.sg

Stephanie Plenchette Apoptosis Research Centre, Children’s Hospital of Eastern

Ontario, Ottawa, Ontario K1H 8L1; Department of Biochemistry, Microbiology,

and Immunology, University of Ottawa, Ottawa K1H 8M5, Canada.

Dianne C. Purves Department of Developmental and Cell Biology, School

of Biological Sciences, University of California, Irvine, CA 92697-2300, USA

Wei Qiu Department of Pharmacology and Pathology, University

of Pittsburgh Cancer Institute and University of Pittsburgh School

of Medicine, Pittsburgh, PA 15213, USA

Jeffrey C. Rathmell Department of Pharmacology and Cancer Biology, Sarah

W. Stedman Nutrition and Metabolism Center; Department of Immunology,

Duke University Medical Center, Durham, NC 27710, USA,

jeff.rathmell@duke.edu

Markus Rehm Systems Biology Group, Department of Physiology and

Medical Physics, Royal College of Surgeons in Ireland, RCSI York House,

York Street, Dublin 2, Ireland, mrehm@rcsi.ie

Stefan J. Riedl Program of Apoptosis and Cell Death Research, Burnham

Institute for Medical Research, La Jolla, CA 92037, USA,

sriedl@burnham.org

Stefan W. Ryter Pulmonary and Critical Care Medicine, Brigham and

Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA,

sryter@partners.org

Luca Scorrano Department of Cell Physiology and Metabolism, University

of Geneva, 1Rue Michel Servet, 1211, Geneva 4-CH, Switzerland,

luca.scorrano@unige.ch

Fiona L. Scott Apoptos Inc., San Diego, CA 92121, USA

Ekihiro Seki Department of Medicine, University of California, San Diego

School of Medicine, La Jolla, California 92093, USA

Han-Ming Shen Department of Community Occupational and Family

Medicine, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore 117597, Singapore, cofshm@nus.edu.sg

Contributors xvii

Yigong Shi Department of Molecular Biology, Lewis Thomas Laboratory,

Princeton University, Princeton, NJ 08544, USA, yshi@molbio.princeton.edu

Armando P. Signore Department of Neurology, University of Pittsburgh

School of Medicine, Pittsburgh, PA 15237, USA

R. Anne Stetler Department of Neurology, University of Pittsburgh School

ofMedicine, Pittsburgh, PA 15237, USA; National Key Laboratory ofMedical

Neurobiology, Shanghai Medical School, Fudan University, Shanghai, China,

stetlerra@upmc.edu

Quanhong Sun Department of Pharmacology and Pathology, University

of Pittsburgh Cancer Institute andUniversity of Pittsburgh School ofMedicine,

Pittsburgh, PA 15213, USA

Franky Van Herreweghe Molecular Signaling and Cell Death Unit,

Department for Molecular Biomedical Research, VIB, 9052 Ghent;

Department of Molecular Biology, Ghent University, 9052 Ghent,

Belgium

Tom Vanden Berghe Molecular Signaling and Cell Death Unit, Department

for Molecular Biomedical Research, VIB, 9052 Ghent; Department

of Molecular Biology, Ghent University, 9052 Ghent, Belgium

Peter Vandenabeele Molecular Signaling and Cell Death Unit, Department

for Molecular Biomedical Research, VIB; Department of Biomedical

Molecular Biology, Ghent University, B-9052 Zwijnaarde (Ghent), Belgium,

peter.vandenabeele@dmbr.vib-UGent.be

Nele Vanlangenakker Molecular Signaling and Cell Death Unit, Department

for Molecular Biomedical Research, VIB, 9052 Ghent; Department

of Molecular Biology, Ghent University, 9052 Ghent, Belgium

Naohide Watanabe Department of Plant Biology and Pathology,

Biotechnology Center for Agriculture and the Environment, Rutgers

University, Foran Hall, 59 Dudley Road, New Brunswick, NJ 08901, USA

Qingqing Wei Department of Cellular Biology and Anatomy, Medical

College of Georgia and Charlie Norwood VA Medical Center, Augusta, GA

30912, USA

Yi-Chun Wu Department of Zoology, National Taiwan University, Taipei

10617, Taiwan, yichun@ccms.ntu.edu.tw

Ding Xue Department of Molecular, Cellular and Developmental Biology,

University of Colorado, Boulder, CO 80309-0347, USA,

ding.xue@colorado.edu

Xiao-Ming Yin Department of Pathology, University of Pittsburgh School

of Medicine, Pittsburgh, PA 15261, USA, xmyin@pitt.edu

xviii Contributors

Jian Yu Department of Pharmacology and Pathology, Universityof Pittsburgh Cancer Institute andUniversity of Pittsburgh School ofMedicine,Pittsburgh, PA 15213, USA

Zahra Zakeri Department of Biology, Queens College and Graduate Centerof the City University of New York, Flushing, NY 11367, USA

Jianhua Zhang Department of Pathology, University of Alabama,Birmingham, AL 35294, USA

Lin Zhang Department of Pharmacology and Chemical Biology, Universityof Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine,Pittsburgh, PA 15213, USA, zhanglx@upmc.edu

Yuxing Zhao Department of Pharmacology and Cancer Biology,Sarah W. Stedman Nutrition and Metabolism Center, Duke UniversityMedical Center, Durham, NC 27710, USA

Contributors xix

Color Plates

Color Plate1 Schematic representation of the amplificatory loop at the

mitochondrial level in response to an apoptotic stimulus.

Three main interconnected mitochondrial steps are repre-sented: (1) oligomerization of Bax and Bak, which generatesa physical pathway for the efflux of proteins across the outermitochondrial membrane; (2) Opa1-controlled remodelingof the cristae, leading to the redistribution of cytochrome cin the intermembrane space; (3) activation of mechanismsthat cause mitochondrial fragmentation, following calci-neurin (CnA)-dependent dephosphorylation of Drp1, orinteraction of the latter with TIMMP8a, a component ofthe import machinery of mitochondria that is releasedtogether with cytochrome c. (Chapter 6, Fig. 1; seediscussion on p. 167)

Color Plate 2 History of apoptotic systems modeling. Published systemsbiology studies were classified into four methodologicalcategories and ordered chronologically. Arrows indicatean influence or logical connection between different stu-dies with respect to the adoption of biological or metho-dological information (Chapter 12, Fig. 2; see discussionon p. 287)

Color Plate 3 The molecular machinery of yeast apoptosis. Exogenous andendogenous induction of yeast apoptosis leads to the activa-tion of the basic molecular machinery of cell death, which isconfigured by conserved apoptotic key players such as theyeast caspase Yca1p, the yeast homologue of mammalianHtrA2/OMI (Nma111p), or the apoptosis-inducing factorAif1p. Furthermore, it involves complex processes likehistone modification, mitochondrial fragmentation, cyto-chrome c release, and cytoskeletal perturbations(Chapter 14, Fig. 1; see discussion on p. 334)

xxi

Color Plate 4 Physiological scenarios and yeast apoptosis. A wild-typeyeast population promotes its own long-term survival andspreading of the clone by eliminating unfertile, damaged, orgenetically unadapted individuals. Death in the populationmay also be triggered by toxins from nonclonal enemystrains or higher eukaryotes that hijack the PCDmachineryof yeast (Chapter 14, Fig. 2; see discussion on p. 340)

Color Plate 5 The molecular model for the cell corpse engulfment process.Two partially redundant pathways mediate the engulfmentprocess. CED-1 and CED-7 act on the surface of engulfingcells to mediate cell corpse recognition and to transduce theengulfing signal through CED-6 to the cellular machinery ofthe engulfing cells for engulfment. CED-7 also acts in dyingcells. DYN-1 acts in the CED-1 pathway to promote thedelivery of intracellular vesicles to the phagocytic cups andthe maturation of phagosomes. RAB-2 and RAB-7 mediatelysosome fusion with phagosomes and are important for thedegradation of internalized apoptotic cells. The CED-2/CED-5/CED-12 ternary complex mediates the signalingevents from externalized phosphatidylserine (PS)/PSR-1and other unidentified engulfing signal(s) and receptor(s) toactivate CED-10 during phagocytosis (Chapter 15, Fig. 5; seediscussion on p. 364)

Color Plate 6 Molecular model of PS externalization during apoptosis.Phospholipid asymmetry is maintained through the actionof three classes of proteins: scramblases, ABC transporters,and aminophospholipid translocases. In a living cell, scram-blases are not activated and show little to no activity, ABCtransporters are only used tomaintain lipid balance betweenthe two bilayers, and the aminophospholipid translocasestransport any externalized PS and PE to the inner leaflet.During apoptosis, scramblases are activated, for example,by WAH-1 released from mitochondria, randomly scram-bling phospholipids on the membrane, ABC transportersmay be activated to transport specific lipids to the outerleaflet, and the aminophospholipid translocase is inacti-vated, leading to PS externalization on the outer leaflet(Chapter 15, Fig. 6; see discussion on p. 366)

Color Plate 7 Live imaging demonstrates that cell death in the pupal eye istemporally and spatially regulated. (A–D) Pupal retinae ofdevelopmental ages as shown with cell boundaries outlinedin white. For clarity, an ommatidium is shaded (primarypigment cells are dark gray and cone cells are light gray) ineach panel and bristle groups are indicated by green arrows.IOCs account for the remainder of cells. In (D), the

xxii Color Plates

remaining IOCs after death are colored pink. (E) The per-centage of cells observed dying graphed relative to specificregions. The shading correlates to the regions in (F). Twopink asterisks indicate that no cells were observed to die inthese positions. (F) Schematic of the pupal retina, with eachshaded region corresponding to a position in which cells willeither be more likely to live (pink and green) or die (orangeand yellow). Figure adapted from (139). (Chapter 16, Fig. 4;see discussion on p. 389)

Color Plate 8 Bcl-2 family proteins regulate multiple aspects of hematopoiesis.Schematic representation of the non-lymphoid hematopoie-tic hierarchy. Proposed pro-apoptotic (?) and antiapopto-tic (") functions for Bcl-2 family members are indicated.Hematopoietic stem cell (HSC), common myeloid progeni-tor (CMP), megakaryocyte/erythroid progenitor (MEP),granulocyte/macrophage progenitor (GMP), burst-formingunit-erythroid (BFU-E), megakaryocyte progenitor (MkP),colony-forming unit granulocyte (CFU-G), colony-formingunit macrophage (CFU-M) (Chapter 19, Fig. 1; seediscussion on p. 443)

Color Plate 9 Progression of cell death in multiple models of acute neuronal

injury. The progression of cell death varies across cell deathmodels. Focal ischemia and traumatic brain injury displayacute injury within hours of the insult, marked by the pre-sence of necrotic morphology within the core of the injury(black regions), and progressing rapidly to inflammation(dark red regions). Delayed cell death, with morphologicalfeatures of apoptosis or mixed morphologies, is found inregions proximal to the core of the injury and occurs overdays toweeks (red regions).Global ischemia and kainic acid-induced epilepsy affect similar overall regions in bothhemispheres of the brain, but in differing subregions. Celldeath is more delayed compared to focal ischemia or trau-matic brain injury and presents hallmarks of programmedcell death, with limited necrotic phenotypes. The figurerepresents moderate injury models; the range and severityof cell death are highly dependent on the degree of toxicityand the species or method used to induce injury.(Chapter 20, Fig. 1; see discussion on p. 462)

Color Plate 10 Imaging cardiac muscle apoptosis in vivo. Confocal image ofTUNEL-positive cardiomyocyte labeling in the mouseheart after aortic banding, with TUNEL-positive nucleus(red), sarcomeric actin (green), and nuclei counterstainedwith DAPI (blue) (Chapter 22, Fig. 1; see discussion onp. 506)

Color Plates xxiii

Color Plate 11 Function of Bcl-2 family proteins. Named after the foundingmember of the family, which was isolated as a gene involvedin B-cell lymphoma (hence the name bcl), the Bcl-2 family iscomprised of well over a dozen proteins, which have beenclassified into three functional groups. Members of the firstgroup, such as Bcl-2 and Bcl-xL, are characterized by fourshort, conserved Bcl-2 homology (BH) domains(BH1–BH4). They also possess a C-terminal hydrophobictail, which localizes the proteins to the outer surface ofmitochondria, with the bulk of the protein facing the cyto-sol. The key feature of group I members is that they allpossess antiapoptotic activity and protect cells from death.In contrast, group II consists of Bcl-2 family members withproapoptotic activity. Members of this group, whichincludes Bax and Bak, have a similar overall structure togroup I proteins, containing the hydrophobic tail and all butthe most N-terminal, BH4 domain. Group III consists of alarge and diverse collection of proteins whose only commonfeature is the presence of the �12- to 16-amino-acid BH3domain. The Bcl-2 family of proteins function primarily toprotect or disrupt the integrity of the mitochondrial mem-brane and control themitochondrial release of proapoptoticproteins like cytochrome c, AIF, and Smac/DIABLO. Anti-apoptotic Bcl-2 members (Bcl-2, Bcl-xL) protect the mito-chondrial membrane. In response to environmental cues,these antiapoptotic proteins engage another set of proapop-totic proteins of the Bax subfamily (which includes Bax,Bak), normally loosely residing on the mitochondrial outermembranes or the cytosol. The interaction between Bak andBax proteins results in oligomerization and insertion intothe mitochondrial membrane of the complete complex(Chapter 22, Fig. 2; see discussion on p. 508)

Color Plate 12 Activation of DISC by cigarette smoke. MRC-5 cells at 70%confluence were exposed to 20% CSE in serum-free media.Immunofluorescence images ofMRC-5 double-labeled withindicated antibodies (anti-FAS in blue and anticaspase-8 ingreen) are shown. The cyan pseudocolor (arrow, top panel)indicates a co-localization of Fas and caspase-8. The sameimages with either the green or blue color removed areshown for clarity (middle and bottom panels). Data in thisfigure are representative of 20–49 cells analyzed for eachtime point. All panels are the same scale (97). [Figure repro-duced from Park et al. (97) with permission from the Amer-ican Association of Immunologists.]

xxiv Color Plates

The cartoon illustrates the potential role of protein kinase-c(PKC) isoforms in regulating DISC formation. PKC� dis-played an antiapoptotic effect in CSE-treated cells and inchronic cigarette smoke-exposed mice, whereas PKC� dis-played a proapoptotic effect. PKC� potentially inhbitsDISC trafficking by activating the PI3K pathway in fibro-blasts. PKC� promoted DISC trafficking by inhibiting thePI3K pathway (97) (Chapter 23, Fig. 3; see discussion onp. 536)

Color Plate 13 Schematic representation of liver diseases. Hepatocyteapoptosis is initiated by various stimuli via direct effectsand/or inflammatory responses. Massive hepatocyteapoptosis with impairment of hepatocyte regenerationresults in acute liver failure. Chronic hepatocyte apoptosisleads to liver cirrhosis and liver cancer. Kupffer cellsengulf apoptotic bodies of hepatocytes and release fibro-genic cytokines, which trigger collagen production byhepatic stellate cells. Chronic hepatocyte apoptosis alsostimulates hepatocyte regeneration, and dysregulation ofthe balance between hepatocyte proliferation and celldeath causes hepatocarcinogenesis (Chapter 24, Fig. 1;see discussion on p. 547)

Color Plate 14 (a) A model that describes how chromosome loss or non-disjunction occurs in spindle checkpoint-defective cells(MAD2-depleted cells or complete BUB1-depleted cells).In spindle checkpoint-mutant cells, the spindle checkpointis not activated even if there are defects in kinetochore–mi-crotubule attachment. No mitotic delay occurs, whichresults in the premature exit from mitosis. Thus, there issubstantial chromosome loss or nondisjunction, and pre-sumably cell death follows. (b) A model that describes thesame scenario in partial BUB1-depleted cells. Here, defectsin kinetochore–microtubule attachment induce lethal DNAfragmentation (CIMD). Because cells are still arrested inmitosis, the mitotic index remains unchanged. Therefore, thespindle checkpoint appears to be active.(Chapter 28, Fig. 2;see discussion on p. 637)

Color Plate 15 CIMD occurs in BUB1-depleted cells in the presence ofmicrotubule inhibitors or 17-AAG. HeLa cells that areBUB1-depleted and 17-AAG-treated exhibit DNA frag-mentation (TUNEL-positive; red) during mitosis (top row,prometaphase; bottom, metaphase). Forty-eight hours afterHeLa cells were transfected with BUB1 siRNA, they wereincubated with 17-AAG (þ17AAG, 500 nM) for 24 hours at37 8C. Fixed samples were stained by using an in situ cell

Color Plates xxv

death detection system that contained TMR red (TUNEL-signal; red), an antiphosphorylated histone H3 mousemonoclonal antibody, and FITC-conjugated secondaryantibodies (green). DNA was stained with DAPI (blue) tovisualize (Chapter 28, Fig. 3; see discussion on p. 638)

xxvi Color Plates

Part I

Molecules and Pathways of Apoptosis

Chapter 1

Caspases: Activation, Regulation, and Function

Stefan J. Riedl and Fiona L. Scott

Abstract The main effectors of apoptosis are proteases belonging to the cas-pase family. Caspases represent key mediators in the initiation and execution ofthe apoptotic program. The apoptotic caspases constitute a minimal two-stepsignaling pathway that culminates in the controlled demise of the affected cell.At the center of intense research for more than a decade and a half, a thoroughpicture of these regulatory proteases has emerged. A plethora of recent reportsshed exciting new and refined light on their activation, regulation, and function.In addition to an advanced understanding of caspases in the apoptoticprogram, additional functions of these proteases in other pathways and theirintriguing regulation by new signaling platforms have surfaced. With caspasesaffecting biological processes extending from apoptosis to other forms ofcell death and inflammation, a closer look at these regulatory proteases isparamount for our understanding of cell signaling.

Keywords Apoptosis � Caspase � Protease � Activation � Inhibition �Signaling platforms � Caspase-substrates � Mechanism � Apoptosome �DISC � Inflammasome

Introduction

As the evolution of multicellular organisms took place, the issue of maintainingboth body and organ size conducive with health and viability became a chal-lenge. In addition, constant cell proliferation had to be counterbalanced by amechanism of cell deletion compatible with the newly developing innateimmune system. The evolutionarily derived answer to the problem came inthe form of programmed cell death or apoptosis. The term ‘‘apoptosis’’ literally

S.J. Riedl (*)Program of Apoptosis and Cell Death Research, Burnham Institute for MedicalResearch, La Jolla, CA 92037, USAe-mail: sriedl@burnham.org

X.-M. Yin, Z. Dong (eds.), Essentials of Apoptosis,DOI 10.1007/978-1-60327-381-7_1,� Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009

3

means ‘‘the dropping of leaves from a tree’’ in Greek and was coined to describe

the morphological features observed during this coordinated deconstruction andpackaging of the cell for elimination by phagocytes and neighboring cells (1).

The importance of apoptosis during development and in the adult can beseen inmany disease states (discussed in detail in Part III of this book). Ischemic

injury, neurodegenerative disorders, and AIDS are the result of excessiveapoptosis in the absence of sufficient cell replenishment (2, 3). In contrast, thepathogenesis of autoimmune diseases and cancer results from a deficit inapoptosis (2, 4–7). Recent drug design strategies to combat chronic and acutediseases linked to abnormalities in cell death involve directly targeting theapoptotic cellular machinery (8–10).

The past 15 years have seen a fever of research activity aimed toward under-standing the mechanisms of programmed cell death. As a result, many of thegenes involved have been well characterized at the genomic, biochemical, andprotein structural levels and are conserved across species including nematodes,flies, and mammals (11–13). Apoptosis is triggered by diverse stimuli that canbe classified as part of either the extrinsic pathway (discussed further inChapter 5)or the intrinsic pathway (discussed further in Chapter 6), as depicted in Fig. 1.1.Central to both pathways are the cysteine-dependent aspartate-specific proteases,

Fig. 1.1 Simplified extrinsic and intrinsic apoptosis pathways. Upon engagement of the cellsurface death receptor by a ligand, adaptor molecules are recruited that in turn recruitcaspase-8 to form the death-inducing signaling complex (DISC). Caspase-8 is activated bydimerization at the DISC. This is termed the extrinsic pathway. In response to cell stress, themitochondria release cytochrome c from the intermembrane space into the cytosol. Thistriggers the formation of the apoptosome, which activates caspase-9 by dimerization. Activecaspase-8 and -9 can proteolytically activate the executioner caspases-3 and -7. XIAP inhibitscaspases-3, -7, and -9

4 S.J. Riedl and F.L. Scott

or caspases. These proteolytic enzymes are the suicide weapons of the apoptoticpathway, and their activity directly eradicates the cell. We will discuss recentadvances in our understanding of caspase activation, regulation, and their cel-lular substrates, both in apoptosis and in nonapoptotic biology.

Caspases—A Historical Perspective

In 1992, the cysteine protease responsible for maturation of interleukin-1b wascloned by two independent groups (14, 15). Initially termed ‘‘interleukin-1b-converting enzyme’’ (ICE), it has since been renamed ‘‘caspase-1.’’ Caspase-1cleaves pro-interleukin-1b at a critical aspartate residue, converting it to themature form, interleukin-1b. During the same time, through extensive studiesof cell deletion during development in Caenorhabditis elegans, Horvitz andcolleagues identified three central genes critical for normal programmed celldeath—ced-3, ced-4, and ced-9 (Fig. 1.2; discussed further in Chapter 13). Theced-3 gene product was found to share greater than 24% identity with ICE (16).These exciting findings prompted researchers to look for other cysteine pro-teases with catalytic preference for aspartic acid residues, anticipating that they,too, would have a role in cell death. A huge body of research has led to theclassification of 11 human caspases. Only seven of these are involved in apop-tosis. The apoptotic caspases are further divided into those involved in theinitiation phase of apoptosis (caspases-2, -8, -9, and -10) and those involvedin the execution phase (caspases-3, -6, and -7). Interestingly, the functionalorthologue of CED3 is not caspase-1 (Fig. 1.2). Caspase-1 and the otherremaining caspases are cytokine activators and are important in inflammation

Fig. 1.2 The caspase cascade. Functional alignment of apoptosis regulators in the vertebratesDrosophila melanogaster and Caenorhabditis elegans

1 Caspases: Activation, Regulation, and Function 5

(caspases-1, -4, and -5) or are involved in keratinocyte differentiation andmaintenance of barrier function of the skin (caspase-14) (17). The key tocaspase involvement in such a diversity of biological processes lies in theirmechanism, and more specifically their intriguing routes of activation.

Activation of Caspases

As described above, caspases are highly evolved signaling proteases that parti-cipate in a stunning variety of cellular functions and pathways (18–20). At theheart of their extraordinary capabilities lies their flexibility to be activated indifferent manners (21–23). In the absence of an activating stimulus, caspasesexist as latent forms, or zymogens. Activation can occur by two means, namelyactivation by limited proteolysis or activation through binding to so-calledactivation platforms, each of which is used by different caspase groups(20, 24–26). The executioner caspases, responsible for the execution phase ofthe apoptotic program, become activated by limited proteolysis. Since thesecaspases operate at the bottom of the apoptotic cascade (Fig. 1.2), it is feasiblethat their zymogen forms are cleaved and activated by caspases higher in thehierarchy or other proteases such as granzyme B (26–29). The existence ofinactive zymogens and their activation by limited proteolysis is a feature com-mon to proteases (30). The cleavage-based activation of executioner caspasesthus quickly became an accepted paradigm. However, the question soon aroseas to how the apical caspases become activated. That is, how do caspases at theapex of the pathways sense a nonproteolytic signal, become activated, and thustranslate this signal into a proteolytic signal? The answer lies in the unusualproperties and architecture of caspases, which set them apart from most otherproteases. This architecture is very simple at first glance [Fig. 1.3(A)]. Allcaspases possess a catalytic domain, which shows a characteristic caspase fold(24). Additionally, many members of the family possess one or more adaptordomains belonging to the DEATH domain family, namely CARD and DED(31). In fact, it is this architecture, slight differences in the catalytic domain, andthe presence or absence of an adaptor domain that facilitate different activationin different caspase groups. While the executioner caspases rely solely onactivation by cleavage, the initiator caspases become activated by binding toactivation platforms (26, 32, 33). The latter triggers self-association (dimeriza-tion) of the initiator caspases leading to their activation as postulated by the so-called induced-proximity model, which is discussed ahead (34, 35).

Activation of Apoptotic Executioner Caspases

The executioner caspase zymogens follow the paradigmof activation by cleavage.The activity of their zymogens is essentially zero, but once cleaved, executionercaspases such as caspase-7 are highly functional legitimate proteases (36, 37).Typically, they are expressed as single-chain proteins, which exist in the cell as

6 S.J. Riedl and F.L. Scott

Fig. 1.3 Caspases: domain organization and activation mechanisms. (A). Domain organiza-tion. All caspases possess a catalytic domain, with a characteristic organization of a largesubunit connected to a small subunit by a linker region, which carries caspase/protease cleavagesites. Additionally, caspases exhibit anN-terminal region comprising only a short peptide in thecase of executioner caspases or one or two adaptor domains belonging to the DEATH domainfamily. Cleavage sites are also found between the N-terminal region and catalytic domain withthe exception of caspase-9. (B). Apoptotic executioner caspase activation. Shown are thezymogen and the active form of caspase-7 in surface representation, with the linker regionand loop regions important for active site formation also shown (PDB entries: 1GQF, 1F1J).Executioner caspases are constitutive dimers and become activated by cleavage of the linkerregion, which allows for the formation of an active site. (C). Apoptotic initiator caspaseactivation. Shown schematically are the inactive monomeric forms of caspase-9, which needto bind to an activation platform, the apoptosome, via their adaptor domains. This leads todimerization and activation of the caspase according to the induced-proximity model. Cleavageof the linker region can occur but is not necessary for activation

1 Caspases: Activation, Regulation, and Function 7

constituent homodimers [Fig. 1.3(B)]. Induction of the apoptotic program leadsto cleavage of their short N-terminal regions and, most importantly in terms oftheir activation, a cleavage within the catalytic domain generating new terminiand a large and small subunit (38, 39) [Fig. 1.3(A)].

Various studies on the catalytic domain of executioner caspases, amongthem three-dimensional crystal structures at atomic resolution, now pro-vide a relatively proficient picture of their activation mechanism (24).Their catalytic domains are built by a solid frame consisting of a centralbeta sheet flanked by alpha helices adopting the characteristic caspase fold.Interestingly, crucial features for the catalytic function of the caspases liein loop regions between these scaffolding elements. These loop regions alsoprovide the key to caspase activation. As shown in Fig. 1.3(B) forcaspase-7, one of the best-studied caspases in terms of activation, boththe zymogen and the cleaved/active caspase exist as dimers. Yet distinctloop regions differ between the zymogen and the active caspase, withdrastic consequences. One of these regions is known as the linker region.It is in this region that the actual activation cleavage of the executionercaspase occurs [Figure 1.3(A)]. The highly specific cleavage by a moreapical caspase or granzymeB results in the generation of a large (�20-kDA) and a small (�10-kDa) subunit. This cleavage has significantmechanistic consequences—in essence, it allows for the formation of theactive site in the caspase. The detailed events have been described andreviewed elsewhere (22–24) and are also described in Chapter 4, but ageneral picture of the activation events is outlined in the following (Fig.1.3). Cleavage in the linker region of the zymogen dimer releases the newlyformed termini to move outward. A consequence of this is that anotherloop switches from a random conformation in the zymogen to a well-defined position in the active caspase. This loop now represents the bottomof the active site cleft and provides central residues that the active caspaseutilizes to bind its substrate (39, 40). Cleavage of the linkers followed bythese two major rearrangements converts a practically nonexistent activesite into a well-defined active site cleft with substrate binding sites andwell-ordered catalytic machinery. Again, various studies describe thisprocess in more detail and also discuss the more subtle involvement ofadditional loop regions using a specifically tailored nomenclature (41). Yetthe principles outlined here illustrate the fundamental nature of this pro-cess, namely that activation cleavage effectively generates a genuine andwell-defined active site in executioner caspases.

Interestingly, in the cleaved active form of the executioner caspases, eachnewly formed terminus of one caspase interacts with the new terminus of theother caspase of the dimer. Thus, the fact that executioner caspases are con-stitutive dimers is actually a prerequisite for their activation mechanism bylimited proteolysis. This is different in the case of the initiator caspases, whichexist as inactivemonomers prior to activation and require binding to oligomericsignaling platforms to become activated.

8 S.J. Riedl and F.L. Scott