Ashkenazi / Wells / Yuan Regulated Cell Death Part B

Necroptotic, Autophagic and other Non-apoptotic Mechanisms
1. Auflage 2014
ISBN: 978-0-12-801619-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 545, 352 Seiten

Reihe: Methods in Enzymology

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. - 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

Autoren/Hrsg.

Ashkenazi, Avi

Wells, Jim

Yuan, Junying

Inhaltsverzeichnis

1;Front Cover;1
2;Regulated Cell Death Part B: Necroptotic, Autophagic and other Non-apoptotic Mechanisms;4
3;Copyright;5
4;Contents;6
5;Contributors;10
6;Preface;14
7;Chapter One: Assays for Necroptosis and Activity of RIP Kinases;16
7.1;1. Introduction;17
7.1.1;1.1. Distinguishing features of necroptotic cell death;17
7.1.2;1.2. Pathways and mediators of necroptosis;18
7.2;2. Cellular Models of Necroptosis;20
7.2.1;2.1. Cell types (Table 1.1);20
7.2.2;2.2. Inducers of necroptosis;22
7.2.3;2.3. Inhibitors of necroptosis;23
7.3;3. Measurement of Necroptotic Cell Death;24
7.3.1;3.1. Analysis of viability of FADD-deficient Jurkat cells treated with TNFa using CellTiter-Glo assay (Fig. 1.1);24
7.3.2;3.2. Determination of specific cell death using SYTOX Green assay (Fig. 1.2);25
7.3.3;3.3. Annexin V/PI assay (Fig. 1.3);26
7.3.4;3.4. Analysis of ROS increase (Fig. 1.4);27
7.3.5;3.5. Mitochondrial membrane depolarization (Fig. 1.3);29
7.3.6;3.6. Analysis of TNFa gene expression changes by qPCR (Fig. 1.5);29
7.4;4. Recapitulation of RIP1 Kinase Expression in RIP1-Deficient Jurkat Cells;31
7.4.1;4.1. Transient transfection (Fig. 1.6);31
7.4.2;4.2. Generation of stable-inducible cell lines (Fig. 1.7);32
7.5;5. Analysis of Necrosome Complex Formation;33
7.5.1;5.1. Immunoprecipitation of necrosome complex (Fig. 1.8);33
7.5.2;5.2. Immunoprecipitation of TNFR1 complex;35
7.5.3;5.3. Assessment of necrosome formation by fluorescence microscopy;36
7.6;6. Endogenous RIPK Autophosphorylation Assays (Fig. 1.9);36
7.7;7. Analysis of Recombinant RIPK1 Kinase Activity and Inhibition by Necrostatins;38
7.7.1;7.1. Expression and purification of recombinant RIP1 and RIP3;38
7.7.2;7.2. Kinase-Glo assay (Fig. 1.10);38
7.7.3;7.3. HTRF KinEASE assay (Fig. 1.11);40
7.7.4;7.4. Fluorescence polarization assay (Fig. 1.12);41
7.7.5;7.5. Thermomelt assay (Fig. 1.13);42
7.8;8. Conclusions;43
7.9;Acknowledgments;44
7.10;References;44
8;Chapter Two: IAP Family of Cell Death and Signaling Regulators;50
8.1;1. Identification of IAPs, Structure, and Domain Function;51
8.1.1;1.1. Discovery;51
8.1.2;1.2. Domain structure—BIRs;53
8.1.3;1.3. Domain structure—RING and UBA;54
8.1.4;1.4. Domain structure—NACHT and enigmatic CARD;54
8.2;2. IAP Proteins and Cell Death Pathways;54
8.2.1;2.1. XIAP—Inhibitor of the intrinsic Bcl-2 blockable pathway;54
8.2.2;2.2. XIAP—Caspase inhibitor;55
8.2.3;2.3. Inhibition of cell death by c-IAP1 and c-IAP2;58
8.2.4;2.4. IAP proteins and ubiquitin;61
8.2.5;2.5. Regulation of signaling pathways by IAP proteins;65
8.2.6;2.6. Targeting IAP proteins;67
8.3;References;70
9;Chapter Three: Activation of the NLRP3 Inflammasome by Proteins That Signal for Necroptosis;82
9.1;1. Introduction;83
9.2;2. Altered Expression or Function of Enzymes That Control Induction of Necroptosis Results in Altered Generation of IL-1ß...;84
9.2.1;2.1. Generation of mouse bone marrow-derived DCs;85
9.2.2;2.2. Use of transgenic mice to obtain DCs deficient in caspase-8 or RIPK3;86
9.2.3;2.3. Knockdown of proteins signaling for necroptosis in DCs;87
9.2.4;2.4. Induction of cytokines in DCs by agents inducing and activating the NLRP3 inflammasome;87
9.2.5;2.5. Quantification of the induced cytokines;87
9.3;3. Signaling Proteins Controlling Necroptosis Affect Assembly of the NLRP3 Inflammasome;87
9.3.1;3.1. Assessment of the proteolytic processing of caspase-1 and of the IL-1ß precursor protein;88
9.3.2;3.2. Confirmation of the requirement for NLRP3 and ASC for IL-1ß generation;88
9.3.3;3.3. Assessment of the assembly of the inflammasome by measuring the detergent solubility of the inflammasome components;89
9.3.4;3.4. Assessment of the assembly of the inflammasome by the use of cross-linking reagents;89
9.4;4. Does the Similarity Between the Regulation of Necroptosis and of Assembly of the NLRP3 Inflammasome Reflect Activation...;90
9.4.1;4.1. Viability tests applied to DCs;91
9.4.2;4.2. Assessment of ROS generation in the DCs;91
9.4.3;4.3. Assessment of the release of inflammasome-activating agents by the DCs;92
9.4.3.1;4.3.1. ATP release by the DCs;92
9.4.3.2;4.3.2. Assessment of permeability changes in the DCs as a marker for exposure to ATP;92
9.4.3.3;4.3.3. Assessment of the ability of DC lysates to activate IL-1ß generation;92
9.4.3.4;4.3.4. Coculturing of wild-type and caspase-8-deficient cells;93
9.5;5. Concluding Remarks;93
9.6;Acknowledgments;94
9.7;References;94
10;Chapter Four: Characterization of the Ripoptosome and Its Components: Implications for Anti-inflammatory and Cancer Therapy;98
10.1;1. Introduction;99
10.2;2. The Ripoptosome: Cellular Model Systems to Study Its Formation;102
10.2.1;2.1. Induction of ripoptosome formation by IAP antagonists (SMAC mimetics);103
10.2.1.1;2.1.1. Experimental procedure;103
10.2.2;2.2. Induction of ripoptosome formation via RIP1 over expression;104
10.2.2.1;2.2.1. Experimental procedure;105
10.2.3;2.3. Quantitative and qualitative analysis of ripoptosome-mediated cell death;105
10.2.3.1;2.3.1. Screening test: Crystal violet cell death assay;105
10.2.3.1.1;2.3.1.1. Experimental procedure;106
10.2.3.2;2.3.2. Specific test to assay the quality of cell death: Annexin V/propidium iodide staining followed by FACS analysis;106
10.2.3.2.1;2.3.2.1. Experimental procedure;107
10.2.3.3;2.3.3. Morphological analysis: Sytox Green/Hoechst staining followed by fluorescent microscopy;107
10.2.3.3.1;2.3.3.1. Experimental procedure;108
10.2.4;2.4. Signaling pathway analysis: siRNA knockdown of target proteins involved in ripoptosome-mediated cell death;108
10.2.4.1;2.4.1. Experimental procedure;108
10.3;3. Biochemical Analysis of the Ripoptosome: Analysis of Ripoptosome Formation and Identification of Novel Components via ...;109
10.3.1;3.1. Caspase-8 immunoprecipitation;109
10.3.1.1;3.1.1. Experimental procedure;109
10.3.2;3.2. Ripoptosome purification by tandem-affinity purification;110
10.3.3;3.3. Complex gel filtration combined with mass spectrometry;111
10.3.4;3.4. Advantages and disadvantages of these procedures;111
10.4;4. Outlook: Future Implications of the Function and Regulation of the Ripoptosome;112
10.5;References;115
11;Chapter Five: Tools and Techniques to Study Ligand–Receptor Interactions and Receptor Activation by TNF Superfamily Members;118
11.1;1. Introduction;119
11.2;2. Methods;121
11.2.1;2.1. Tagged ligands and receptors for interaction and functional studies;121
11.2.1.1;2.1.1. Tagged ligands;121
11.2.1.2;2.1.2. Tagged receptors;122
11.2.1.3;2.1.3. Purification and storage of ligands and receptors;122
11.2.2;2.2. The measure of ligand–receptor interactions by ELISA;123
11.2.2.1;2.2.1. Measure of ligand–receptor interactions with crude or purified tagged proteins;123
11.2.2.1.1;2.2.1.1. Materials;123
11.2.2.1.2;2.2.1.2. Method;123
11.2.2.2;2.2.2. Measure interactions of untagged proteins or inhibitors by competition in the receptor-Fc and Flag-ligand ELISA;126
11.2.2.2.1;2.2.2.1. Method;126
11.2.3;2.3. The measure of ligand-receptor interactions by immunoprecipitation;126
11.2.3.1;2.3.1. Reagents;127
11.2.3.2;2.3.2. Method;127
11.2.3.2.1;2.3.2.1. Precipitations with receptors-Fc;127
11.2.3.2.2;2.3.2.2. Precipitations with heparin-Sepharose;127
11.2.4;2.4. The measure of ligand–receptor interactions by FACS;128
11.2.4.1;2.4.1. Interactions of tagged ligands with GPI-anchored receptors;128
11.2.4.1.1;2.4.1.1. Reagents;128
11.2.4.1.2;2.4.1.2. Method;128
11.2.4.1.2.1;Cell transfection and preparation;128
11.2.4.1.2.2;For Fc-ligands;129
11.2.4.1.2.3;For Flag-ligands using unconjugated anti-Flag (human cells only);129
11.2.4.1.2.4;For Flag-ligands using biotinylated anti-Flag (for any cell);129
11.2.4.1.2.5;For anti-GPI;129
11.2.4.1.2.6;Final steps;129
11.2.4.1.2.7;Additional remarks;130
11.2.4.2;2.4.2. Interactions of tagged receptors with BAFFN-fusion ligands;130
11.2.4.2.1;2.4.2.1. Reagents;130
11.2.4.2.2;2.4.2.2. Method;130
11.2.4.3;2.5. The measure of ligand activity using reporter cells;131
11.2.4.3.1;2.5.1. Generation of receptor: Fas-expressing reporter cell lines;131
11.2.4.3.1.1;2.5.1.1. Reagents;131
11.2.4.3.1.2;2.5.1.2. Method;131
11.2.4.3.1.2.1;Preparation of retrovirus;131
11.2.4.3.2;2.5.2. Monitoring ligand activity by apoptosis induction in reporter cell lines;134
11.2.4.3.2.1;2.5.2.1. Reagents;134
11.2.4.3.2.2;2.5.2.2. Method;134
11.2.4.3.3;2.5.3. Monitoring inhibitors or activators of ligands and receptors in reporter cell lines;135
11.2.4.3.4;2.5.4. Monitoring ligand activity with NF-.B reporter cells;135
11.2.4.3.4.1;2.5.4.1. Reagents;135
11.2.4.3.4.2;2.5.4.2. Method;136
11.2.4.4;2.6. The measure of ligand-independent receptor interactions by Förster resonance energy transfer;136
11.2.4.4.1;2.6.1. Method;138
11.3;3. Conclusions;138
11.4;Acknowledgments;139
11.5;References;139
12;Chapter Six: Necrotic Cell Death in Caenorhabditis elegans;142
12.1;1. Introduction;143
12.1.1;1.1. Characteristics of necrotic cells;143
12.1.2;1.2. Caenorhabditis elegans as a model to study necrosis;144
12.1.3;1.3. The apoptotic machinery in C. elegans;145
12.2;2. Necrotic Cell Death Paradigms During C. elegans Development;145
12.2.1;2.1. Death of the linker cell;145
12.2.2;2.2. Death of mis-specified uterine-vulval (uv1) cells;146
12.3;3. Nondevelopmental Necrotic Death;149
12.3.1;3.1. Cell death induced by ionic imbalance;149
12.3.1.1;3.1.1. Degenerins;149
12.3.1.2;3.1.2. Other ion channels;150
12.3.2;3.2. Heat-induced necrotic death;152
12.3.3;3.3. Bacterial infection-induced necrosis;154
12.3.4;3.4. Hypo-osmotic shock-induced cell death;154
12.4;4. Execution of Necrosis;155
12.5;5. C. elegans as a Model for Human Diseases Entailing Necrosis;157
12.5.1;5.1. Hypoxia;157
12.5.2;5.2. Parkinson´s disease;158
12.5.3;5.3. Tau toxicity: Modeling Alzheimer´s disease in C. elegans;160
12.6;6. Concluding Remarks;162
12.7;Acknowledgments;164
12.8;References;164
13;Chapter Seven: Noncanonical Cell Death in the Nematode Caenorhabditis elegans;172
13.1;Highlights;173
13.2;1. Introduction;173
13.3;2. Pathological Cell Death Induced by Genome Lesions and Environmental Stress;174
13.3.1;2.1. Ion channel mutations;174
13.3.2;2.2. NAD metabolism defects;177
13.3.3;2.3. Cell differentiation mutations;178
13.3.4;2.4. lin-24/lin-33 mutants;179
13.3.5;2.5. A latent apoptotic pathway in Pn.p cells?;180
13.3.6;2.6. Cell shedding in caspase mutants;182
13.4;3. Developmental Cell Deaths That Do not Follow the Canonical Apoptotic Pathway;183
13.4.1;3.1. Germline cell death;183
13.4.2;3.2. Tail-spike cell death;184
13.4.3;3.3. Sex-specific death of CEM neurons;185
13.4.4;3.4. The use of alternate caspases in dying cells;186
13.5;4. Nonapoptotic, Caspase-Independent Linker Cell Death;187
13.6;5. Conclusion;190
13.7;Acknowledgments;190
13.8;References;190
14;Chapter Eight: Autophagy and Cell Death in the Fly;196
14.1;1. Introduction;197
14.1.1;1.1. Drosophila as a biological system for studying autophagic cell death;197
14.1.2;1.2. Genetic approaches to study autophagic cell death in Drosophila;198
14.2;2. Materials and Methods;199
14.2.1;2.1. Fly food;199
14.2.2;2.2. Staging of animals;200
14.2.3;2.3. Histology;200
14.2.3.1;2.3.1. Preparation of samples;201
14.2.3.2;2.3.2. Sectioning;201
14.2.3.3;2.3.3. Staining;202
14.2.4;2.4. Immunochemistry;202
14.2.4.1;2.4.1. Immunoblotting;203
14.2.4.2;2.4.2. Immunofluorescence;203
14.2.5;2.5. Terminal deoxynucleotidyl transferase dUTP nick end labeling;204
14.2.6;2.6. Transmission electron microscopy;205
14.2.7;2.7. Atg8 tagged fluorescence;206
14.3;3. Data Analysis and Interpretation;206
14.3.1;3.1. Interpreting histological sections;206
14.3.2;3.2. Quantifying and interpretation of TUNEL;209
14.3.3;3.3. Quantifying and interpretation of immunochemistry and fluorescently tagged Atg8;209
14.3.3.1;3.3.1. Immunoblotting;209
14.3.3.2;3.3.2. Immunofluorescence and fluorescently tagged Atg8;210
14.3.4;3.4. Quantifying and interpretation of TEM;212
14.3.5;3.5. Caveats to autophagy markers and flux through the pathway;212
14.4;Acknowledgments;213
14.5;References;213
15;Chapter Nine: Structural Studies of Death Receptors;216
15.1;1. Introduction. Signaling by the Tumor Necrosis Receptor Superfamily;217
15.2;2. Outline Death Ligand and DR Domain Structure;219
15.3;3. DR Ectodomain Structure;221
15.3.1;3.1. The TNFR1 ectodomain;224
15.3.2;3.2. The TRAIL-R2 ectodomain;225
15.3.3;3.3. CD95 ectodomain;227
15.4;4. Physiological Complexes of Death Ligands with DRs;228
15.5;5. A Decoy Receptor-Ligand Complex;229
15.6;6. The DR Preligand Association Domain;230
15.7;7. Death Ligand Structure-Activity Relationships;232
15.8;8. Structural Analysis of AntiTNF Agents;234
15.9;9. Structural Analysis of the Blockade of DR Function;235
15.10;10. DR Cytoplasmic Domains;236
15.11;11. DD Structure;237
15.12;12. The DD Superfamily;240
15.13;13. DD Assembly Revealed by the Structure of the PIDDosome Core;241
15.14;14. Structural Characterization of CD95:FADD-DD Complexes;243
15.15;15. Relevance of CD95:FADD-DD Assemblies to Physiological CD95 Signaling;245
15.16;16. Unanswered Questions and Future Prospects;248
15.17;Acknowledgments;249
15.18;References;249
16;Chapter Ten: Use of E2Ubiquitin Conjugates for the Characterization of Ubiquitin Transfer by RING E3 Ligases Such as the ...;258
16.1;1. Introduction;259
16.2;2. Synthesis of E2Ub Conjugates;261
16.2.1;2.1. Purification of the E1;262
16.2.2;2.2. Purification of the E2;262
16.2.3;2.3. Purification of ubiquitin;263
16.2.4;2.4. Formation of disulfide-linked E2ubiquitin conjugate;263
16.2.4.1;Experimental procedure:;263
16.2.5;2.5. Formation of oxyester- and isopeptide-linked conjugate;265
16.2.5.1;Experimental procedure:;265
16.3;3. Characterization of RING-E2Ub Complexes;266
16.3.1;3.1. Binding studies;267
16.3.1.1;3.1.1. Pulldown assays;268
16.3.1.2;3.1.2. Analytical SEC;269
16.3.1.3;3.1.3. SPR and ITC;270
16.3.2;3.2. Discharge assays;271
16.3.3;3.3. Structural studies of RING-E2Ub complexes;272
16.4;4. Conclusion;274
16.5;Acknowledgments;275
16.6;References;275
17;Chapter Eleven: Multidimensional Profiling in the Investigation of Small-Molecule-Induced Cell Death;280
17.1;1. Introduction;281
17.2;2. Gene Expression Profiling;286
17.2.1;2.1. Comparing small-molecule profiles;286
17.2.2;2.2. Protocol for the use of the Connectivity Map database;287
17.2.3;2.3. Applications in cell death;288
17.2.4;2.4. Advantages and limitations in the study of cell death;290
17.3;3. Protein Quantification;292
17.3.1;3.1. Comparing small-molecule profiles;292
17.3.2;3.2. Application in cell death;293
17.3.3;3.3. Advantages and limitations in the study of cell death;294
17.4;4. Gene-Small-Molecule Interactions;295
17.4.1;4.1. Chemical–genetic profiling in yeast;295
17.4.2;4.2. Applications of yeast profiling in cell death;295
17.4.3;4.3. Chemical–genetic profiling in mammalian cells;296
17.4.4;4.4. Advantages and limitations in the study of cell death;296
17.5;5. Small-Molecule Combination Interactions;297
17.5.1;5.1. Profiles based on small-molecule interactions;297
17.5.2;5.2. Advantages and limitations in the study of cell death;298
17.6;6. Cell Line Viability Profiling;299
17.6.1;6.1. NCI60 screen;299
17.6.1.1;6.1.1. Notable applications of the NCI60 screen;299
17.6.2;6.2. Use of molecularly characterized cell lines;301
17.6.2.1;6.2.1. Molecular characterization of NCI60 cell lines;301
17.6.2.2;6.2.2. Expanded cell line databases;301
17.6.3;6.3. Advantages and limitations in the study of cell death;302
17.7;7. Quantitative Imaging;303
17.7.1;7.1. High-content imaging in cell culture;303
17.7.2;7.2. Advantages and limitations of image-based profiles in studying cell death;304
17.8;8. Modulatory Profiling;305
17.8.1;8.1. Design and validation;305
17.8.2;8.2. Modulatory profiling protocol;308
17.8.3;8.3. Application of modulatory profiling to the investigation of ferroptosis;310
17.8.4;8.4. Advantages and limitations in the study of cell death;310
17.9;9. Conclusions;311
17.10;References;312
18;Author Index;318
19;Subject Index;346
20;Color Plate;354

Leseproben

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
1 Corresponding author: email address: [e-mail] 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. Keywords Necrosis Necroptosis Necrosome RIP1 RIP3 MLKL Apoptosis Cell death 1 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 pan-caspase 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 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 zVAD-induced 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-1ß/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; 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 to reduce the requirement for RIPK1 in necroptosis initiation (Moujalled et al., 2013). Interferons were also found to be efficient inducers of necroptosis, utilizing kinase PKR to initiate necrosome formation (Thapa et al., 2013). While RIPK3 clearly plays an indispensable role in necroptosis, RIPK1 appears to serve a critical role as a master regulator controlling multiple cell fate decisions, including cell survival, apoptosis, and necroptosis. RIPK1 is a 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 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...