Methods in enzymology, volume 545

METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N ABELSON and MELVIN I SIMON Division of Biology California Institute of Technology Pasadena, California ANNA MARIE PYLE Departments of Molecular, Cellular and Developmental Biology and Department of Chemistry Investigator Howard Hughes Medical Institute Yale University Founding Editors SIDNEY P COLOWICK and NATHAN O KAPLAN Academic Press is an imprint of Elsevier 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK 32 Jamestown Road, London NW1 7BY, UK First edition 2014 Copyright © 2014, Elsevier Inc All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) Notices Knowledge and best practice in this field are constantly changing As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein ISBN: 978-0-12-801430-1 ISSN: 0076-6879 For information on all Academic Press publications visit our website at store.elsevier.com CONTRIBUTORS Eric H Baehrecke Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA Rhesa Budhidarmo Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Catherine L Day Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Alexei Degterev Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts, USA Paul C Driscoll Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, London, United Kingdom Peter Geserick Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg, Germany Tae-Bong Kang Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel, and Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chung-Ju, Republic of Korea Maxime J Kinet Laboratory of Developmental Genetics, The Rockefeller University, New York, USA Andrew Kovalenko Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Martin Leverkus Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg, Germany Jenny L Maki Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts, USA Adam J Middleton Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand ix x Contributors Charles Nelson Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA Vassiliki Nikoletopoulou Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Heraklion, Greece Ramon Schilling Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg, Germany Pascal Schneider Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Shai Shaham Laboratory of Developmental Genetics, The Rockefeller University, New York, USA John Silke The Walter and Eliza Hall Institute of Medical Research, and Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia Cristian R Smulski Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Brent R Stockwell Department of Biological Sciences; Department of Chemistry, and Howard Hughes Medical Institute, Columbia University, New York, USA Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Heraklion, Greece Beata Toth Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Domagoj Vucic Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, California, USA David Wallach Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Laure Willen Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Adam J Wolpaw Residency Program in Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA Seung-Hoon Yang Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Contributors Junying Yuan Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA Wen Zhou Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA xi PREFACE Cell turnover is a fundamental feature of metazoan biology Severe damage to cellular integrity usually causes passive, nonregulated cell death In contrast, more confined disruption can lead to more deliberate cell elimination, through specific mechanisms of Regulated Cell Death In these two volumes of Methods in Enzymology, we aim to highlight the current molecular understanding of the major processes of Regulated Cell Death and to illustrate basic and advanced methodologies to study them Volume A focuses on the most extensively studied mode of cell death—apoptosis Volume B covers several nonapoptotic mechanisms These include necroptosis, which shares certain signal transduction aspects with apoptosis but is unique in its execution phase, and autophagic cell death, which is an offshoot of autophagy—a more basic prosurvival metabolic adaptation mechanism Chapters 1–4 cover how to measure necroptosis and various molecular components and complexes that signal this process Chapter discusses approaches to interrogating interactions between tumor necrosis factor superfamily ligands and receptors Chapters 6–8 highlight nonapoptotic cell death mechanisms in the model organisms, C elegans and D melanogaster Chapters and 10 discuss structural aspects of death receptor complexes and strategies to study posttranslational modification of downstream signaling components by RING E3 ubiquitin ligases Finally, Chapter 11 describes a multidimensional profiling approach to studying smallmolecule-induced cell death We hope these chapters will be both conceptually informative and practically useful for readers interested in the current understanding and the key open questions in each area, as well as in experimental strategies and techniques to interrogate nonapoptotic regulated cell death mechanisms AVI ASHKENAZI JAMES A WELLS JUNYING YUAN xiii CHAPTER ONE Assays for Necroptosis and Activity of RIP Kinases Alexei Degterev*, Wen Zhou†, Jenny L Maki*, Junying Yuan†,1 *Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts, USA † Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA Corresponding author: e-mail address: junying_yuan@hms.harvard.edu Contents Introduction 1.1 Distinguishing features of necroptotic cell death 1.2 Pathways and mediators of necroptosis Cellular Models of Necroptosis 2.1 Cell types 2.2 Inducers of necroptosis 2.3 Inhibitors of necroptosis Measurement of Necroptotic Cell Death 3.1 Analysis of viability of FADD-deficient Jurkat cells treated with TNFa using CellTiter-Glo assay 3.2 Determination of specific cell death using SYTOX Green assay 3.3 Annexin V/PI assay 3.4 Analysis of ROS increase 3.5 Mitochondrial membrane depolarization 3.6 Analysis of TNFa gene expression changes by qPCR Recapitulation of RIP1 Kinase Expression in RIP1-Deficient Jurkat Cells 4.1 Transient transfection 4.2 Generation of stable-inducible cell lines Analysis of Necrosome Complex Formation 5.1 Immunoprecipitation of necrosome complex 5.2 Immunoprecipitation of TNFR1 complex 5.3 Assessment of necrosome formation by fluorescence microscopy Endogenous RIPK Autophosphorylation Assays Analysis of Recombinant RIPK1 Kinase Activity and Inhibition by Necrostatins 7.1 Expression and purification of recombinant RIP1 and RIP3 7.2 Kinase-Glo assay 7.3 HTRF KinEASE assay 7.4 Fluorescence polarization assay 7.5 Thermomelt assay Methods in Enzymology, Volume 545 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-801430-1.00001-9 # 2014 Elsevier Inc All rights reserved 2 5 9 10 11 12 14 14 16 16 17 18 18 20 21 21 23 23 23 25 26 27 Alexei Degterev et al Conclusions Acknowledgments References 28 29 29 Abstract Necrosis is a primary form of cell death in a variety of human pathologies The deleterious nature of necrosis, including its propensity to promote inflammation, and the relative lack of the cells displaying necrotic morphology under physiologic settings, such as during development, have contributed to the notion that necrosis represents a form of pathologic stress-induced nonspecific cell lysis However, this notion has been challenged in recent years by the discovery of a highly regulated form of necrosis, termed regulated necrosis or necroptosis Necroptosis is now recognized by the work of multiple labs, as an important, drug-targetable contributor to necrotic injury in many pathologies, including ischemia–reperfusion injuries (heart, brain, kidney, liver), brain trauma, eye diseases, and acute inflammatory conditions In this review, we describe the methods to analyze cellular necroptosis and activity of its key mediator, RIP1 kinase INTRODUCTION 1.1 Distinguishing features of necroptotic cell death Discovery of regulated necrosis originates from the observations that “canonical” inducers of apoptosis, such as agonists TNFa family of death domain receptors (DRs), can trigger cell death morphologically resembling necrosis in cells either intrinsically deficient in caspase activation (e.g., mouse fibrosarcoma L929 cells) or under conditions when caspase activation is inhibited (e.g., caspase-8-deficient Jurkat cells or cells treated with pancaspase inhibitor zVAD.fmk) (Holler et al., 2000; Matsumura et al., 2000; Vercammen, Vandenabeele, Beyaert, Declercq, & Fiers, 1997) The lack of caspase activation as well as the absence of other typical features of apoptosis, such as cytochrome c release, membrane blebbing, phosphatidylserine (PS) exposure, and intranucleosomal DNA cleavage, served as important initial differentiators between necroptosis and apoptosis (Tait & Green, 2008) Electron microscopy has also proved very useful in distinguishing necroptosis from apoptosis in morphology Necroptotic cells are characterized by the lack of typical nuclear fragmentation, swelling of cellular organelles especially mitochondria, and the loss of plasma membrane integrity, whereas apoptotic cells exhibit shrinkage, blebbing, nuclear fragmentation, and chromatin condensation (Degterev et al., 2005) Robust activation of Necroptosis Assays autophagy is another feature of necroptosis which provides useful means to distinguish this form of cell death in vitro and in vivo both morphologically (e.g., by EM) and at the molecular level (e.g., by measuring of LC3II formation) (Degterev et al., 2005; Yu et al., 2004) This leads to necroptosis in some cases being referred to as “autophagic cell death,” such as zVADinduced death of L929 cells (Yu et al., 2004) It should be noted, however, that functional role of autophagy varies greatly depending on the specifics of necroptosis activation, with instances where this process promotes, inhibits, or does not affect cell death (Degterev et al., 2005; Shen & Codogno, 2012; Yu et al., 2004) Furthermore, activation of necroptosis-inducing necrosome complex (discussed below) can also happen downstream from autophagosome formation (Basit, Cristofanon & Fulda, 2013) A detailed comparison of TNF-induced necroptosis and H2O2-induced necrosis was performed by Vanden Berghe et al (2010) Despite the different kinetics of cellular events including ROS production, mitochondrial polarization changes, and lysosomal membrane permeabilization, the major hallmarks of necroptosis and oxidant-induced necrosis were remarkably similar, leading to an important conclusion that necroptosis is a subtype of necrosis, morphologically indistinguishable from other types of necrosis but defined by a specific mode of activation (discussed below) Generation of DAMPs as a result of cell lysis is an important consequence of necroptotic death both in vitro and in vivo (Duprez et al., 2011; Murakami et al., 2013) In addition, recent evidence suggests that synthesis of TNFa occurs independently of cell death as a result of specific signaling by key necroptosis initiator RIP1 kinases (RIPK1) (Christofferson et al., 2012; Kaiser et al., 2013; McNamara et al., 2013) Autocrine TNFa can promote cell death dependent on a cytosolic complex “ripoptosome” consisting of RIPK1, FADD, and caspase-8 (Biton & Ashkenazi, 2011; Hitomi et al., 2008; Kaiser et al., 2013; Tenev et al., 2011) Several instances have also been reported where RIPK1 and RIPK3 promote inflammatory signaling through the production of IL-1a and IL-1b/IL-18 in the absence of cell death (Kang, Yang, Toth, Kovalenko, & Wallach, 2013; Lukens et al., 2013) These data highlight complex interrelationship between necroptosis and inflammation 1.2 Pathways and mediators of necroptosis We refer the readers to a number of in-depth reviews on the subject (Christofferson et al., 2012; Christofferson, Li, & Yuan, 2014; Alexei Degterev et al Christofferson & Yuan, 2010b; Fulda, 2013; Zhou, Han, & Han, 2012) We will just briefly summarize some of the key findings Initiation of necroptosis is best understood in the context of TNFa signaling Engagement of TNFR1 leads to the formation of a membrane-bound complex named Complex I, containing RIPK1, TRADD, and TRAF2 as key components (Micheau & Tschopp, 2003) Ubiquitination of Lys377 of RIPK1 within this complex leads to the assembly of NF-kB-activating complexes involving TAK1 and IKK kinases (Ea, Deng, Xia, Pineda, & Chen, 2006) Dissociation of the components from TNFR1 is followed by the assembly of cytosolic signaling complexes: either Complex IIa/DISC including RIPK1, FADD, and caspase-8 which leads to apoptosis (Micheau & Tschopp, 2003), or Complex IIb/necrosome including FADD, RIPK1, and RIPK3 which leads to necroptosis in the absence of caspase activity (summarized in Galluzzi, Kepp, & Kroemer, 2009) Activation of necroptosis requires cross-phosphorylation of RIPK1 and RIPK3, utilizing Ser/Thr kinase domains of both proteins (Cho et al., 2009) RIPK1 and RIPK3 kinases further form amyloid-like fibers (Li et al., 2012), and RIPK3 recruits and phosphorylates pseudokinase MLKL on Thr357/Ser358, which serves as a critical gateway to necroptosis execution (Murphy et al., 2013; Sun et al., 2012; Wu et al., 2013) Downstream events are currently less well understood As discussed above, oxidative stress mediated by mitochondrial Complex I and NADPH oxidase was found to play a role in some cell types Other factors, such as Ca2+, ceramide, activation of autophagy, and HtrA2 and UCH-L1 proteases (Sosna et al., 2013), have also been proposed to play a role However, connections between these factors and necrosome remain unknown Other signals were also shown to promote necrosome activation, but the mechanisms may differ For example, multiple Toll-like receptors (TLRs) were found to induce necroptosis (He, Liang, Shao, & Wang, 2011; Kaiser et al., 2013) The mechanisms differ depending on the specific signals and cell types TLR3 and TLR4 act through adaptor TRIF to directly recruit RIPK1 and RIPK3 through their RHIM domains, while other TLRs signaling through MyD88 adaptor trigger necroptosis through an autocrine TNFa loop Furthermore, while RIPK1 is required for TRIF-mediated necroptosis in macrophages, it is dispensable in epithelial and fibroblast cells Additional signals directly triggering RIPK3, such as activation of viral DNA sensor DAI (Upton, Kaiser, & Mocarski, 2012), have also been described, and overexpression of RIPK3 was shown Author Index Romani, N., 70–71 Rong, Y S., 183 Rooney, T M., 173–174 Ross, D T., 286 Ross, K N., 271–272 Rossi, L., 280–281 Rossi, R M., 274–275 Rossin, A., 233–234 Rotello, R., 266–267 Rothe, M., 36–38, 244–245 Rothman, J H., 164, 166–167 Rothstein, J D., 136 Roumier, C., 143 Rousseeuw, P J., 294 Roy, N., 36–38, 40–42 Royal, D C., 159 Royal, M A., 159 Ru, H., 209 Ruan, Q., 144 Rubartelli, A., 77 Ruberti, G., 215–216 Rubin, G M., 36, 183–184 Rubin, L L., 11–12 Rubinstein, L., 269t, 284 Ruel, R., 39–40 Ruggieri, S., 133 Rui, L., 169–170 Ruschak, A M., 227–228 Russell, S J., 210–211 Russo, C., 293 Ryan, B M., 52–53 S Saha, A., 257 Saha, S., 144, 147t Sakata, E., 247, 250, 251–252 Saleh, A., 41–42 Sali, A., 269t, 277, 278 Salinas, L S., 168–169 Salsbury, F R., 224–225 Salvesen, G S., 36–38, 39–42, 75–76 Salvioli, S., 14 Samara, C., 140–141, 161–162 Sanches, M., 260 Sancho-Martinez, I., 232–233 Sandu, C., 225–226 Sankar, A., 224–225, 227–228, 231–233 Santi, C M., 142–143 323 Santonicola, P., 168–169 Santoro, M M., 39 Saras, J., 221–222 Sato, R., 142–143 Sato, Y., 248 Satoh, T., 247, 250, 251–252 Saulle, E., 43–45 Sausville, E A., 285–286 Savig, E S., 277 Savino, W., 75–76, 77 Sawa, E., 221–222 Scaffidi, C., 203–204 Scarpa, A., 277 Schaefer, G I., 269t, 286–287 Schapira, A H., 144 Schavemaker, J M., 133 Scheidereit, C., 5, 50–51, 52 Schellenberg, G D., 145–147, 147t Schena, M., 271 Scherf, U., 286 Scherle, P A., 220–221 Schickel, R., 232–234 Schierenberg, E., 129–130, 158 Schile, A J., 42 Schimmer, A D., 53 Schindelin, H., 247 Schlak, I., 166 Schleich, K., 233–234 Schliess, F., 232–233 Schlis, K., 274 Schmidt, M L., 143 Schmukle, A C., 43, 47–48, 49, 50–51, 88, 95–96 Schnabel, R., 170 Schneider, P., 43, 46–47, 50–51, 55, 104–106, 116, 123–124, 204–205, 213–214, 225–226, 274–275 Schneider-Brachert, W., 233–234 Schoeffler, A J., 55, 245 Schoeneberger, H., Schoenfeld, H J., 206–207, 207f, 213 Scholz, R P., 52–53 Schop, R., 49–50 Schramek, D., 104 Schreiber, S L., 271 Schroder, K., 70, 72–73 Schrofelbauer, B., 202–203 Schuchmann, M., 71, 98 324 Schulman, B A., 46–47 Schultz, P G., 54–55 Schumacher, F.-R., 47–48, 49, 245, 255, 258–259, 260 Schutze, S., 230–231, 232–234 Schwartz, H T., 170–171 Schweitzer, B., 277 Scott, B A., 142–143, 147t Scott, F L., 40–41, 223f, 228–229, 231–233 Screaton, G., 215–216 Scuderi, P., 213–214 Scudiero, D A., 269t, 284 Scupoli, M T., 277 Seaman, J E., 278 Sebbagh, M., 232–233 Seed, B., 7, 20–21 Sekine, K., 39 Seldin, M F., 222 Selvin, P R., 121 Sendoel, A., 168–169 Sergent, O., 232–233 Serniwka, S A., 251–252 Seshagiri, S., 36 Seslija, L., 274–275 Setola, V., 276–277 Settleman, J., 288 Sevecka, M., 269t, 277–278 Sevier, C S., 280, 281–282 Shaham, S., 130, 158–159, 160f, 169, 171, 172–173, 174, 266–267 Shalev, A., 293 Shalon, D., 271 Shankavaram, U T., 286 Shao, F., 4–5, 7–8 Shao, J., 5–7, 68–69 Shao, W., 277 Sharifnia, T., 280, 281–282 Sharipo, A., 225–226 Sharma, S V., 288 Shaw, G S., 251–252, 254–255 Shaw, N., 209 Shaw, T J., 53 Sheikh, B., 269t, 280, 281–282 Shekdar, K., 159 Shen, H M., 2–3 Shen, L., 203–204 Shepel, P N., 140 Sherman, F., 43–45 Author Index Shevchenko, A., 69, 203–204 Shi, Y., 23, 28–29, 52, 171 Shi, Y G., 225–226 Shibasaki, T., 142–143 Shibata, H., 217–218 Shimada, K., 269t, 284, 285f, 290, 291f, 292f, 293–295 Shimbo, K., 278 Shimizu, Y., Shin, H., 40–41, 47–48 Shin, H C., 218–219 Shin, N K., 206, 213–214, 218–219 Shindo, R., 12–14 Shiozaki, E., 41–42 Shirley, S., 47–48, 49, 55, 244–245, 247–248, 253–254, 256 Shisler, J., 20–21 Shoemaker, R H., 269t, 284 Shravage, B., 182–183, 192f Shravage, B V., 184f, 195f, 196f, 197f Shreffler, W., 147t, 159 Shu, H B., 98 Shutler, G., 36–38 Sibbet, G J., 49, 250, 255–256, 257, 259–260 Sidhu, S S., 210–211 Siegel, R M., 215–216, 222–224, 233–234 Siemer, A B., 3–4, 21–23 Sigl, V., 104 Signore, A., 215–216 Signorelli, P., Silke, J., 38, 39–42, 46–47, 49, 50–51, 52–53, 244–245, 260 Silva, N., 168–169 Silverman, G A., 139–140 Sim, W S., 224–225 Simard, C., 43, 50–51, 55 Simeonov, A., 26–27 Simin, R., 182–183, 192f Simin, R T., 184f, 195f, 196f, 197f Simon, G M., 269t, 277, 278 Simon, H U., 232–233 Simonds, E F., 279 Simpkin, J C., 68–69 Sims, J J., 253–254 Singleton, A B., 143 Sirvio, J., 144, 147t Sivonova, M., 138–139 Author Index Skaper, S D., 143 Skaug, B., 47–48 Skidan, I., 3, 6t, 14–16 Skouta, R., 268, 269t, 284, 285f, 290, 291f, 292f, 293–295 Slack, M D., 269t, 288–289 Slingsby, C., 137 Slone, S R., 144 Smith, A M., 280 Smith, A S., 219 Smith, C A., 47–48, 49, 245, 255, 258–259, 260 Smith, C C., 68–69 Smith, E E., 26–27, 26f Smith, J B., 40–41 Smith, K G., 203, 222 Smith, L., 40–41 Smith, L H., 286 Smith-Raska, M R., 71 Smits, C., 38 Smulski, C R., 121–123 Solomon, K M., 47–48 Sommer, R J., 166 Song, Y L., 213–214 Sookhareea, S., 163 Sorger, P K., 279, 287–288, 294 Sosna, J., 3–4 Soss, S E., 256, 258–259 Soulas, P., 49–50 Spear, E D., 280, 281–282 Speckmann, C., 49–50, 244–245 Speliotes, E K., 36–38 Spellman, P., 286 Spencer, S L., 279 Spillantini, M G., 143, 145 Spitzer, M., 280–281 Sprang, S R., 205–206, 205f, 207–208, 208f, 209–210, 215–216, 224–225 Spratt, D E., 254–255 Sprick, M R., 89, 203–204 Sridharan, H., 3, 4–5, 6t, 7–8, 14–16 Srinivasula, S M., 40–42, 43–45, 225–226 SriRamaratnam, R., 295 St Onge, R P., 280–281 Stadt, U Z., 244–245 Staes, A., 49, 88 Stam, R W., 274 Standaert, D G., 144 325 Stanger, B Z., 98 Starace, G., 215–216 Starovasnik, M A., 204, 221–222 Starr, D A., 163–164 Staub, E., 225–226 Stebe, E., 216–217 Stec, B., 223f, 228–229, 231–233 Steiner, Q G., 115 Steller, H., 42 Stennicke, H R., 36–38, 40–42 Sternberg, P W., 130–133, 166, 174 Stevens, C., 286 Stilmann, M., 52 Stirnimann, C., 208–209, 212–213 Stockwell, B R., 268, 271, 282, 293 Stoll, K E., 251–252 Stoughton, R., 269t, 271 Strahm, Y., 43–45 Stransky, N., 269t, 286–287 Strasser, A., 42, 104, 203–204, 222 Strathearn, K E., 145 Stuart, D I., 205–206, 213–214 Stumpel, D J., 274–275 Stupack, D., 232–233 Stuurman, N., 167–168 Su, H., 2–3 Su, L J., 145 Su, L S., 219–220 Su, Y., 54–55, 54f Subramanian, A., 280, 281–282 Sukits, S F., 224–225 Sulston, J E., 129–131, 158, 170, 172 Sun, C., 38 Sun, H., 99 Sun, L., 3–4, 6t, 8, 21, 45–46, 84, 97–98 Sun, L., 68, 69 Sun, P D., 257–258 Sun, X., 41–42, 71 Sun, X M., 85 Sung, B J., 206, 213–214 Suresh, S., 280 Surprenant, A., 75–76, 77 Suzuki, H., 43–45 Suzuki, Y., 42 Swee, L K., 115, 116, 123–124 Syntichaki, P., 132f, 134–135, 140–141, 161–162 326 T Tai, L., 204–205, 230–231, 233–234 Tait, S W., 2, 267, 282 Takahashi, K., 42 Takahashi, M., 221–222 Takahashi, N., 3, 68–69 Takahashi, R., 39–42 Takayama, H., 269t, 277–278 Takio, K., 42 Tamai, K., 36–38 Tamm, I., 40–41 Tanabe, L., 286 Tang, E D., 47–48, 49 Tang, Q Y., 143 Tao, C Y., 269t, 288–289 Tardiff, D F., 145 Tardivel, A., 104–107, 113, 115, 116, 123–124, 230–231, 233–234 Tartaglia, L A., 20–21, 221–222 Tassi, S., 77 Tatarkova, Z., 138–139 Tatham, M H., 49, 250–251, 256, 257, 259–260 Taupin, J L., 215–216 Tavernarakis, N., 132f, 133, 134–135, 136, 137, 139, 140–142, 147t, 159–160, 161–162 Taylor, R C., 267 Tazunoki, T., 221–222 Tchikov, V., 230–231, 232–234 Tchoghandjian, A., 52–53 Telliez, J B., 224–225 Temkin, V., 14 Tenev, T., 3, 5, 42, 45–47, 49, 52–53, 85, 88, 92–93, 94, 96, 97–98 Teng, X., 6t, 7, 8, 16, 21–23, 22f, 26–27, 26f, 27f, 68, 267 Tepper, C G., 222 Tepperink, J., 215–216 Terskikh, A., 213–214 Testi, R., 215–216 Teuliere, J., 167–168 Tewari, M., 222, 231–232 Than, M E., 209 Thapa, R J., 4–5, 6t, 7, 18–20, 19f Thijssen, R., 87–88 Thomas, D L., 275 Thomas, J H., 144, 145–146, 147t Author Index Thomas, L R., 224–225 Thomas, R M., 43–45 Thomas, T., 43, 49–50, 51, 88 Thome, M., 2, 6t, 7–8, 68, 69, 225–226 Thompson, C B., 129f Thompson, J R., 255 Thompson, L A., 220–221 Thomson, J N., 129–131, 158 Thorburn, A., 224–225 Thornberry, N A., 39–40 Tian, Y., 53–55 Ting, A T., 43, 50–51 Tishbi, N., 169–170 Tomasi, T., 170 Tong, J K., 271 Toth, B., 3, 69–70, 78 Totpal, K., 205f, 206, 210–211 Totty, N F., 42 Towb, P., 224–225 Trad, A., 3–4 Treinin, M., 135–136, 147t, 159 Tres Brazell, J., 23, 27f Trinidad, J C., 269t, 277, 278 Trojanowski, J Q., 142, 143, 145–146, 147t Tromp, J M., 87–88 Troulinaki, K., 141–142 Truong, L., 170 Tschopp, J., 3–4, 20–21, 39, 50–51, 70, 72–73, 104–106, 225–227, 227f, 233–234 Tschumi, A I., 269t, 282–283 Tseng, P H., 50–51, 55 Tsokos, G C., 215–216 Tsunoda, M., 143, 144, 147t Tsunoda, S., 213 Tsuruo, T., 36–38, 39 Tugarinov, V., 227–228 Turmaine, M., 276–277 Turner, V M., 49–50, 55 Twomey, D., 274 Tynan, G A., 97–98 U Uchiyama, Y., Uldrijan, S., 245 Ultsch, M., 213 Ultsch, M H., 205f, 206 Unlu, M., 277 Upperman, C., 3–4, 7–8, 14–16 327 Author Index Upton, J W., 3, 4–5, 6t, 7–8, 14–16, 68–69, 87, 98 Uren, A G., 36–38, 39–40, 244–245 Usenovic, M., 145 V Vale, R D., 167–168 Valitutti, S., 2, 6t, 7–8, 68, 69 Vallabhapurapu, S., 50–51, 55 Van Arsdale, T., 40–42 van der Horst, A., 133 van der Linden, A M., 135–136 Van Gilst, M R., 168–169 Van Hauwermeiren, F., 3, 68–69 Van Laar, J., 87–88 Van Montfort, R., 137 van Swieten, J C., 145 Vanags, D M., 11–12 Vance, R E., 79 Vanden Berghe, T., 3, 7–8, 12–14, 45–46, 68–69, 75–76, 85–87 Vandenabeele, P., 2, 7–8, 45–46, 75–76, 85–87, 97–98, 244–245 Vandendriessche, B., 3, 68–69 Vanderhyden, B C., 53 Vandongen, J L., 36–38, 39–40 Vanlangenakker, N., 3, 5, 7–8, 12–14 Varfolomeev, E., 38, 39, 42, 43, 46–49, 50–51, 53–55, 54f, 244–245, 260 Varfolomeev, E E., 71, 88, 98 Varshavsky, A., 244 Vartiainen, S., 144, 147t Vattem, K., 188 Vaux, D L., 36–38, 39–40, 41–42, 46–48, 49, 244–245, 255, 258–259, 260 Velazquez-Campoy, A., 255 Velds, A., 280 Venkatesan, K., 269t, 286–287 Vercammen, D., 2, Verhagen, A., 43–45 Verhagen, A M., 36–38, 42 Vierling, E., 137 Vihinen, H., 197 Vince, J E., 4–5, 18–20, 38, 39–40, 43, 46–47, 49, 50–51, 55, 88, 89, 244–245 Vincenz, C., 221–222, 226–227 Vinsant, S., 172–173 Virchow, R L K., 266 Virginio, C., 75–76, 77 Viswanathan, V S., 295 Vitale, I., 128, 266–267 Vittal, V., 260 Vlachos, M., 133 Vogel, P., Voigt, S., 3–4 von Moltke, J., 79 von Schweinitz, D., Voss, A K., 43, 49–50, 51, 88 Voss, M E., 220–221 Votteler, J., 123 Vousden, K H., 245 Vrablik, T L., 133, 147t, 162–163 Vucic, D., 36–38, 39, 43, 46–47, 50–51, 52–54, 54f, 260 W Wade Harper, J., 184f, 195f, 196f, 197f Wagner, G., 225–226, 227–228 Wagner, K W., 54–55, 216–217 Wagner, L., 5, 7–8, 99 Wakeham, A., 98 Walczak, H., 50–51, 88, 203–204 Walker, G., 41–42, 85 Walker, N I., 128, 134 Walker, N P., 205–206 Wallace, H A., 52 Wallace, I M., 280 Wallach, D., 3, 68–70, 78 Wallberg, F., 3, 5, 45–46, 85, 88, 92–93, 94, 96, 97–98 Waltham, M., 277, 279, 286 Walz, T., 226–227, 227f Wan, F Y., 225–226 Wang, C., 278 Wang, C Y., 47–48, 49 Wang, H., 3–4, 6t, 8, 21, 38, 45–46, 49, 50–51, 55, 68, 69, 84, 97–98, 162 Wang, J., 53–55, 222–224 Wang, L., 5–7, 18–20, 21, 43, 53–55, 54f, 68–69, 203, 204, 221–222, 224–226 Wang, L W., 225–226, 229, 230f, 231–232 Wang, M., 277 Wang, S X., 221–222 Wang, T., 5–7, 18–20, 21, 68–69, 260 Wang, X., 3–5, 7–8, 38, 42, 43–45, 69, 220 Wang, Y., 38, 46–47, 55, 159 Wang, Z., 3–4, 6t, 8, 21, 45–46, 68, 69, 84, 97–98 328 Wang, Z W., 142–143 Warmer, M., 123 Warnken, U., 233–234 Wasielewski, E., 255 Wassarman, D A., 36 Wassell, R., 42, 43–45 Wasserman, S A., 224–225 Watanabefukunaga, R., 221–222 Wayson, S M., 38, 46–47, 50–51, 54f, 55, 88, 244–245 Webb, C P., 284–285 Weber, C H., 221–222, 226–227 Wei, A., 142–143 Wei, G., 274 Wei, W.-L., 162 Wei, Y., 224–225 Wei, Y F., 224–226 Weinberg, R A., 288 Weinlich, R., 5, 45–46, 68, 69, 75–76, 87, 98, 99 Weinstein, J N., 269t, 286 Weinstein, M., 164 Weissman, A M., 47, 247–248 Weist, B M., Weller, M., 43–45, 53–54 Wells, J A., 49, 269t, 277, 278 Welsch, M E., 269t, 290, 291f, 292f, 293–295 Welsh, K., 40–41, 54–55, 54f Welsh, S., 2–3 Wenzel, D M., 251 Wertz, I E., 39, 47, 50–51, 260 West, A P., 220 West, D C., 275 West, K., 42, 53–54 White, J G., 129–130, 131–133, 158 White, J Q., 170 Whitty, A., 219 Wick, W., 43–45, 53–54 Wider, G., 225–226 Wiener, R., 250, 252–253, 259–260 Wieringa, B., 221–222 Wiestler, B., 232–233 Wijsman, E., 145 Wildenhain, J., 280–282 Wilkinson, J C., 47–48 Willen, L., 115, 116, 123–124 Williams, D E., 269t, 280 Author Index Williams, O., 274–275 Williams, R., 251, 258–259 Williams, R T., 281 Wilson, N S., 203 Wilson, S., 144, 147t Windheim, M., 255 Wing, S S., 47 Winoto, A., 98 Wittkopf, N., 99 Witze, E S., 166–167 Woelfel, M., 215–216 Wolberger, C., 250, 251–252, 259–260 Wolff, P., 121–123 Wolinsky, E., 134, 147t, 159 Wolpaw, A J., 269t, 290, 291f, 292f, 293–295 Wong, D., 139, 147t Wong, E T., 52 Wong, G H., 20–21 Wong, H., 42, 53–54 Wong, L., 36–38, 39 Wong, S C., 36–38 Wong, V., 145 Wong, W W., 18–20, 39–40, 41–42, 43–47, 49–51, 55, 84, 88, 89, 95–96, 97–98, 99 Wong, W W.-L., 244–245 Woo, M S., Workman, L M., 43, 50–51 Woronicz, J D., 224–225 Wright, A T., 277 Wright, P., 260 Wrobel, M J., 269t, 271 Wu, G., 225–226 Wu, H., 38, 53–55, 203, 204, 221–222, 224–226 Wu, J., 3–4, 7, 11–12, 23, 28–29, 217, 267 Wu, J L., 224–225 Wu, K., 254–255 Wu, L F., 269t, 288–289 Wu, M., 52 Wu, T Y., 54–55 Wu, X., 28 Wu, Z H., 52 Wyllie, A H., 128, 266 X Xavier, R J., 3, 5, 6t, 7–8, 14–16 Xia, Z P., 3–4 329 Author Index Xiao, T., 224–225 Xie, T., 8, 23, 28–29 Xiong, Y., 47–48, 49 Xiong, Z.-G., 162 Xu, G Y., 224–225 Xu, K., 140, 141, 147t, 159–160, 161 Xu, M., 47–48 Xu, N., 38 Xu, T., 183–184 Xu, X., 164 Xu, X N., 213–214 Xuan, J Y., 36–38 Xue, D., 158–159, 170–171 Xue, W., 39 Xue, Y N., 217 Y Yabal, M., 49–50, 51, 244–245 Yacoubian, T A., 144, 147t Yagi-Utsumi, M., 247, 250, 251–252 Yamagata, A., 248 Yamagishi, J., 213–214 Yamaguchi, H., 145–146 Yamaguchi, Y., 247, 250, 251–252 Yamamoto, S., 247, 250, 251–252 Yamashima, T., 141 Yamashita, M., 248 Yamayoshi, M., 213–214 Yamori, T., 36–38, 39 Yan, C., 8, 23, 28–29 Yan, N., 158–159, 171 Yan, Q R., 202–203, 214–215 Yanagisawa, J., 221–222 Yang, G., 46–47, 55 Yang, J., 173–174 Yang, J K., 203, 204, 221–222, 224–226, 229, 230f, 231–232 Yang, Q H., 46–47 Yang, S H., 3, 69–70, 78 Yang, W S., 284, 285f, 293, 295 Yang, X., 158–159, 164, 280, 281–282 Yang, Y., 46–47, 49, 169–170 Yang, Z R., 220 YanoYanagisawa, H., 221–222 Yao, J., 3, 5, 6t, 7–8, 14–16 Yao, Y., 46–47, 55 Yaraghi, Z., 36–38 Yeger-Lotem, E., 145 Yeh, E T., 233–234 Yeh, P., 269t, 282–283 Yeh, W C., 46–47, 98 Yellon, D M., 68–69 Yin, J P., 204, 221–222 Yin, Q., 5355, 254, 256 Ylaă-Anttila, P., 197 Yogev, N., 71 Yoon, J B., 46–47, 49 Yoshikawa, A., 248 Yoshikawa, M., 213 Yoshimura, S., 172–173 Yoshina, S., 171 Yoshioka, Y., 213, 217–218 Young, D W., 269t, 288–289 Young, F., 274–275 Young, L., 277, 279, 286 Young, S S., 36–38 Yousefi, S., 232–233 Yu, G L., 221–222 Yu, I W., 250, 259–260 Yu, J., 52 Yu, J W., 225–226, 267 Yu, L., 2–3, 280–281 Yu, Y., 52 Yuan, A., 142–143 Yuan, J., 3–4, 8, 11–12, 39–40, 130 Yuan, J Y., 266–267 Yun, H., 162 Z Zacharias, D A., 215–216 Zaitsev, E M., 268, 295 Zajonc, D M., 211–212, 211f Zappe, A., 215–216 Zarkower, D., 163, 170–171 Zarnegar, B J., 46–47, 55 Zaru, R., 2, 6t, 7–8, 68, 69 Zender, L., 39 Zeng, W., 47–48 Zenteno, E., 165 Zhan, C Y., 202–203, 214–215 Zhan, F., 49–50 Zhang, B., 145–146, 147t Zhang, C., 269t, 280, 281–282 Zhang, D., 52, 220 Zhang, D P., 219–220 Zhang, D W., 5–7, 68–69 330 Zhang, H., 98 Zhang, J., 45–46 Zhang, J G., 3–4, 7, 97–98 Zhang, L., 43, 50–51 Zhang, M., 255 Zhang, N., 5–7, 68–69 Zhang, W., 50–51, 55 Zhang, X., 252–253, 260 Zhang, Z., 3–4, 7, 11–12, 42, 43–45, 143 Zhao, J., 45–46, 68, 69, 84 Zhao, L., 5–7, 18–20, 21, 68–69 Zhao, L X., 209 Zhao, Y., 49–50 Zheng, C., 38 Zheng, L., 20–21, 215–216 Zheng, L X., 215–216, 222–224, 225–226 Zheng, X., 28 Zhong, C Q., 28 Zhou, P., 227–228 Author Index Zhou, Q., 40–42 Zhou, Q H., 171 Zhou, W., 3–4, 7–8, 14–16 Zhou, X., 98, 162 Zhou, Z., 3–4, 28, 68, 84, 174 Zhu, H., 3–4, 7–8, 14–16, 266–267 Zhu, X.-M., 162 Zhuang, M., 49 Zimmermann, G R., 269t, 282–283 Zipperlen, P., 158 Zischka, H., 128 Zitvogel, L., 128 Zobel, K., 5, 7–8, 42, 43, 46–47, 50–51, 53–55, 54f, 99 Zolla, L., 277 Zornig, M., 203, 222 Zuger, S., 208–209, 212–213 Zumsteg, A., 115 Zunder, E R., 277 zur Stadt, U., 49–50 SUBJECT INDEX Note: Page numbers followed by “f ” indicate figures and “t ” indicate tables A Adenosine 50 -triphosphate (ATP) application, 70 DCs, 77 and LPS, 70 Alzheimer’s disease aex-3 promoter, 145–146 aminothienopyridazine class, 146 autosomal-dominant mutations, 145 tau-mediated neurotoxicity, 146–147 transgenic worms, 146 Annexin V/PI assay FADD-deficient Jurkat cells, 12 and mitochondrial membrane potential assays, 11–12, 11f AntiTNF agents, 219–220 Apoptosis in ALL cells, 274 caspase-independent death, 267 and necrosis (see Necrosis) phosphorylation, 278 and pyroptosis, 267 ATP See Adenosine 50 -triphosphate (ATP) Autophagic cell death, Drosophila See also Drosophila biological system caspase-independent manner, 182–183 catabolic process, 182 genetic approaches, 183–184 Autophagy cell death, 128 and endocytosis, 142 heat-stroke-induced cell death, 137 morphological features, 128, 129f B Baculovirus IAP repeat (BIR) caspases, 38 NAIP, 39 structural properties, 54–55 XIAP and c-IAPs, 38 Bone marrow-derived cell (BMDC), 69–70 C Caenorhabditis elegans apoptotic cell death, 158–159 apoptotic genes, 158–159 apoptotic machinery, 130 cell death programs, 158, 175 developmental cell deaths, 168–171 EGL-1 protein, 158–159 genetic studies, 159 as model, human diseases, 142–147 necrosis studies, 129–130 nonapoptotic, caspase-independent linker cell death, 172–175 pathological cell death, 159–168 Calpain papain-like cysteine proteases, 141 signaling and metabolic processes, 141 CARD See Caspase recruitment domain (CARD) Caspase-8 immunoprecipitation experimental procedure, 94–95 intracellular and extracellular stimuli, 94 Caspase-independent linker cell death, nonapoptotic apoptotic cell death genes, 172 degeneration, Drosophila salivary glands, 174 distal neurite degeneration, 173–174 dying linker cells, 172–173 genetics, 172, 173f nuclear crenellation, 172–173 Caspase inhibitor apoptotic pathway, 41–42 fluorogenic peptide, 40–41 XIAP, 42 Caspase recruitment domain (CARD) in c-IAP1 and c-IAP2, 39 and NACHT, 39 Cathepsin aspartyl proteases, 140–141 human lysosomal enzyme, 144 331 332 CDRs See Complementarity-determining regions (CDRs) Cell death necrosis (see Necrosis) small-molecule-induced (see Smallmolecule-induced death) Cell line viability profiling cell death, advantages and limitations, 287–288 databases, 286–287 NCI60 screen (see The US National Cancer Institute (NCI) 60 screen) Chemical-genetic profiling in mammalian cells, 281 in yeast, 280 Complementarity-determining regions (CDRs) CDR-H3 loop, 210–211 CRD1 and CRD2, 210–211 tyrosines, 210–211 Complex gel filtration, 96 Connectivity Map database, 272–273 Conversion of membrane-bound Fas (CD95) binding surface, FADD, 230–231 ectodomain, 212–213 FADD-DD complexes C-helix hairpins, 228–229 chimeric complex, 229 Huh7 cells, 228–229 low-resolution crystal structure, 229, 230f physiological signaling, 230–233 tetrameric arrangement, 228–229 in vitro interactions, 232–233 ligand-dependent clustering, 231–232 signaling, 230–233 TRAIL-R1 and TRAIL-R2, 232–233 Tyr291 side-chain hydroxyl, 232–233 CRD See Cysteine-rich domains (CRD) Cysteine-rich domains (CRD), 104–106 D Damage-associated molecular pattern (DAMPs) ATP, 75–76 caspase-8-deficient cells, 79 and trigger inflammation, 77 Subject Index Death domain (DD) CD95 and crystallographic complex, 222–224, 223f and DR (see Death receptor (DR)) DR adaptor protein, 224–225 3D structure, 205–206, 205f α-helical secondary structure, 204 homologous protein family, 204–205 in vitro and in vivo binding assays, 224–225 and NMR, 222–224 PIDDosome core, 226–228 superfamily, 225–226 thermodynamic stability, 222–224 Death-inducing signaling complex (DISC) autoproteolysis, 203–204 ligand-bound receptor, 230–231 receptor-accreted assembly, 203–204 Death receptor (DR) antiTNF agents, 219–220 blockade, 220–221 CD95 and FADD-DD complexes, 228–229 cytoplasmic domains, 221–222 and DD (see Death domain (DD)) decoy receptor-ligand complex, 214–215 domain and ligand structure, 204–206 ectodomain structure, 206–213 ligand structure-activity relationships, 217–219 physiological complexes, ligands, 213–214 preligand association domain, 215–217 TNFR (see Tumor necrosis factor receptor (TNFR)) Dendritic cells (DCs) ATP release, 77 caspase-8, 69 necroptosis, 72 ROS generation, 76–77 Developmental cell deaths, C elegans caspases, dying cells, 171 germline cell death, 168–169 sex-specific death, CEM neurons, 170–171 tail-spike cell death, 169–170 DISC See Death-inducing signaling complex (DISC) DR See Death receptor (DR) 333 Subject Index Drosophila Atg8 tagged fluorescence, 191 autophagic cell death, 182–183 data analysis and interpretation caveats, autophagy markers and flux, 197–198 histological sections, 191–193 immunoblotting, 194–195 immunofluorescence and fluorescently tagged Atg8, 195–197 TEM, 197, 197f TUNEL, 194, 194f fly food, 184–185 immunoblotting, 188 immunofluorescence, 188–189 preparation, 186 sectioning, 186–187 staging, animals, 185 staining, 187 TEM, 190–191 TUNEL, 189–190 E Ectodomain structure, DR CD95, 212–213 CRDS, 207–208 DR6 disulfide ladder, 209 3D structure, 207–208, 208f homotrimeric ligand, 206–207 TNFR1, 209–210 TRAIL-R2, 210–212 ELISA See Enzyme-linked immunosorbent assay (ELISA) Endogenous RIPK autophosphorylation assays immunoprecipitation, 22 Jurkat cells, 22 MBP, 21–23 Enzyme-linked immunosorbent assay (ELISA) crude/purified tagged proteins materials, 108 method, 108–110 immunoprecipitations, 108, 109f ligand-receptor interactions detection, 108, 109f untagged proteins/inhibitors, 111 E2Ub conjugates disulfide-linked formation, 248–250 E1 purification, 247 E2 purification, 247–248 oxyester-and isopeptide-linked conjugate formation, 250–251 ubiquitin purification, 248 F Fas-associated death domain (FADD) deficient Jurkat cells, 12 MEFs, Fas-expressing reporter cell lines, 116–119 Fluorescence-activated cell sorting (FACS) tagged ligands, GPI-anchored receptors flow cytometry, 113 method, 113–115 reagents, 113 tagged receptors, BAFFN-fusion ligands EGFP, 115 reagents, 115 Fluorescence polarization assay, 26–27 F€ orster resonance energy transfer (FRET) CD40, 121–123, 122f ECFP and EYFP proteins, 121 flow cytometry, 121–123 method, 123 signals, 121–123, 122f FRET See F€ orster resonance energy transfer (FRET) G Gene expression profiling cell death applications, 273–275 Connectivity Map database, 272–273 on-and off-target, 276–277 RNA-seq, 275–276 small-molecule, 269t, 271–272 Germline cell death, 168–169 Glycosyl-phosphatidylinositol (GPI), 107 H Heat-induced necrotic death core body temperature, 137 mammalian PMR-1, 138–139 mechanisms, 137, 138f nonlethal temperature, 137 Homogeneous time-resolved fluorescence (HTRF) KinEASE assay, 25–26 Hypoxia, 142–143 334 I IAPs See Inhibitors of apoptosis proteins (IAPs) Immunoprecipitation, ligand-receptor interactions Flag-tagged ligands, 111–112 precipitations heparin-Sepharose, 112–113 receptors-Fc, 112 reagents, 112 Inhibitors of apoptosis proteins (IAPs) antagonists experimental procedure, 88, 89f and TLR, 88 BIRs, 38, 54–55 caspase inhibitor, 40–42 cell death, c-IAP1 and c-IAP2, 43–46 chromosomal deletions, 36 c-IAP1 and c-IAP2 proteins, 55 description, 36 human pathologies, 52–53 human, proteins, 36–38, 37f intrinsic Bcl-2 blockable pathway, 39–40 NACHT and enigmatic CARD, 39 oligonucleotides, 53 proteins and ubiquitin, 43–46 RING and UBA, 39 signaling pathways, 50–52 Smac-mimicking small-molecule, 53–54 structure, 53–54, 54f X-chromosome-linked, 36–38 Ionic imbalance degenerins, 134–135 excitotoxicity, 136 glutamate transporters, 136 vacuolar degeneration, 135–136 K Kinase-Glo assay description, 23–25 luminescence signal, 24 recombinant GST-RIP1 kinase activity, 24f L Ligand-receptor interactions apoptosis induction, 119 Subject Index BAFFN, 115–116 CRD, 104–106 ELISA, 108–111 FACS, 113–116 Fas-expressing reporter cell lines, 116–119 FRET, 121–123 immunoprecipitation, 111–113 monitoring inhibitor, 120 NF-kB reporter cells, 120–121 purification and storage, 107–108 reagents and assay principles, 104–106, 105f TNF and TNFR superfamilies, 104 transmembrane proteins, 106 Ligands BAFFN, 107 Fc-ligands, 107 Flag-ACRP-ligands, 106–107 Flag-ligands, 106 Linker cell death See Caspase-independent linker cell death, nonapoptotic M MBP See Myelin basic protein (MBP) Mis-specified uterine-vulval (uv1) cells egg laying, 131–133 ku212 allele, 133 LET-23 EGF receptor, 131–133 Mitochondrial membrane depolarization, 11f, 14 Mixed lineage kinase domain-like protein (MLKL) phosphorylation and necroptotic execution, 97–98 role, 84 Modulatory profiling cell death study, 295–296 comparison and clustering, 290, 292f creation, 290, 291f ferroptosis, 295 protocol, 293–294 Myelin basic protein (MBP) exogenous substrate, 23–25 and histone, 21–23 N Necroptosis Annexin V/PI assay, 11–12 cellular models, 5–7, 6t 335 Subject Index CYLD, DAMPs, domain receptors, electron microscopy, 2–3 FADD-deficient Jurkat cells, 9, 9f inducers, 7–8 inhibitors, Jurkat cells, mitochondrial membrane depolarization, 14 necrosome complex, 2–3 qPCR, TNFα gene expression changes, 14–16 RIPK3, RIPK1, TRADD and TRAF2, 3–4 ROS increase, 12–14 signaling, TNFα, 3–4 SYTOX green assay, 10–11, 10f TLRs, 4–5 TNF-induced, Necrosis blue fluorescence, 128–129 Caenorhabditis elegans, 129–130 calcium-binding chaperones, 140 calpains, 141 cell death paradigms, 130–133 clathrin-mediated endocytosis, 141–142 cytoplasmic acidification, 140–141 description, cell death, 128 innexin INX-16, 128–129 intracellular calcium, 140 morphological features, 128, 129f mutations, 141 nondevelopmental necrotic death, 134–140 synergistic protection, 142 triggers and paradigms, 147–149, 147t Necrosome complex formation FADD-deficient Jurkat cells, 19 fluorescence microscopy, 21 lysate, 19 protease inhibitor, 19 protein concentrations, 19 RIPK1/RIPK3, 18–20 TNFR1 complex, 20–21 NF-kB See Nuclear factor-kappaB (NF-kB) NLRP3 inflammasome ATP release, DCs, 77 BMDC, 69–70, 77 caspase-8 deficiency, 70, 78 cellular ROS, 76–77 cytokines, DCs, 72 DC lysates, 77 IL-1β, 70 inflammatory mediators, 69 Itgax gene, 71–72 mice deficient, RIPK3, 71 mouse bone marrow-derived DCs, 70–71 necroptosis, 69 necrotic death, 68–69 protein phosphorylation, 68 proteins signaling, necroptosis, 72 ROS, 75–76 signaling proteins controlling necroptosis, 72–75 viability tests, DCs, 76 Nonapoptotic cell death, 295 Nondevelopmental necrotic death bacterial infection, 139 gain-of-function mutations, 134 heat-induced, 137–139 hypo-osmotic shock-induced cell death, 139–140 ionic imbalance, 134–136 Nuclear factor-kappaB (NF-kB) description, 120 dual luciferase reporter assay system, 120 method, 121 plasmids, 120 P Parkinson’s disease ATP13A2, 145 C elegans models, 144 dopaminergic neuron, 144 hVPS41 overexpression, 144 mitochondrial dysfunction, 144 SNARE machinery, 143 α-synuclein toxicity, 145 Pathological cell death, C elegans apoptotic pathway, Pn.p cells, 165–167 cell differentiation mutations, 163–164 cell shedding, caspase mutants, 167–168 ion channel mutations calcium homeostasis, 159–160, 161f cathepsin activation and cell demise, 161 336 Pathological cell death, C elegans (Continued ) cytoplasmic cathepsins, 161 dying neurons accumulation, 159 ENaC-type cation channels, 159 glutamate-induced toxicity, 162 morphological features, 159, 160f neuronal cell death, 162 nicotinic acetylcholine receptor, 159 lin-24/lin-33 mutants, 164–165 NAD metabolism defects, 162–163 Protein quantification advantages and limitations, cell death, 279–280 cell death application, 278 mRNA levels, 277 small-molecule profiles, 277–278 R Reactive oxygen species (ROS), 76–77 Really interesting new gene (RING) carboxy-terminal, 39 E2Ub complexes, (symbol must be inserted) analytical SEC, 254–255 characterization, 254f, 257 chemical shift perturbations, 258–259 crystallization efforts, 258–259 discharge assays, 256–257 isopeptide-linked and oxyester-linked conjugate, 257 NMR, 258–259 open and closed conformations, 251, 252f polyubiquitin chains, 252 pulldown assays, 253–254 SPR and ITC, 255–256 structure, 257, 258f thioester-linked conjugate, 251 IAP proteins, 47 structure, 49 and UBA, 39 ubiquitin ligases bind, 46–47 Receptors Fas chimeric proteins, 107 GPI, 107 receptors-Fc, 107 Recombinant RIPK1 kinase activity and inhibition Subject Index fluorescence polarization assay, 26–27 HTRF KinEASE assay, 25–26 Kinase-Glo assay, 23–25 and RIP3, 23 thermomelt assay, 27–28 RIP1 kinase expression centrifugation, 17 RHIM domain mutants, 16 stable-inducible cell lines, 17–18 transfections, 16 Ripoptosome advantages and disadvantages, 96–97 Annexin V/propidium iodide staining, 91–92 biochemical analysis caspase-8 immunoprecipitation, 94–95 compounds, 94 cancer cells, 99 cell death, 85 cFLIP, 98 complex gel filtration, 96 components, 99–100 experimental procedure, 91 formation, cancer therapy, 87–88 HaCaT cells, 90, 90f heterodimer, 85–87 IAP antagonists, 88 kinase activities, 85, 86f MLKL, 84 multicellular organisms, 84 necroptosis, 87 procaspase-8, 85–87 proteins involvement, 93–94 purification, tandem-affinity purification, 95–96 RIP1, 89–90 stress-responsive signaling, 97–98 Sytox Green/Hoechst staining, 92–93 TNF-blocking receptor, 97–98 ROS See Reactive oxygen species (ROS) S Sex-specific death, CEM neurons ced-3 transcription, 170–171 and tail-spike cell, 171 Signaling proteins controlling necroptosis caspase-1 and IL-1β precursor protein, 73 chemical cross-linking reagent, 74–75 IL-1β precursor protein, 72–73 337 Subject Index inflammasome, 74 LPS and ATP, 72–73 NLRP3 and ASC, 73 Small-molecule-induced death bioactivity, 268 Caenorhabiditis elegans, 266–267 cell death pathways diversity, 267, 267f cell line viability profiling, 284–288 compounds, profiles, 282–283 descriptions, 266 gene expression profiling, 271–277 gene, interactions, 280–282 high-content imaging, cell culture, 288–289 higher-order combinations, 283 image-based profiles, 289 modulatory profiling, 290–296 protein quantification, 277–280 RNAi, 283 technologies, profiling, 268, 268f Stable-inducible cell lines, 17–18 T Tail-spike cell death, 169–170 Tau toxicity, 145–147 TEM See Transmission electron microscopy (TEM) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) DNA fragmentation, 189–190 staining, 194, 194f Thermomelt assay, 27–28 TLRs See Toll-like receptors (TLRs) TNF See Tumor necrosis factor (TNF) TNF apoptosis-inducing ligand receptor (TRAIL-R2) ectodomain CDRs, 210–211 human cytomegalovirus, 211–212 UL141 and TRAIL-R1, 211–212 TNFR See Tumor necrosis factor receptor (TNFR) Toll-like receptors (TLRs), 4–5 Transmission electron microscopy (TEM), 190–191, 197, 197f Tumor necrosis factor (TNF) cDNA, 15 CellTiter-Glo assay, gene expression changes, qPCR, 14–16 ligand-receptor interactions (see Ligand-receptor interactions) MEFs, 14 MyD88-dependent autocrine, 7–8 necroptosis, 3–4 qPCRs, 15 RIPK1 activation, 14–16 TZ vs control samples, 16 Tumor necrosis factor receptor (TNFR) DcR3, 202–203 DD-containing proteins, 203 description, 203 DR signaling, 203–204 ligand-dependent clustering, 203–204 TNFR1 ectodomain, 209–210 TUNEL See Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) U Ubiquitylation biochemically defined assays, 260 E2-binding site, 245 E2Ub conjugates, 246–251 IAP proteins, 244–245 mutagenesis, 245 nondegradative ubiquitin signals, 244–245 nucleotide-binding, 244–245 oligomerization domain signaling, 244–245 protein-protein interaction module, 244–245 RING domain function, 245–246 RING-E2UB complexes, 251–259 tumor suppressor proteins, 260 The US National Cancer Institute (NCI) 60 screen applications, 284–286 description, 284 molecular characterization, 286 X X-chromosome-linked IAP (XIAP) caspase inhibitor, 40–42 intrinsic Bcl-2 blockable pathway, 39–40 Y Yeast profiling, 280–281 ... multidomain protein, which contains N-terminal Ser/Thr kinase, followed by intermediate domain including K377 ubiquitination site and RHIM motif, and C-terminal death domain mediating binding to... experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety... DRs E3 ubiquitin ligases cIAP1/2 in concert with TRAF2 ubiquitinates RIPK1 in Complex I, providing conditions for TAK1 and IKK kinase complex binding, activating the downstream proinflammatory