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Regulated Cell Death Part A - 1st Edition - ISBN: 9780124171589, 9780124171756

Regulated Cell Death Part A, Volume 544

1st Edition

Apoptotic Mechanisms

Editors: Avi Ashkenazi Junying Yuan Jim Wells
Hardcover ISBN: 9780124171589
eBook ISBN: 9780124171756
Imprint: Academic Press
Published Date: 24th June 2014
Page Count: 504
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Table of Contents

  • Preface
  • Chapter One: Examining the Molecular Mechanism of Bcl-2 Family Proteins at Membranes by Fluorescence Spectroscopy
    • Abstract
    • 1 Introduction
    • 2 An In Vitro Fluorescence-Based Liposome System
    • 3 Membrane Permeabilization Assay
    • 4 Fluorescence Resonance Energy Transfer
    • 5 Tracking the Conformation Changes of a Protein
    • 6 Determining the Topology of Proteins within Membranes
    • 7 Conclusion
    • Acknowledgments
  • Chapter Two: Photoreactive Stapled Peptides to Identify and Characterize BCL-2 Family Interaction Sites by Mass Spectrometry
    • Abstract
    • 1 Introduction
    • 2 Overview of Photoreactive Stabilized Alpha-Helices Methodology
    • 3 Design and Synthesis of Photoreactive Stapled Peptides
    • 4 Photoaffinity Labeling and Retrieval
    • 5 Mass Spectrometry Analysis
    • 6 Computational Docking
    • 7 Mapping Novel Binding Sites
    • 8 Conclusions and Future Directions
    • Acknowledgments
  • Chapter Three: The Structural Biology of BH3-Only Proteins
    • Abstract
    • 1 Introduction
    • 2 What Constitutes a BH3-Only Protein?
    • 3 Specificity and Interactions with the Bcl-2 Family
    • 4 Structures of BH3-Only Proteins and Their Complexes
    • 5 Interaction of BH3-Only Proteins with Viral Bcl-2 Homologues
    • 6 Interaction with Non-Bcl-2 Proteins: Beyond the BH3-Motif
    • 7 Regulation
    • 8 BH3-Only Proteins and Disease
    • 9 Conclusion
    • Acknowledgments
  • Chapter Four: How to Analyze Mitochondrial Morphology in Healthy Cells and Apoptotic Cells in Caenorhabditis elegans
    • Abstract
    • 1 Introduction
    • 2 Why Address These Questions in C. elegans
    • 3 How to Inactivate or Overexpress a Particular Candidate Gene in C. elegans
    • 4 How to Visualize Mitochondria in C. elegans
    • 5 Potential Outcomes and Future Experiments
    • 6 Concluding Remarks and Future Challenges
    • Acknowledgments
  • Chapter Five: Apoptosis Initiation Through the Cell-Extrinsic Pathway
    • Abstract
    • 1 Introduction
    • 2 Ligand-Induced Receptor Clustering
    • 3 Control of Caspase-8 Activation
    • 4 Death Receptor Stimulation of Necroptosis
    • 5 Conclusion
  • Chapter Six: Using RNAi Screening Technologies to Interrogate the Extrinsic Apoptosis Pathway
    • Abstract
    • 1 Introduction
    • 2 RNAi-Mediated Gene Knockdown in Mammalian Cells
    • 3 Concluding Remarks and Perspectives
  • Chapter Seven: Caspase Enzymology and Activation Mechanisms
    • Abstract
    • 1 Introduction
    • 2 Activating Initiator Caspases
    • 3 Investigating Caspase Activity and Inhibition
    • 4 Conclusions
    • Acknowledgments
  • Chapter Eight: Turning ON Caspases with Genetics and Small Molecules
    • Abstract
    • 1 Use of Chemical-Induced Dimerizers to Activate Caspases
    • 2 Use of Cre-LoxP and a Self-Activating Caspase-3TevS for Conditional Apoptosis of Neurons
    • 3 Small-Molecule Activators of Caspases
    • Acknowledgments
  • Chapter Nine: A Multipronged Approach for Compiling a Global Map of Allosteric Regulation in the Apoptotic Caspases
    • Abstract
    • 1 Introduction
    • 2 Zinc-Mediated Allosteric Inhibition of Caspases
    • 3 Using the Embedded Record of Functionally Important Posttranslational Modifications to Identify Allosterically Sensitive Sites
    • 4 Synthetic Small-Molecule Inhibitors to Identify Allosteric Sites
    • 5 Exploiting Structural Differences Among Caspases
    • 6 Future of Allosteric Site Discovery
    • Acknowledgments
  • Chapter Ten: Measuring Caspase Activity In Vivo
    • Abstract
    • 1 Introduction
    • 2 Small Molecule Reagents
    • 3 Protein Indicators
    • 4 Future Directions
  • Chapter Eleven: Single-Molecule Sensing of Caspase Activation in Live Cells via Plasmon Coupling Nanotechnology
    • Abstract
    • 1 Imaging of Caspase Activation
    • 2 Gold Nanoparticles and Their Potential as Plasmon Rulers
    • 3 Live Cell Imaging Using Crown Nanoparticles
    • 4 Methods
    • 5 Conclusions
    • Highlights
    • Acknowledgments
  • Chapter Twelve: In Vivo Monitoring of Caspase Activation Using a Fluorescence Resonance Energy Transfer-Based Fluorescent Probe
    • Abstract
    • 1 Introduction
    • 2 Monitoring Caspase Activities in Living Cells: Principle and Tips for Using a SCAT3 Probe
    • 3 Monitoring Caspase Activation with SCAT3 in Drosophila
    • 4 Monitoring Caspase Activation with SCAT3 in Mice
    • 5 Concluding Remarks
  • Chapter Thirteen: Global Analysis of Cellular Proteolysis by Selective Enzymatic Labeling of Protein N-Termini
    • Abstract
    • 1 Introduction
    • 2 Basic Features of Subtiligase Method
    • 3 Subtiligase-Based Labeling Method
    • 4 Experimental Applications of Subtiligase-Based N-Terminomics
    • 5 Limitations to the Subtiligase Labeling Method
    • 6 Summary of Findings from Subtiligase-Based N-Terminomics
    • 7 Future Directions
    • Acknowledgments
  • Chapter Fourteen: Complementary Methods for the Identification of Substrates of Proteolysis
    • Abstract
    • 1 Introduction
    • 2 Combined Fractional Diagonal Chromatography
    • 3 Protein Topography and Migration Analysis Platform
    • 4 Global Analyzer of SILAC-Derived Substrates of Proteolysis
    • 5 Immuno-Affinity Enrichment of C-Terminal Aspartic Acid Containing Peptides for the Identification of Caspase substrates
    • 6 Summary
  • Chapter Fifteen: Phospholipid Scrambling on the Plasma Membrane
    • Abstract
    • 1 Introduction
    • 2 Assays for Calcium-Induced Phospholipid Scrambling
    • 3 Assays for Apoptosis-Induced Phospholipid Scrambling
  • Chapter Sixteen: Studying Apoptosis in the Zebrafish
    • Abstract
    • 1 Introduction
    • 2 Detecting and Quantifying Apoptosis in Zebrafish
    • 3 Approaches to Manipulating Apoptosis Pathways in Zebrafish
    • 4 Mutant Lines and Morpholinos
    • 5 Summary
    • Acknowledgments
  • Author Index
  • Subject Index


Regulated Cell Death Part A & Part B of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in apoptosis focusing on the important areas of intrinsic pathway, extrinsic pathway, caspases, cellular assays and post-apoptotic effects and model organisms; as well as topics on necroptosis and screening approaches.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers research methods in biomineralization science
  • Regulated Cell Death Part A & Part B contains sections on such topics as apoptosis focusing on the important areas of intrinsic pathway, extrinsic pathway, caspases, cellular assays and post-apoptotic effects and model organisms; as well as topics on necroptosis and screening approaches


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.


No. of pages:
© Academic Press 2014
24th June 2014
Academic Press
Hardcover ISBN:
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Ratings and Reviews

About the Editors

Avi Ashkenazi

Avi Ashkenazi

Dr Avi Ashkenazi received his PhD in Biochemistry in 1986 from the Hebrew University of Jerusalem, Israel. From 1986 to 1989 he trained as a postdoctoral fellow at the University of California, San Francisco, and the biotechnology company, Genentech. As a postdoc, he helped identify the muscarinic acetylcholine receptor gene family and deciphered its interactions with specific G proteins – work that in 1988 earned him and two of his colleagues the first prize of the Boehringer Ingelheim Award. In 1989 Dr Ashkenazi joined Genentech as s Scientist and progressed through the ranks to become Senior Staff Scientist and Director, most recently within the Research Oncology division. In the early 1990’s he contributed to the development and translation of a technology to fuse immunoglobulin Fc domains to other proteins, now used in numerous research laboratories and in important biotechnology drugs including Enbrel® and Eylea®. Subsequently, Dr Ashkenazi’s laboratory discovered several novel members of the tumor necrosis factor (TNF) superfamily, most notably, the apoptosis-inducing ligand Apo2L/TRAIL and its “death” and “decoy” receptors. Dr Ashkenazi’s basic research has focused on the mechanisms of apoptosis signaling by death receptors, and his translational work in this area pioneered the clinical development of pro-apoptotic receptor agonists (PARAs) for cancer therapy. To date, Dr Ashkenazi has published 110 research papers and 31 review articles and has co-edited a book on antibody fusion proteins. His top 5 publications have been cited in sum over 11,000 times. His work on death receptors is highlighted in the textbook: “The biology of Cancer” by Robert A. Weinberg. He has presented 99 lectures at scientific institutions and conferences and is a named inventor on 59 issued US patents. Dr Ashkenazi co-chaired the international TNF conference in 2007 and has served or serves on the editorial boards of Current Biology, Clinical Cancer Research, Nature Reviews Cancer, Journal of Biological Chemistry, Cancer Biology & Therapy, Cell Death & Differentiation and Molecular Cancer Therapy.

Affiliations and Expertise

Oncology, Genentech Inc, CA, USA

Junying Yuan

Affiliations and Expertise

Department of Cell Biology, Harvard Medical School, MA, USA

Jim Wells

Jim Wells

James A. Wells, PhD, focuses on development of enabling technologies for engineering proteins and for identifying small molecules to aid in drug discovery for challenging targets such as allosteric regulation and protein-protein interactions. He is interested in the discovery and design of small molecules and enzymes that trigger or modulate cellular processes in inflammation and cancer. Using small molecules and engineered proteins, the Wells lab is studying how activation of particular signaling nodes involving protease, kinases, or ubiquitin ligases drives cell biology. The lab has focused much on a set of proteases, known as caspases, responsible for fate determining cellular decisions involved in apoptosis and innate inflammation among others. These enzymes act as cellular remodelers and help us understand the essential protein struts that support life. These targets also provide leads for developing new cancer therapeutics and biomarkers for cancer treatment.

Wells is a professor and chair of the Department of Pharmaceutical Chemistry in the UCSF School of Pharmacy. He holds a combined appointment as professor in the Department of Cellular & Molecular Pharmacology in the School of Medicine. He joined UCSF in 2005 as holder of the Harry Wm. and Diana V. Hind Distinguished Professorship in Pharmaceutical Sciences. Wells also founded and directs the Small Molecule Discovery Center (SMDC) located at UCSF’s Mission Bay campus. He earned a PhD degree in biochemistry from Washington State University with Professor Ralph Yount in 1979 and completed postdoctoral work at Stanford University School of Medicine with Professor George Stark in 1982. Before joining UCSF, Wells was a founding scientist in Genentech’s Protein Engineering Department and in 1998 co-founded Sunesis Pharmaceuticals.

Wells is a recipient of the Hans Neurath Award by the Protein Society, the Pfizer Award and Smissman Award given by the American Chemical Society, the Perlman Lecture Award given by the ACS Biotechnology Division, the du Vigneaud Award given by the American Peptide Society, the Merck Award from the ASBMB and in 1999 a member of the National Academy of Sciences.

Affiliations and Expertise

Departments of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, The Wells Lab, UCSF Mission Bay, CA, USA