125 results on '"Silke, John"'
Search Results
2. A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.
- Author
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Garnish SE, Martin KR, Kauppi M, Jackson VE, Ambrose R, Eng VV, Chiou S, Meng Y, Frank D, Tovey Crutchfield EC, Patel KM, Jacobsen AV, Atkin-Smith GK, Di Rago L, Doerflinger M, Horne CR, Hall C, Young SN, Cook M, Athanasopoulos V, Vinuesa CG, Lawlor KE, Wicks IP, Ebert G, Ng AP, Slade CA, Pearson JS, Samson AL, Silke J, Murphy JM, and Hildebrand JM
- Subjects
- Humans, Animals, Mice, Phosphorylation, Cell Membrane metabolism, Mutation, Transcription Factors metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Protein Kinases genetics, Protein Kinases metabolism, Apoptosis
- Abstract
Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL
S132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease., (© 2023. Springer Nature Limited.)- Published
- 2023
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3. Apoptotic cell death in disease-Current understanding of the NCCD 2023.
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Vitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, Agostini M, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW, Aqeilan RI, Arama E, Baehrecke EH, Balachandran S, Bano D, Barlev NA, Bartek J, Bazan NG, Becker C, Bernassola F, Bertrand MJM, Bianchi ME, Blagosklonny MV, Blander JM, Blandino G, Blomgren K, Borner C, Bortner CD, Bove P, Boya P, Brenner C, Broz P, Brunner T, Damgaard RB, Calin GA, Campanella M, Candi E, Carbone M, Carmona-Gutierrez D, Cecconi F, Chan FK, Chen GQ, Chen Q, Chen YH, Cheng EH, Chipuk JE, Cidlowski JA, Ciechanover A, Ciliberto G, Conrad M, Cubillos-Ruiz JR, Czabotar PE, D'Angiolella V, Daugaard M, Dawson TM, Dawson VL, De Maria R, De Strooper B, Debatin KM, Deberardinis RJ, Degterev A, Del Sal G, Deshmukh M, Di Virgilio F, Diederich M, Dixon SJ, Dynlacht BD, El-Deiry WS, Elrod JW, Engeland K, Fimia GM, Galassi C, Ganini C, Garcia-Saez AJ, Garg AD, Garrido C, Gavathiotis E, Gerlic M, Ghosh S, Green DR, Greene LA, Gronemeyer H, Häcker G, Hajnóczky G, Hardwick JM, Haupt Y, He S, Heery DM, Hengartner MO, Hetz C, Hildeman DA, Ichijo H, Inoue S, Jäättelä M, Janic A, Joseph B, Jost PJ, Kanneganti TD, Karin M, Kashkar H, Kaufmann T, Kelly GL, Kepp O, Kimchi A, Kitsis RN, Klionsky DJ, Kluck R, Krysko DV, Kulms D, Kumar S, Lavandero S, Lavrik IN, Lemasters JJ, Liccardi G, Linkermann A, Lipton SA, Lockshin RA, López-Otín C, Luedde T, MacFarlane M, Madeo F, Malorni W, Manic G, Mantovani R, Marchi S, Marine JC, Martin SJ, Martinou JC, Mastroberardino PG, Medema JP, Mehlen P, Meier P, Melino G, Melino S, Miao EA, Moll UM, Muñoz-Pinedo C, Murphy DJ, Niklison-Chirou MV, Novelli F, Núñez G, Oberst A, Ofengeim D, Opferman JT, Oren M, Pagano M, Panaretakis T, Pasparakis M, Penninger JM, Pentimalli F, Pereira DM, Pervaiz S, Peter ME, Pinton P, Porta G, Prehn JHM, Puthalakath H, Rabinovich GA, Rajalingam K, Ravichandran KS, Rehm M, Ricci JE, Rizzuto R, Robinson N, Rodrigues CMP, Rotblat B, Rothlin CV, Rubinsztein DC, Rudel T, Rufini A, Ryan KM, Sarosiek KA, Sawa A, Sayan E, Schroder K, Scorrano L, Sesti F, Shao F, Shi Y, Sica GS, Silke J, Simon HU, Sistigu A, Stephanou A, Stockwell BR, Strapazzon F, Strasser A, Sun L, Sun E, Sun Q, Szabadkai G, Tait SWG, Tang D, Tavernarakis N, Troy CM, Turk B, Urbano N, Vandenabeele P, Vanden Berghe T, Vander Heiden MG, Vanderluit JL, Verkhratsky A, Villunger A, von Karstedt S, Voss AK, Vousden KH, Vucic D, Vuri D, Wagner EF, Walczak H, Wallach D, Wang R, Wang Y, Weber A, Wood W, Yamazaki T, Yang HT, Zakeri Z, Zawacka-Pankau JE, Zhang L, Zhang H, Zhivotovsky B, Zhou W, Piacentini M, Kroemer G, and Galluzzi L
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- Animals, Humans, Cell Death, Carcinogenesis, Mammals metabolism, Apoptosis genetics, Caspases genetics, Caspases metabolism
- Abstract
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)
- Published
- 2023
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4. Targeting the Extrinsic Pathway of Hepatocyte Apoptosis Promotes Clearance of Plasmodium Liver Infection.
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Ebert G, Lopaticki S, O'Neill MT, Steel RWJ, Doerflinger M, Rajasekaran P, Yang ASP, Erickson S, Ioannidis L, Arandjelovic P, Mackiewicz L, Allison C, Silke J, Pellegrini M, and Boddey JA
- Subjects
- Administration, Oral, Animals, Biological Availability, Caspase 3 metabolism, Culicidae parasitology, Dipeptides administration & dosage, Dipeptides pharmacology, Hepatocytes drug effects, Immunity drug effects, Indoles administration & dosage, Indoles pharmacology, Inhibitor of Apoptosis Proteins antagonists & inhibitors, Inhibitor of Apoptosis Proteins metabolism, Life Cycle Stages drug effects, Malaria immunology, Plasmodium drug effects, Plasmodium growth & development, Plasmodium metabolism, Protozoan Proteins metabolism, Sporozoites drug effects, Sporozoites physiology, Thiazoles pharmacology, Tumor Necrosis Factor-alpha metabolism, Apoptosis drug effects, Hepatocytes pathology, Liver parasitology, Liver pathology, Malaria parasitology, Malaria pathology
- Abstract
Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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5. Targeting XIAP and PPARγ in Granulosa Cell Tumors Alters Metabolic Signaling.
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Leung DTH, Rainczuk A, Nguyen T, Stephens A, Silke J, Fuller PJ, and Chu S
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- Cell Line, Tumor, Cell Proliferation drug effects, Female, Humans, Proteome analysis, Proteome drug effects, Proteome metabolism, Proteomics, Rosiglitazone pharmacology, Signal Transduction drug effects, Apoptosis drug effects, Granulosa Cell Tumor metabolism, PPAR gamma metabolism, X-Linked Inhibitor of Apoptosis Protein antagonists & inhibitors, X-Linked Inhibitor of Apoptosis Protein metabolism
- Abstract
Ovarian granulosa cell tumors (GCTs) are hormonally active cancers characterized by indolent growth and late, invasive relapse. No therapies have yet proven to be efficacious. We previously reported that the inhibition of the antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) removes transrepression of the pro-proliferative nuclear receptor, peroxisome proliferator-activated receptor (PPAR)-γ, in a GCT-derived cell line, KGN. Both PPARγ and XIAP are overexpressed in human GCT. The inhibition of XIAP with the restoration of PPARγ signaling using a SMAC-mimetic (Compound A (CmpdA)) and rosiglitazone (RGZ)/retinoic acid (RA), respectively, reduced cell proliferation and induced apoptosis in the KGN cells. Utilizing stable isotope labeling with amino acids in cell culture, we identified 32 differentially expressed proteins in the KGN cells following the CmpdA/RGZ/RA-treatment, 22 of which were upregulated by ≥1.5 fold. Of these, stearoyl-CoA desaturase (SCD; 4.5-fold induction) was examined for putative binding sites for PPARγ using in silico screening. Chromatin immunoprecipitation confirmed the direct binding of PPARγ on the promoter region of SCD, with increased binding in the CmpdA/RGZ/RA-treated KGN cells. Because PPARγ plays a pivotal role in lipid and glucose metabolism, the upregulation of proteins associated with metabolic processes such as SCD is consistent with the restoration of PPARγ activity.
- Published
- 2019
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6. The Mitochondrial Apoptotic Effectors BAX/BAK Activate Caspase-3 and -7 to Trigger NLRP3 Inflammasome and Caspase-8 Driven IL-1β Activation.
- Author
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Vince JE, De Nardo D, Gao W, Vince AJ, Hall C, McArthur K, Simpson D, Vijayaraj S, Lindqvist LM, Bouillet P, Rizzacasa MA, Man SM, Silke J, Masters SL, Lessene G, Huang DCS, Gray DHD, Kile BT, Shao F, and Lawlor KE
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- Animals, Caspase 3 metabolism, Caspase 7, Caspase 8 metabolism, Enzyme Activation, Macrophages metabolism, Mice, Protein Aggregates, Proteolysis, Signal Transduction, Apoptosis, Caspases metabolism, Inflammasomes metabolism, Interleukin-1beta metabolism, Mitochondria metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, bcl-2 Homologous Antagonist-Killer Protein metabolism, bcl-2-Associated X Protein metabolism
- Abstract
Intrinsic apoptosis resulting from BAX/BAK-mediated mitochondrial membrane damage is regarded as immunologically silent. We show here that in macrophages, BAX/BAK activation results in inhibitor of apoptosis (IAP) protein degradation to promote caspase-8-mediated activation of IL-1β. Furthermore, BAX/BAK signaling induces a parallel pathway to NLRP3 inflammasome-mediated caspase-1-dependent IL-1β maturation that requires potassium efflux. Remarkably, following BAX/BAK activation, the apoptotic executioner caspases, caspase-3 and -7, act upstream of both caspase-8 and NLRP3-induced IL-1β maturation and secretion. Conversely, the pyroptotic cell death effectors gasdermin D and gasdermin E are not essential for BAX/BAK-induced IL-1β release. These findings highlight that innate immune cells undergoing BAX/BAK-mediated apoptosis have the capacity to generate pro-inflammatory signals and provide an explanation as to why IL-1β activation is often associated with cellular stress, such as during chemotherapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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7. A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.
- Author
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Murai S, Yamaguchi Y, Shirasaki Y, Yamagishi M, Shindo R, Hildebrand JM, Miura R, Nakabayashi O, Totsuka M, Tomida T, Adachi-Akahane S, Uemura S, Silke J, Yagita H, Miura M, and Nakano H
- Subjects
- Animals, Cell Membrane metabolism, Cells, Cultured, Endosomal Sorting Complexes Required for Transport genetics, Endosomal Sorting Complexes Required for Transport metabolism, Gene Silencing, HMGB1 Protein metabolism, Histones metabolism, Humans, Luminescent Proteins genetics, Mice, Molecular Imaging, Necrosis physiopathology, Phosphorylation, Protein Kinases genetics, Protein Transport, Receptor-Interacting Protein Serine-Threonine Kinases genetics, Time Factors, Alarmins metabolism, Apoptosis physiology, Biosensing Techniques, Necrosis metabolism, Protein Kinases metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism
- Abstract
Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.
- Published
- 2018
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8. The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.
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Davies KA, Tanzer MC, Griffin MDW, Mok YF, Young SN, Qin R, Petrie EJ, Czabotar PE, Silke J, and Murphy JM
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- Amino Acid Sequence, Animals, Cell Line, Doxycycline, Humans, Mice, Mutagenesis, Site-Directed, Necrosis, Phosphorylation, Protein Domains, Protein Kinases chemistry, Protein Kinases genetics, Protein Multimerization, Protein Structure, Tertiary, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Scattering, Small Angle, Sequence Alignment, Ultracentrifugation, X-Ray Diffraction, Apoptosis drug effects, Protein Kinases metabolism
- Abstract
The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.
- Published
- 2018
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9. Necroptotic signaling is primed in Mycobacterium tuberculosis-infected macrophages, but its pathophysiological consequence in disease is restricted.
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Stutz MD, Ojaimi S, Allison C, Preston S, Arandjelovic P, Hildebrand JM, Sandow JJ, Webb AI, Silke J, Alexander WS, and Pellegrini M
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- Animals, Humans, Macrophages pathology, Mice, Mice, Knockout, Necrosis, Tuberculosis pathology, Apoptosis, Macrophages metabolism, Macrophages microbiology, Mycobacterium tuberculosis metabolism, Signal Transduction, Tuberculosis metabolism
- Abstract
Mixed lineage kinase domain-like (MLKL)-dependent necroptosis is thought to be implicated in the death of mycobacteria-infected macrophages, reportedly allowing escape and dissemination of the microorganism. Given the consequent interest in developing inhibitors of necroptosis to treat Mycobacterium tuberculosis (Mtb) infection, we used human pharmacologic and murine genetic models to definitively establish the pathophysiological role of necroptosis in Mtb infection. We observed that Mtb infection of macrophages remodeled the intracellular signaling landscape by upregulating MLKL, TNFR1, and ZBP1, whilst downregulating cIAP1, thereby establishing a strong pro-necroptotic milieu. However, blocking necroptosis either by deleting Mlkl or inhibiting RIPK1 had no effect on the survival of infected human or murine macrophages. Consistent with this, MLKL-deficiency or treatment of humanized mice with the RIPK1 inhibitor Nec-1s did not impact on disease outcomes in vivo, with mice displaying lung histopathology and bacterial burdens indistinguishable from controls. Therefore, although the necroptotic pathway is primed by Mtb infection, macrophage necroptosis is ultimately restricted to mitigate disease pathogenesis. We identified cFLIP upregulation that may promote caspase 8-mediated degradation of CYLD, and other necrosome components, as a possible mechanism abrogating Mtb's capacity to coopt necroptotic signaling. Variability in the capacity of these mechanisms to interfere with necroptosis may influence disease severity and could explain the heterogeneity of Mtb infection and disease.
- Published
- 2018
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10. Methods for Studying TNF-Mediated Necroptosis in Cultured Cells.
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Liu Z, Silke J, and Hildebrand JM
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- Animals, Cells, Cultured, Dermis drug effects, Fibroblasts drug effects, Mice, Signal Transduction, Apoptosis, Dermis pathology, Fibroblasts pathology, Necrosis, Protein Kinases metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Necroptosis is a caspase-independent form of programmed cell death that is induced by a variety of different signalling cascades-all culminating in the activation of the pseudokinase mixed lineage kinase domain-like (MLKL). TNF-induced necroptosis is the most intensively studied of these pathways. Here we describe reagents and cell-based techniques that can be used to investigate TNF-mediated necroptosis in the lab.
- Published
- 2018
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11. PD-L1 and IAPs co-operate to protect tumors from cytotoxic lymphocyte-derived TNF.
- Author
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Kearney CJ, Lalaoui N, Freeman AJ, Ramsbottom KM, Silke J, and Oliaro J
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- Animals, Antineoplastic Agents pharmacology, Cell Line, Tumor, Dipeptides pharmacology, Humans, Indoles pharmacology, Lymphocytes drug effects, Lymphocytes metabolism, Mice, Mitochondrial Proteins metabolism, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Apoptosis drug effects, B7-H1 Antigen metabolism, Inhibitor of Apoptosis Proteins metabolism, Tumor Necrosis Factor-alpha metabolism
- Abstract
Smac-mimetics are emerging as promising anti-cancer agents and are being evaluated in clinical trials for a variety of malignancies. Smac-mimetics can induce TNF production from a subset of tumor cells and simultaneously sensitize them to TNF-induced apoptosis. However, TNF derived from other cellular sources, such as cytotoxic lymphocytes (CLs) within the tumor, may also contribute to the anti-tumor activity of SMs. Here, we show that CD8
+ T cells and NK cells potently kill tumor cells in the presence of the SM, birinapant. Enhanced CL killing occurred through TNF secretion upon tumor antigen recognition or NK-activating receptor ligation. Importantly, the perforin/granzyme route to CL-mediated tumor cell killing was dispensable for the efficacy of birinapant, emphasizing the importance of the TNF-mediated apoptosis pathway. Time-lapse microscopy revealed that birinapant sensitized tumor cells to apoptosis as bystanders and to membrane-bound TNF delivered to tumor cells within the immunological synapse. Furthermore, PD-L1 expression on tumor cells suppressed antigen-driven TNF production by CD8+ T cells, which could be antagonized through PD-1 blockade. Importantly, the elevated levels of TNF produced upon PD-1 blockade further enhanced tumor cell killing when combined with birinapant. The combined anti-tumor activity of IAP antagonism and PD-1 blockade occurred independently of perforin-mediated tumor cell death. Taken together, we identify CL-derived TNF as a potent effector of birinapant mediated anti-tumor immunity and opportunity for combination therapy through co-inhibition of immune checkpoints.- Published
- 2017
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12. MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death.
- Author
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Jaco I, Annibaldi A, Lalaoui N, Wilson R, Tenev T, Laurien L, Kim C, Jamal K, Wicky John S, Liccardi G, Chau D, Murphy JM, Brumatti G, Feltham R, Pasparakis M, Silke J, and Meier P
- Subjects
- Animals, Caspase 8 metabolism, Dose-Response Relationship, Drug, Fas-Associated Death Domain Protein metabolism, HT29 Cells, Humans, Intracellular Signaling Peptides and Proteins genetics, MAP Kinase Kinase Kinases metabolism, Mice, Inbred C57BL, Mice, Knockout, Mitogen-Activated Protein Kinase 14 metabolism, Multiprotein Complexes, NF-kappa B metabolism, Necrosis, Phosphorylation, Protein Serine-Threonine Kinases genetics, RNA Interference, Receptor-Interacting Protein Serine-Threonine Kinases genetics, Signal Transduction drug effects, Transfection, Apoptosis drug effects, Intracellular Signaling Peptides and Proteins metabolism, Protein Serine-Threonine Kinases metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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13. Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways.
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Tanzer MC, Khan N, Rickard JA, Etemadi N, Lalaoui N, Spall SK, Hildebrand JM, Segal D, Miasari M, Chau D, Wong WL, McKinlay M, Chunduru SK, Benetatos CA, Condon SM, Vince JE, Herold MJ, and Silke J
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- Animals, CRISPR-Cas Systems genetics, Caspase 10 chemistry, Caspase 10 genetics, Caspase 8 chemistry, Caspase 8 genetics, Caspase 8 metabolism, Caspase Inhibitors pharmacology, Cell Line, Cytokine TWEAK pharmacology, Drug Synergism, HT29 Cells, Humans, Inhibitor of Apoptosis Proteins antagonists & inhibitors, Interferon-gamma genetics, Interferon-gamma metabolism, Mice, Mice, Knockout, Pentanoic Acids pharmacology, Protein Kinases deficiency, Protein Kinases metabolism, Receptor-Interacting Protein Serine-Threonine Kinases deficiency, Receptor-Interacting Protein Serine-Threonine Kinases genetics, Receptors, Tumor Necrosis Factor, Type I deficiency, Receptors, Tumor Necrosis Factor, Type I genetics, Receptors, Tumor Necrosis Factor, Type I metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, Recombinant Proteins pharmacology, Apoptosis drug effects, Caspase 10 metabolism, Inhibitor of Apoptosis Proteins metabolism, Interferon-gamma pharmacology, Receptor-Interacting Protein Serine-Threonine Kinases metabolism
- Abstract
Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.
- Published
- 2017
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14. Inhibitor of Apoptosis Protein-1 Regulates Tumor Necrosis Factor-Mediated Destruction of Intestinal Epithelial Cells.
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Grabinger T, Bode KJ, Demgenski J, Seitz C, Delgado ME, Kostadinova F, Reinhold C, Etemadi N, Wilhelm S, Schweinlin M, Hänggi K, Knop J, Hauck C, Walles H, Silke J, Wajant H, Nachbur U, W Wei-Lynn W, and Brunner T
- Subjects
- Animals, Baculoviral IAP Repeat-Containing 3 Protein, Cell Death drug effects, Cell Line, Tumor, Cytokine TWEAK, Humans, Intestinal Mucosa cytology, Intestinal Mucosa drug effects, Liver drug effects, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, Organoids, Receptors, Tumor Necrosis Factor, Type I genetics, Receptors, Tumor Necrosis Factor, Type II genetics, Thiazoles pharmacology, Tumor Necrosis Factor-alpha pharmacology, Tumor Necrosis Factors pharmacology, Apoptosis drug effects, Apoptosis genetics, Epithelial Cells drug effects, Inhibitor of Apoptosis Proteins genetics, Inhibitor of Apoptosis Proteins metabolism, Tumor Necrosis Factor-alpha metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism
- Abstract
Background and Aims: Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids., Methods: RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice., Results: YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1., Conclusions: In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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15. EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.
- Author
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Pearson JS, Giogha C, Mühlen S, Nachbur U, Pham CL, Zhang Y, Hildebrand JM, Oates CV, Lung TW, Ingle D, Dagley LF, Bankovacki A, Petrie EJ, Schroeder GN, Crepin VF, Frankel G, Masters SL, Vince J, Murphy JM, Sunde M, Webb AI, Silke J, and Hartland EL
- Subjects
- Animals, Cell Death, Citrobacter rodentium pathogenicity, Cysteine Proteases metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Enteropathogenic Escherichia coli enzymology, Enteropathogenic Escherichia coli metabolism, Escherichia coli Proteins genetics, HEK293 Cells, Humans, Lipopolysaccharides pharmacology, Mice, Phosphorylation, Receptor-Interacting Protein Serine-Threonine Kinases genetics, Signal Transduction drug effects, Tumor Necrosis Factor-alpha metabolism, Type III Secretion Systems, Apoptosis, Escherichia coli Proteins metabolism, Inflammation, Necrosis, Receptor-Interacting Protein Serine-Threonine Kinases metabolism
- Abstract
Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling
1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.- Published
- 2017
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16. The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis.
- Author
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Alvarez-Diaz S, Dillon CP, Lalaoui N, Tanzer MC, Rodriguez DA, Lin A, Lebois M, Hakem R, Josefsson EC, O'Reilly LA, Silke J, Alexander WS, Green DR, and Strasser A
- Subjects
- Animals, Caspase 8 metabolism, Mice, Mice, Inbred C57BL, Necrosis metabolism, Apoptosis physiology, Autoimmune Diseases metabolism, Cell Death physiology, Fas-Associated Death Domain Protein metabolism, Protein Kinases metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism
- Abstract
The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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17. The intersection of cell death and inflammasome activation.
- Author
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Vince JE and Silke J
- Subjects
- Animals, Caspase 1 metabolism, Caspase 8 metabolism, Humans, Inflammation pathology, Interleukin-18 metabolism, Interleukin-1beta metabolism, Mice, Protein Kinases metabolism, Reactive Oxygen Species metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Apoptosis physiology, Inflammasomes metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Pyroptosis physiology, Signal Transduction physiology
- Abstract
Inflammasomes sense cellular danger to activate the cysteine-aspartic protease caspase-1, which processes precursor interleukin-1β (IL-1β) and IL-18 into their mature bioactive fragments. In addition, activated caspase-1 or the related inflammatory caspase, caspase-11, can cleave gasdermin D to induce a lytic cell death, termed pyroptosis. The intertwining of IL-1β activation and cell death is further highlighted by research showing that the extrinsic apoptotic caspase, caspase-8, may, like caspase-1, directly process IL-1β, activate the NLRP3 inflammasome itself, or bind to inflammasome complexes to induce apoptotic cell death. Similarly, RIPK3- and MLKL-dependent necroptotic signaling can activate the NLRP3 inflammasome to drive IL-1β inflammatory responses in vivo. Here, we review the mechanisms by which cell death signaling activates inflammasomes to initiate IL-1β-driven inflammation, and highlight the clinical relevance of these findings to heritable autoinflammatory diseases. We also discuss whether the act of cell death can be separated from IL-1β secretion and evaluate studies suggesting that several cell death regulatory proteins can directly interact with, and modulate the function of, inflammasome and IL-1β containing protein complexes.
- Published
- 2016
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18. TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1.
- Author
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Etemadi N, Chopin M, Anderton H, Tanzer MC, Rickard JA, Abeysekera W, Hall C, Spall SK, Wang B, Xiong Y, Hla T, Pitson SM, Bonder CS, Wong WW, Ernst M, Smyth GK, Vaux DL, Nutt SL, Nachbur U, and Silke J
- Subjects
- Animals, Disease Models, Animal, Mice, Psoriasis pathology, Signal Transduction, Tumor Necrosis Factor-alpha metabolism, Apoptosis, Inflammation pathology, NF-kappa B metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism, Skin pathology, TNF Receptor-Associated Factor 2 metabolism
- Abstract
TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1.
- Published
- 2015
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19. Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation.
- Author
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Lim EJ, Chin R, Nachbur U, Silke J, Jia Z, Angus PW, and Torresi J
- Subjects
- Adult, Aged, Aged, 80 and over, Animals, Antineoplastic Combined Chemotherapy Protocols pharmacology, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Caspase 3 metabolism, Cell Survival drug effects, Cyclosporine pharmacology, Cyclosporine urine, Drug Therapy, Combination methods, Female, Fluorouracil pharmacology, Fluorouracil therapeutic use, Hepatocytes metabolism, Humans, Immunosuppressive Agents pharmacology, Liver metabolism, Liver Cirrhosis drug therapy, Liver Cirrhosis metabolism, Liver Transplantation methods, Male, Mice, Mice, Inbred C57BL, Middle Aged, Mitomycin pharmacology, Mitomycin therapeutic use, Semustine pharmacology, Semustine therapeutic use, Sirolimus pharmacology, Sirolimus therapeutic use, Tacrolimus pharmacology, Tacrolimus therapeutic use, Apoptosis drug effects, Hepatocytes drug effects, Immunosuppressive Agents therapeutic use, Liver drug effects, Liver Transplantation adverse effects
- Abstract
Introduction: Immunosuppressants are used ubiquitously post-liver transplantation to prevent allograft rejection. However their effects on hepatocytes are unknown. Experimental data from non-liver cells indicate that immunosuppressants may promote cell death thereby driving an inflammatory response that promotes fibrosis and raises concerns that a similar effect may occur within the liver. We evaluated apoptosis within the liver tissue of post-liver transplant patients and correlated these findings with in vitro experiments investigating the effects of immunosuppressants on apoptosis in primary hepatocytes., Methods: Hepatocyte apoptosis was assessed using immunohistochemistry for M30 CytoDEATH and cleaved PARP in human liver tissue. Primary mouse hepatocytes were treated with various combinations of cyclosporine, tacrolimus, sirolimus, or MMF. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved PARP and cleaved caspase 3., Results: Post-liver transplant patients had a 4.9-fold and 1.7-fold increase in M30 CytoDEATH and cleaved PARP compared to normal subjects. Cyclosporine and tacrolimus at therapeutic concentrations did not affect hepatocyte apoptosis, however when they were combined with MMF, cell death was significantly enhanced. Cell viability was reduced by 46% and 41%, cleaved PARP was increased 2.6-fold and 2.2-fold, and cleaved caspase 3 increased 2.2-fold and 1.8-fold following treatment with Cyclosporine/MMF and Tacrolimus/MMF respectively. By contrast, the sirolimus/MMF combination did not significantly reduce hepatocyte viability or promote apoptosis., Conclusion: Commonly used immunosuppressive drug regimens employed after liver transplantation enhance hepatocyte cell death and may thus contribute to the increased liver fibrosis that occurs in a proportion of liver transplant recipients.
- Published
- 2015
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20. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.
- Author
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Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier JM, Dobson RC, Webb AI, Tripaydonis A, Babon JJ, Mulcair MD, Scanlon MJ, Alexander WS, Wilks AF, Czabotar PE, Lessene G, Murphy JM, and Silke J
- Subjects
- Adenosine Triphosphate metabolism, Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Cell Membrane metabolism, Enzyme Activation, Inhibitory Concentration 50, Mice, Mice, Transgenic, Molecular Sequence Data, Mutation, Phosphorylation, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Transport, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Apoptosis, Necrosis, Protein Kinases metabolism
- Abstract
Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.
- Published
- 2014
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21. Hepatitis C virus-induced hepatocyte cell death and protection by inhibition of apoptosis.
- Author
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Lim EJ, El Khobar K, Chin R, Earnest-Silveira L, Angus PW, Bock CT, Nachbur U, Silke J, and Torresi J
- Subjects
- Amino Acid Chloromethyl Ketones metabolism, Animals, Caspases analysis, Cell Survival, Enzyme Inhibitors metabolism, Hepatocytes drug effects, Humans, Imidazoles metabolism, Indoles metabolism, Mice, Mice, Inbred C57BL, Quinolines metabolism, Apoptosis, Hepacivirus physiology, Hepatitis C pathology, Hepatocytes physiology, Hepatocytes virology, Necrosis
- Abstract
Chronic hepatitis C virus (HCV) infection results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for cytokeratin 18 (M30 CytoDEATH) and cleaved poly(ADP-ribose) polymerase (PARP). In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, primary mouse hepatocytes (PMoHs) and primary human hepatocytes (PHHs). Cell viability and apoptosis were studied using crystal violet assays and Western blots probed for cleaved caspase-3 and cleaved PARP, with and without treatment with the pan-caspase inhibitor Q-VD-OPh and necrostatin-1. Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared with HCV-negative patients. rAdHCV infection reduced cell viability compared with uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoHs and PHHs infected with rAdHCV showed significantly increased levels of apoptotic markers compared with uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-OPh and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoHs. Hepatocyte apoptosis is known to be increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes, and this was inhibited by Q-VD-OPh and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis, and provide new insights into the mechanisms of HCV-induced liver injury., (© 2014 The Authors.)
- Published
- 2014
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22. The polycomb repressive complex 2 governs life and death of peripheral T cells.
- Author
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Zhang Y, Kinkel S, Maksimovic J, Bandala-Sanchez E, Tanzer MC, Naselli G, Zhang JG, Zhan Y, Lew AM, Silke J, Oshlack A, Blewitt ME, and Harrison LC
- Subjects
- Animals, Cell Survival immunology, Enhancer of Zeste Homolog 2 Protein, Female, Humans, Interferon-gamma immunology, Interleukin-10 immunology, Listeria monocytogenes immunology, Listeriosis immunology, Listeriosis pathology, Male, Mice, T-Lymphocytes, Helper-Inducer cytology, Apoptosis immunology, Cell Differentiation immunology, Gene Silencing immunology, Polycomb Repressive Complex 2 immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets., (© 2014 by The American Society of Hematology.)
- Published
- 2014
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23. Ars Moriendi; the art of dying well - new insights into the molecular pathways of necroptotic cell death.
- Author
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Murphy JM and Silke J
- Subjects
- Amino Acid Sequence, Animals, Humans, Molecular Sequence Data, Receptor-Interacting Protein Serine-Threonine Kinases chemistry, Apoptosis, Necrosis, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Signal Transduction
- Abstract
When our time comes to die most people would probably opt for a quick, peaceful and painless exit. But the manner and timing are rarely under our direct control. Hence the Ars moriendi, literally, "The Art of Dying", two texts written in Latin around the 15th century that offered advice on how to die well according to the Christian ideals of the time. In contrast, for individual cells, the death process is frequently under their control and several signaling pathways that cause cell death, including apoptosis, pyroptosis and necroptosis, have been described. Furthermore the manner in which cells die can have good or bad consequences for the organism. In this review we will discuss how cells die via the necroptotic signaling pathway, with emphasis on recent structural work and place this work in a biological context by discussing relevant studies with knock-out animals.
- Published
- 2014
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24. IAP family of cell death and signaling regulators.
- Author
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Silke J and Vucic D
- Subjects
- Humans, Inhibitor of Apoptosis Proteins chemistry, Inhibitor of Apoptosis Proteins genetics, NF-kappa B genetics, Protein Structure, Tertiary, Signal Transduction, Ubiquitin-Protein Ligases metabolism, Ubiquitination genetics, X-Linked Inhibitor of Apoptosis Protein chemistry, X-Linked Inhibitor of Apoptosis Protein genetics, Apoptosis genetics, Inhibitor of Apoptosis Proteins metabolism, X-Linked Inhibitor of Apoptosis Protein metabolism
- Abstract
Inhibitor of apoptosis (IAP) proteins interface with, and regulate a large number of, cell signaling pathways. If there is a common theme to these pathways, it is that they are involved in the development of the immune system, immune responses, and unsurprisingly, given their name, cell death. Beyond that it is difficult to discover an underlying logic because sometimes IAPs are required to inhibit or prevent signaling, whereas in other cases they are required for signaling to take place. In whatever role they play, they are recruited into signaling complexes and function as ubiquitin E3 ligases, via their RING domains. This review discusses IAP regulation of signaling pathways and focuses on the mammalian IAPs, XIAP, c-IAP1, and c-IAP2, with a particular emphasis on techniques and methods that were used to uncover their roles. We also provide a perspective on targeting IAP proteins for therapeutic intervention and methods used to define the clinical relevance of IAP proteins., (© 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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25. Lymphotoxin α induces apoptosis, necroptosis and inflammatory signals with the same potency as tumour necrosis factor.
- Author
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Etemadi N, Holien JK, Chau D, Dewson G, Murphy JM, Alexander WS, Parker MW, Silke J, and Nachbur U
- Subjects
- Animals, Blotting, Western, Cell Proliferation drug effects, Cells, Cultured, Embryo, Mammalian cytology, Embryo, Mammalian drug effects, Embryo, Mammalian metabolism, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Flow Cytometry, Immunoprecipitation, Inflammation drug therapy, Inflammation metabolism, Lymphotoxin-alpha chemistry, Lymphotoxin-beta chemistry, Mice, Mice, Knockout, Necrosis, Protein Conformation, Receptors, Tumor Necrosis Factor, Type I chemistry, Receptors, Tumor Necrosis Factor, Type II chemistry, Signal Transduction drug effects, Tumor Necrosis Factor-alpha chemistry, Apoptosis, Inflammation pathology, Lymphotoxin-alpha pharmacology, Lymphotoxin-beta pharmacology, Receptors, Tumor Necrosis Factor, Type I metabolism, Receptors, Tumor Necrosis Factor, Type II metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Both of the TNF superfamily ligands, TNF and LTα, can bind and signal through TNFR1 and TNFR2, yet mice mutant for each have different phenotypes. Part of this difference is because LTα but not TNF can activate Herpes Virus Entry Mediator and also heterotrimerise with LTβ to activate LTβR, which is consistent with the similar phenotypes of the LTα and LTβR deficient mice. However, it has also been reported that the LTα3 homotrimer signals differently than TNF through TNFR1, and has unique roles in initiation and exacerbation of some inflammatory diseases. Our modeling of the TNF/TNFR1 interface compared to the LTα3/TNFR1 structure revealed some differences that could affect signalling by the two ligands. To determine whether there were any functional differences in the ability of TNF and LTα3 to induce TNFR1-dependent apoptosis or necroptosis, and if there were different requirements for cIAPs and Sharpin to transmit the TNFR1 signal, we compared the ability of cells to respond to TNF and LTα3. Contrary to our hypothesis, we were unable to discover differences in signalling by TNFR1 in response to TNF and LTα3. Our results imply that the reasons for the conservation of LTα are most likely due either to differential regulation, the ability to signal through Herpes Virus Entry Mediator or the ability of LTα to form heterotrimers with LTβ., (© 2013 FEBS.)
- Published
- 2013
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26. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.
- Author
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Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, Lewis R, Lalaoui N, Metcalf D, Webb AI, Young SN, Varghese LN, Tannahill GM, Hatchell EC, Majewski IJ, Okamoto T, Dobson RC, Hilton DJ, Babon JJ, Nicola NA, Strasser A, Silke J, and Alexander WS
- Subjects
- Animals, Catalytic Domain, Cell Line, Crystallography, X-Ray, Mice, Mice, Inbred C57BL, Mice, Knockout, Necrosis, Phosphoprotein Phosphatases, Phosphoric Monoester Hydrolases metabolism, Phosphorylation, Protein Kinases chemistry, Protein Kinases genetics, Signal Transduction, Apoptosis, Protein Kinases metabolism, Receptor-Interacting Protein Serine-Threonine Kinases metabolism, Tumor Necrosis Factors metabolism
- Abstract
Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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27. Inhibitor of apoptosis (IAP) proteins-modulators of cell death and inflammation.
- Author
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Silke J and Meier P
- Subjects
- Binding Sites, Cell Survival, Immunity, Innate, Inhibitor of Apoptosis Proteins chemistry, Inhibitor of Apoptosis Proteins metabolism, Models, Molecular, Protein Structure, Tertiary, Signal Transduction, Tumor Necrosis Factors metabolism, Apoptosis physiology, Inflammation metabolism, Inhibitor of Apoptosis Proteins physiology
- Abstract
Misregulated innate immune signaling and cell death form the basis of much human disease pathogenesis. Inhibitor of apoptosis (IAP) protein family members are frequently overexpressed in cancer and contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Recent research into IAP biology has unearthed unexpected roles for this group of proteins. In addition, the advances in our understanding of the molecular mechanisms that IAPs use to regulate cell death and innate immune responses have provided new insights into disease states and suggested novel intervention strategies. Here we review the functions assigned to those IAP proteins that act at the intersection of cell death regulation and inflammatory signaling.
- Published
- 2013
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28. Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment.
- Author
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Geserick P, Hupe M, Moulin M, Wong WW, Feoktistova M, Kellert B, Gollnick H, Silke J, and Leverkus M
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Animals, Apoptosis Regulatory Proteins metabolism, CASP8 and FADD-Like Apoptosis Regulating Protein metabolism, CASP8 and FADD-Like Apoptosis Regulating Protein physiology, Caspase Inhibitors, Cysteine Proteinase Inhibitors pharmacology, Death Domain Receptor Signaling Adaptor Proteins metabolism, Fas Ligand Protein metabolism, Humans, Inhibitor of Apoptosis Proteins metabolism, Mice, Protein Isoforms metabolism, Signal Transduction, fas Receptor metabolism, Apoptosis, GTPase-Activating Proteins metabolism, Inhibitor of Apoptosis Proteins physiology, Nuclear Pore Complex Proteins metabolism, RNA-Binding Proteins metabolism, fas Receptor physiology
- Abstract
A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP(L) and not cFLIP(S) interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.
- Published
- 2009
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29. TRAF2 must bind to cellular inhibitors of apoptosis for tumor necrosis factor (tnf) to efficiently activate nf-{kappa}b and to prevent tnf-induced apoptosis.
- Author
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Vince JE, Pantaki D, Feltham R, Mace PD, Cordier SM, Schmukle AC, Davidson AJ, Callus BA, Wong WW, Gentle IE, Carter H, Lee EF, Walczak H, Day CL, Vaux DL, and Silke J
- Subjects
- Amino Acid Motifs physiology, Animals, Cell Line, Inhibitor of Apoptosis Proteins genetics, Mice, Mice, Knockout, NF-kappa B genetics, Protein Binding physiology, Protein Structure, Tertiary physiology, Receptors, Tumor Necrosis Factor, Type I genetics, TNF Receptor-Associated Factor 2 genetics, Apoptosis physiology, Inhibitor of Apoptosis Proteins metabolism, NF-kappa B metabolism, Receptors, Tumor Necrosis Factor, Type I metabolism, TNF Receptor-Associated Factor 2 metabolism, Tumor Necrosis Factors metabolism
- Abstract
Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.
- Published
- 2009
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30. XIAP discriminates between type I and type II FAS-induced apoptosis.
- Author
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Jost PJ, Grabow S, Gray D, McKenzie MD, Nachbur U, Huang DC, Bouillet P, Thomas HE, Borner C, Silke J, Strasser A, and Kaufmann T
- Subjects
- Animals, BH3 Interacting Domain Death Agonist Protein deficiency, BH3 Interacting Domain Death Agonist Protein genetics, Biomimetic Materials pharmacology, Caspase Inhibitors, Enzyme Activation, Fas Ligand Protein metabolism, Female, Hepatitis metabolism, Hepatitis pathology, Hepatocytes cytology, Hepatocytes drug effects, Hepatocytes metabolism, Male, Mice, Mice, Inbred C57BL, Signal Transduction, Thymus Gland cytology, Thymus Gland drug effects, X-Linked Inhibitor of Apoptosis Protein antagonists & inhibitors, X-Linked Inhibitor of Apoptosis Protein deficiency, X-Linked Inhibitor of Apoptosis Protein genetics, fas Receptor antagonists & inhibitors, fas Receptor immunology, Apoptosis, X-Linked Inhibitor of Apoptosis Protein metabolism, fas Receptor metabolism
- Abstract
FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.
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- 2009
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31. Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim.
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Kaufmann T, Jost PJ, Pellegrini M, Puthalakath H, Gugasyan R, Gerondakis S, Cretney E, Smyth MJ, Silke J, Hakem R, Bouillet P, Mak TW, Dixit VM, and Strasser A
- Subjects
- Animals, Bcl-2-Like Protein 11, Mice, Mice, Inbred C57BL, Mice, Knockout, Tumor Necrosis Factor-alpha metabolism, Apoptosis, Apoptosis Regulatory Proteins metabolism, BH3 Interacting Domain Death Agonist Protein metabolism, Caspase 8 metabolism, Chemical and Drug Induced Liver Injury, Hepatocytes pathology, Membrane Proteins metabolism, Proto-Oncogene Proteins metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.
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- 2009
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32. Triggering of apoptosis by Puma is determined by the threshold set by prosurvival Bcl-2 family proteins.
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Callus BA, Moujallad DM, Silke J, Gerl R, Jabbour AM, Ekert PG, and Vaux DL
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- Animals, Caspase Inhibitors, Cell Line, Cell Survival drug effects, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, Humans, Mice, Myeloid Cell Leukemia Sequence 1 Protein, Proteasome Inhibitors, Protein Binding drug effects, bcl-2 Homologous Antagonist-Killer Protein deficiency, bcl-2 Homologous Antagonist-Killer Protein metabolism, bcl-2-Associated X Protein deficiency, bcl-2-Associated X Protein metabolism, Apoptosis drug effects, Apoptosis Regulatory Proteins metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism
- Abstract
Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF(3) cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF(3) cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation.
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- 2008
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33. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis.
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Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, Benetatos CA, Chunduru SK, Condon SM, McKinlay M, Brink R, Leverkus M, Tergaonkar V, Schneider P, Callus BA, Koentgen F, Vaux DL, and Silke J
- Subjects
- Animals, Apoptosis Regulatory Proteins, Autocrine Communication, Benzoquinones metabolism, Brefeldin A metabolism, Caspase 8 metabolism, Caspase Inhibitors, Cell Line, Enzyme Inhibitors metabolism, Humans, Inhibitor of Apoptosis Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Lactams, Macrocyclic metabolism, Mice, Mitochondrial Proteins metabolism, Molecular Mimicry, NF-kappa B metabolism, Proteasome Endopeptidase Complex metabolism, Protein Synthesis Inhibitors metabolism, Receptors, Tumor Necrosis Factor, Type I metabolism, Serpins metabolism, Signal Transduction physiology, TNF Receptor-Associated Factor 2 genetics, TNF Receptor-Associated Factor 2 metabolism, Viral Proteins metabolism, Apoptosis physiology, Inhibitor of Apoptosis Proteins antagonists & inhibitors, Inhibitor of Apoptosis Proteins metabolism, Tumor Necrosis Factor-alpha metabolism
- Abstract
XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.
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- 2007
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34. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die.
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Ekert PG, Read SH, Silke J, Marsden VS, Kaufmann H, Hawkins CJ, Gerl R, Kumar S, and Vaux DL
- Subjects
- Animals, Apoptotic Protease-Activating Factor 1, Caspase 9, Caspases deficiency, Caspases genetics, Cell Line, Cell Survival drug effects, Cytochromes c analysis, Flow Cytometry, Gene Deletion, Interleukin-3 pharmacology, Mice, Mice, Knockout, Proteins genetics, Tumor Cells, Cultured, Apoptosis physiology, Caspases physiology, Cell Survival physiology, Proteins physiology
- Abstract
Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3-dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.
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- 2004
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35. Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis.
- Author
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Silke J, Kratina T, Ekert PG, Pakusch M, and Vaux DL
- Subjects
- Animals, Apoptosis Regulatory Proteins, Binding Sites, Carrier Proteins genetics, Cell Line, Drosophila Proteins genetics, Gene Expression, Humans, Intracellular Signaling Peptides and Proteins, Mitochondrial Proteins genetics, Mutation, Neuropeptides genetics, Proteins chemistry, Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Transfection, X-Linked Inhibitor of Apoptosis Protein, Apoptosis physiology, Carrier Proteins metabolism, Drosophila Proteins metabolism, Mitochondrial Proteins metabolism, Neuropeptides metabolism, Proteins metabolism, Ubiquitin metabolism
- Abstract
Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells.
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- 2004
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36. Programmed cell death: Superman meets Dr Death.
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Meier P and Silke J
- Subjects
- Animals, Autophagy physiology, Caspases metabolism, Humans, Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Tumor Suppressor Protein p53 metabolism, X-Linked Inhibitor of Apoptosis Protein, Apoptosis physiology, Mitochondria enzymology, Signal Transduction physiology
- Abstract
This year's Cold Spring Harbor meeting on programmed cell death (September 17-21, 2003), organised by Craig Thompson and Junying Yuan, was proof that the 'golden age' of research in this field is far from over. There was a flurry of fascinating insights into the regulation of diverse apoptotic pathways and unexpected non-apoptotic roles for some of the key apoptotic regulators and effectors. In addition to their role in cell death, components of the apoptotic molecular machinery are now known to also function in a variety of essential cellular processes, such as regulating glucose homeostasis, lipid metabolism, cell proliferation and differentiation.
- Published
- 2003
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37. Mammalian mitochondrial IAP binding proteins.
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Vaux DL and Silke J
- Subjects
- Animals, Apoptosis Regulatory Proteins, High-Temperature Requirement A Serine Peptidase 2, Inhibitor of Apoptosis Proteins, Intracellular Signaling Peptides and Proteins, Mammals metabolism, Models, Biological, Neoplasms drug therapy, Neoplasms pathology, Serine Endopeptidases metabolism, X-Linked Inhibitor of Apoptosis Protein, Apoptosis, Carrier Proteins metabolism, Mitochondrial Proteins metabolism, Proteins metabolism
- Abstract
Four mitochondrial proteins have been identified that immunoprecipitate with the mammalian inhibitor of apoptosis (IAP) protein XIAP. Each of them interacts via a processed amino terminus that resembles those of the insect pro-apoptotic IAP binding proteins Grim, HID, Reaper, and Sickle. Two, Diablo/Smac and HrtA2/Omi, have been extensively characterized. Both Diablo and HtrA2 can bind to IAPs and promote apoptosis when over-expressed in transfected cells, but unlike the insect IAP antagonists, to date there is scant evidence that they are important regulators of apoptosis in more physiological circumstances.
- Published
- 2003
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38. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome.
- Author
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Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Bouillet P, Adams JM, and Strasser A
- Subjects
- Animals, Apoptotic Protease-Activating Factor 1, B-Lymphocytes cytology, Caspase 9, Cells, Cultured, Enzyme Activation, Hematopoiesis physiology, Mice, Mice, Inbred C57BL, T-Lymphocytes cytology, Apoptosis, Caspases metabolism, Cytochrome c Group metabolism, Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism
- Abstract
Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.
- Published
- 2002
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39. The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3- and caspase 9-interacting sites.
- Author
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Silke J, Hawkins CJ, Ekert PG, Chew J, Day CL, Pakusch M, Verhagen AM, and Vaux DL
- Subjects
- Apoptosis Regulatory Proteins, Binding Sites genetics, Carrier Proteins metabolism, Caspase 3, Caspase 9, Cells, Cultured, Humans, Intracellular Signaling Peptides and Proteins, Mitochondrial Proteins metabolism, Mutagenesis physiology, Point Mutation, X-Linked Inhibitor of Apoptosis Protein, Zinc Fingers genetics, Apoptosis physiology, Caspases metabolism, Proteins genetics, Proteins metabolism
- Abstract
The X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. partial differential RING point mutants of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation.
- Published
- 2002
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40. HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins.
- Author
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Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C, Moritz RL, Simpson RJ, and Vaux DL
- Subjects
- Amino Acid Sequence, Binding Sites, Caspase 3, Caspase Inhibitors, Chromosomal Proteins, Non-Histone metabolism, Cytosol enzymology, High-Temperature Requirement A Serine Peptidase 2, Humans, Inhibitor of Apoptosis Proteins, Mitochondria enzymology, Mitochondrial Proteins, Molecular Sequence Data, Neoplasm Proteins, Proteins chemistry, Serine Endopeptidases chemistry, Serine Endopeptidases radiation effects, Survivin, Ultraviolet Rays, X-Linked Inhibitor of Apoptosis Protein, Apoptosis, Microtubule-Associated Proteins, Proteins antagonists & inhibitors, Serine Endopeptidases physiology
- Abstract
Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.
- Published
- 2002
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41. Apoptotic cell death in disease—Current understanding of the NCCD 2023
- Author
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Vitale, Ilio, Pietrocola, Federico, Guilbaud, Emma, Aaronson, Stuart A, Abrams, John M, Adam, Dieter, Agostini, Massimiliano, Agostinis, Patrizia, Alnemri, Emad S, Altucci, Lucia, Amelio, Ivano, Andrews, David W, Aqeilan, Rami I, Arama, Eli, Baehrecke, Eric H, Balachandran, Siddharth, Bano, Daniele, Barlev, Nickolai A, Bartek, Jiri, Bazan, Nicolas G, Becker, Christoph, Bernassola, Francesca, Bertrand, Mathieu J M, Bianchi, Marco E, Blagosklonny, Mikhail V, Blander, J Magarian, Blandino, Giovanni, Blomgren, Klas, Borner, Christoph, Bortner, Carl D, Bove, Pierluigi, Boya, Patricia, Brenner, Catherine, Broz, Petr, Brunner, Thomas, Damgaard, Rune Busk, Calin, George A, Campanella, Michelangelo, Candi, Eleonora, Carbone, Michele, Carmona-Gutierrez, Didac, Cecconi, Francesco, Chan, Francis K-M, Chen, Guo-Qiang, Chen, Quan, Chen, Youhai H, Cheng, Emily H, Chipuk, Jerry E, Cidlowski, John A, Ciechanover, Aaron, Ciliberto, Gennaro, Conrad, Marcus, Cubillos-Ruiz, Juan R, Czabotar, Peter E, D'Angiolella, Vincenzo, Daugaard, Mads, Dawson, Ted M, Dawson, Valina L, De Maria, Ruggero, De Strooper, Bart, Debatin, Klaus-Michael, Deberardinis, Ralph J, Degterev, Alexei, Del Sal, Giannino, Deshmukh, Mohanish, Di Virgilio, Francesco, Diederich, Marc, Dixon, Scott J, Dynlacht, Brian D, El-Deiry, Wafik S, Elrod, John W, Engeland, Kurt, Fimia, Gian Maria, Galassi, Claudia, Ganini, Carlo, Garcia-Saez, Ana J, Garg, Abhishek D, Garrido, Carmen, Gavathiotis, Evripidis, Gerlic, Motti, Ghosh, Sourav, Green, Douglas R, Greene, Lloyd A, Gronemeyer, Hinrich, Häcker, Georg, Hajnóczky, György, Hardwick, J Marie, Haupt, Ygal, He, Sudan, Heery, David M, Hengartner, Michael O, Hetz, Claudio, Hildeman, David A, Ichijo, Hidenori, Inoue, Satoshi, Jäättelä, Marja, Janic, Ana, Joseph, Bertrand, Jost, Philipp J, Kanneganti, Thirumala-Devi, Karin, Michael, Kashkar, Hamid, Kaufmann, Thomas, Kelly, Gemma L, Kepp, Oliver, Kimchi, Adi, Kitsis, Richard N, Klionsky, Daniel J, Kluck, Ruth, Krysko, Dmitri V, Kulms, Dagmar, Kumar, Sharad, Lavandero, Sergio, Lavrik, Inna N, Lemasters, John J, Liccardi, Gianmaria, Linkermann, Andreas, Lipton, Stuart A, Lockshin, Richard A, López-Otín, Carlos, Luedde, Tom, MacFarlane, Marion, Madeo, Frank, Malorni, Walter, Manic, Gwenola, Mantovani, Roberto, Marchi, Saverio, Marine, Jean-Christophe, Martin, Seamus J, Martinou, Jean-Claude, Mastroberardino, Pier G, Medema, Jan Paul, Mehlen, Patrick, Meier, Pascal, Melino, Gerry, Melino, Sonia, Miao, Edward A, Moll, Ute M, Muñoz-Pinedo, Cristina, Murphy, Daniel J, Niklison-Chirou, Maria Victoria, Novelli, Flavia, Núñez, Gabriel, Oberst, Andrew, Ofengeim, Dimitry, Opferman, Joseph T, Oren, Moshe, Pagano, Michele, Panaretakis, Theocharis, Pasparakis, Manolis, Penninger, Josef M, Pentimalli, Francesca, Pereira, David M, Pervaiz, Shazib, Peter, Marcus E, Pinton, Paolo, Porta, Giovanni, Prehn, Jochen H M, Puthalakath, Hamsa, Rabinovich, Gabriel A, Rajalingam, Krishnaraj, Ravichandran, Kodi S, Rehm, Markus, Ricci, Jean-Ehrland, Rizzuto, Rosario, Robinson, Nirmal, Rodrigues, Cecilia M P, Rotblat, Barak, Rothlin, Carla V, Rubinsztein, David C, Rudel, Thomas, Rufini, Alessandro, Ryan, Kevin M, Sarosiek, Kristopher A, Sawa, Akira, Sayan, Emre, Schroder, Kate, Scorrano, Luca, Sesti, Federico, Shao, Feng, Shi, Yufang, Sica, Giuseppe S, Silke, John, Simon, Hans-Uwe, Sistigu, Antonella, Stephanou, Anastasis, Stockwell, Brent R, Strapazzon, Flavie, Strasser, Andreas, Sun, Liming, Sun, Erwei, Sun, Qiang, Szabadkai, Gyorgy, Tait, Stephen W G, Tang, Daolin, Tavernarakis, Nektarios, Troy, Carol M, Turk, Boris, Urbano, Nicoletta, Vandenabeele, Peter, Vanden Berghe, Tom, Vander Heiden, Matthew G, Vanderluit, Jacqueline L, Verkhratsky, Alexei, Villunger, Andreas, von Karstedt, Silvia, Voss, Anne K, Vousden, Karen H, Vucic, Domagoj, Vuri, Daniela, Wagner, Erwin F, Walczak, Henning, Wallach, David, Wang, Ruoning, Wang, Ying, Weber, Achim, Wood, Will, Yamazaki, Takahiro, Yang, Huang-Tian, Zakeri, Zahra, Zawacka-Pankau, Joanna E, Zhang, Lin, Zhang, Haibing, Zhivotovsky, Boris, Zhou, Wenzhao, Piacentini, Mauro, Kroemer, Guido, Galluzzi, Lorenzo, Vitale, Ilio, Pietrocola, Federico, Guilbaud, Emma, Aaronson, Stuart A, Kumar, Sharad, Galluzzi, Lorenzo, Associazione Italiana per la Ricerca sul Cancro, Italian Institute for Genomic Medicine, Compagnia di San Paolo, Aaronson, Stuart A., Dieter, Adam, Agostini, Massimiliano, Agostinis, Patrizia, Alnemri, Emad S., Altucci, Lucia, Amelio, Ivano, Andrews, David W., Aqeilan, Rami I., Arama, Eli, Balachandran, Siddharth, Bano, Daniele, Bartek, Jiri, Bazan, Nicolas G., Bernassola, Francesca, Bertrand, Mathieu J. M., Bianchi, Marco Emilio, Blander, J. Magarian, Blandino, Giovanni, Blomgren, Klas, Bortner, Carl D., Bove, Pierluigi, Boya, Patricia, Broz, Petr, Damgaard, Rune Busk, Calin, George A., Campanella, Michelangelo, Candi, Eleonora, Carbone, Michele, Carmona-Gutierrez, Didac, Cecconi, Francesco, Chen, Guo‑Qiang, Cheng, Emily H., Chipuk, Jerry E., Cidlowski, John A., Ciechanover, Aaron, Ciliberto, Gennaro, Conrad, Marcus, Czabotar, Peter E., D’Angiolella, Vincenzo, Daugaard, Mads, Dawson, Valina L., De Maria, Ruggero, Debatin, Klaus-Michael, Deberardinis, Ralph J., Degterev, Alexei, Del Sal, Giannino, Deshmukh, Mohanish, Di Virgilio, Francesco, Diederich, Marc, Dixon, Scott J., El-Deiry, Wafik S., Elrod, John W., Engeland, Kurt, Fimia, Gian María, Ganini, Carlo, García-Sáez, Ana J., Garg, Abhishek D., Garrido, Carmen, Gavathiotis, Evripidis, Ghosh, Sourav, Green, Douglas R., Gronemeyer, Hinrich, Häcker, Georg, Hajnóczky, György, Hardwick, J. Marie, Haupt, Ygal, He, Sudan, Heery, David M., Hengartner, Michael O., Hetz, Claudio, Hildeman, David A., Ichijo, Hidenori, Jäättelä, Marja, Janic, Ana, Joseph, Bertrand, Jost, Philipp J., Kanneganti, Thirumala-Devi, Karin, Michael, Kashkar, Hamid, Kaufmann, Thomas, Kelly, Gemma L., Kepp, Oliver, Kimchi, Adi, Klionsky, Daniel J., Kluck, Ruth, Krysko, Dmitri V., Kulms, Dagmar, Lavandero, Sergio, Lavrik, Inna N., Liccardi, Gianmaria, Linkermann, Andreas, Lipton, Stuart A., Lockshin, Richard A., López-Otín, Carlos, Luedde, Tom, MacFarlane, Marion, Madeo, Frank, Malorni, Walter, Manic, Gwenola, Marchi, Saverio, Marine, Jean-Christophe, Martin, Seamus J., Martinou, Jean-Claude, Mastroberardino, Pier G., Medema, Jan Paul, Mehlen, Patrick, Meier, Pascal, Melino, Gerry, Melino, Sonia, Miao, Edward A., Moll, Ute M., Muñoz-Pinedo, Cristina, Murphy, Daniel J., Niklison-Chirou, Maria Victoria, Novelli, Flavia, Oberst, Andrew, Ofengeim, Dimitry, Opferman, Joseph T., Oren, Moshe, Pagano, Michele, Panaretakis, Theocharis, Pasparakis, Manolis, Penninger, Josef M., Pentimalli, Francesca, Pereira, David M., Pervaiz, Shazib, Peter, Marcus E., Pinton, Paolo, Porta, Giovanni, Puthalakath, Hamsa, Rabinovich, Gabriel A., Rajalingam, Krishnaraj, Ravinchandran, Kodi S., Rehm, Markus, Ricci, Jean-Ehrland, Rizzuto, Rosario, Robinson, Nirmal, Rotblat, Barak, Rothlin, Carla V., Rubinsztein, David C., Rufini, Alessandro, Ryan, Kevin M., Sarosiek, Kristopher A., Sawa, Akira, Sayan, Emre, Schroder, Kate, Scorrano, Luca, Sesti, Federico, Shi, Yufang, Sica, Giuseppe, Silke, John, Simon, Hans-Uwe, Sistigu, Antonella, Stockwell, Brent R., Strappazzon, Flavie, Sun, Liming, Sun, Erwei, Szabadkai, G, Tait, Stephen W. G., Tang, Daolin, Tavernarakis, Nektarios, Turk, Boris, Urbano, Nicoletta, Vandenabeele, Peter, Vanden Berghe, Tom, Vander Heiden, Matthew G., Vanderluit, Jacqueline L., Verkhratsky, A., Villunger, Andreas, Von Karstedt, Silvia, Voss, Anne K., Vucic, Domagoj, Vuri, Daniela, Wagner, Erwin F., Walczak, Henning, Wallach, David, Wang, Ruoning, Weber, Achim, Yamazaki, Takahiro, Zakeri, Zahra, Zawacka-Pankau, Joanna E., Zhivotovsky, Boris, Piacentini, Mauro, Kroemer, Guido, Vitale, Ilio [0000-0002-5918-1841], Piacentini, Mauro [0000-0003-2919-1296], Kroemer, Guido [0000-0002-9334-4405], Galluzzi, Lorenzo [0000-0003-2257-8500], Apollo - University of Cambridge Repository, Abrams, John M, Adam, Dieter, Alnemri, Emad S, Andrews, David W, Aqeilan, Rami I, Baehrecke, Eric H, Barlev, Nickolai A, Bazan, Nicolas G, Becker, Christoph, Bertrand, Mathieu J M, Bianchi, Marco E, Blagosklonny, Mikhail V, Blander, J Magarian, Blomgren, Kla, Borner, Christoph, Bortner, Carl D, Brenner, Catherine, Brunner, Thoma, Calin, George A, Chan, Francis K-M, Chen, Guo-Qiang, Chen, Quan, Chen, Youhai H, Cheng, Emily H, Chipuk, Jerry E, Cidlowski, John A, Conrad, Marcu, Cubillos-Ruiz, Juan R, Czabotar, Peter E, D'Angiolella, Vincenzo, Daugaard, Mad, Dawson, Ted M, Dawson, Valina L, De Strooper, Bart, Deberardinis, Ralph J, Dixon, Scott J, Dynlacht, Brian D, El-Deiry, Wafik S, Elrod, John W, Fimia, Gian Maria, Galassi, Claudia, Garcia-Saez, Ana J, Garg, Abhishek D, Gavathiotis, Evripidi, Gerlic, Motti, Green, Douglas R, Greene, Lloyd A, Hardwick, J Marie, Heery, David M, Hengartner, Michael O, Hildeman, David A, Inoue, Satoshi, Jost, Philipp J, Kaufmann, Thoma, Kelly, Gemma L, Kitsis, Richard N, Klionsky, Daniel J, Krysko, Dmitri V, Lavrik, Inna N, Lemasters, John J, Linkermann, Andrea, Lipton, Stuart A, Lockshin, Richard A, López-Otín, Carlo, Macfarlane, Marion, Mantovani, Roberto, Martin, Seamus J, Mastroberardino, Pier G, Miao, Edward A, Moll, Ute M, Murphy, Daniel J, Núñez, Gabriel, Opferman, Joseph T, Panaretakis, Theochari, Pasparakis, Manoli, Penninger, Josef M, Pereira, David M, Peter, Marcus E, Prehn, Jochen H M, Rabinovich, Gabriel A, Ravichandran, Kodi S, Rehm, Marku, Rodrigues, Cecilia M P, Rothlin, Carla V, Rubinsztein, David C, Rudel, Thoma, Ryan, Kevin M, Sarosiek, Kristopher A, Shao, Feng, Sica, Giuseppe S, Stephanou, Anastasi, Stockwell, Brent R, Strapazzon, Flavie, Strasser, Andrea, Sun, Qiang, Szabadkai, Gyorgy, Tait, Stephen W G, Tavernarakis, Nektario, Troy, Carol M, Turk, Bori, Vander Heiden, Matthew G, Vanderluit, Jacqueline L, Verkhratsky, Alexei, Villunger, Andrea, von Karstedt, Silvia, Voss, Anne K, Vousden, Karen H, Wagner, Erwin F, Wang, Ying, Wood, Will, Yang, Huang-Tian, Zawacka-Pankau, Joanna E, Zhang, Lin, Zhang, Haibing, Zhivotovsky, Bori, and Zhou, Wenzhao
- Subjects
Mammals ,genetics [Caspases] ,Cell Death ,Settore BIO/11 ,Carcinogenesis ,Cell death, diseases ,Settore BIO/12 ,metabolism [Mammals] ,Apoptosis ,Cell Biology ,genetic strategies ,regulated cell death (RCD) ,SDG 3 - Good Health and Well-being ,cell loss and tissue damage ,Settore BIO/10 - Biochimica ,Caspases ,Animals ,Humans ,metabolism [Caspases] ,ddc:610 ,Settore BIO/10 ,610 Medizin und Gesundheit ,Molecular Biology ,genetics [Apoptosis] - Abstract
58 p.-5 fig.-7 box., Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., I. Vitale is and has been supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 2017 #20417 and IG 2022 #27685) and by a startup grant from the Italian Institute for Genomic Medicine (Candiolo, Turin, Italy) and Compagnia di San Paolo (Torino, Italy). M. Piacentini, G. Melino, S. Melino, G. Ciliberto are supported by the Ministro dell’Università (Italy) progetto Heal Italia PE6. L. Galluzzi is/has been supported (as a PI unless otherwise indicated) by two Breakthrough Level 2 grants from the US DoD BCRP (#BC180476P1; #BC210945), by a Transformative Breast Cancer Consortium Grant from the US DoD BCRP (#W81XWH2120034, PI: Formenti), by a U54 grant from NIH/NCI (#CA274291, PI: Deasy, Formenti, Weichselbaum), by the 2019 Laura Ziskin Prize in Translational Research (#ZP-6177, PI: Formenti) from the Stand Up to Cancer (SU2C), by a Mantle Cell Lymphoma Research Initiative (MCL-RI, PI: Chen-Kiang) grant from the Leukemia and Lymphoma Society (LLS), by a Rapid Response Grant from the Functional Genomics Initiative (New York, US), by startup funds from the Dept. of Radiation Oncology at Weill Cornell Medicine (New York, US), by industrial collaborations with Lytix Biopharma (Oslo, Norway), Promontory (New York, US) and Onxeo (Paris, France), as well as by donations from Promontory (New York, US), the Luke Heller TECPR2 Foundation (Boston, US), Sotio a.s. (Prague, Czech Republic), Lytix Biopharma (Oslo, Norway), Onxeo (Paris, France), Ricerchiamo (Brescia, Italy), and Noxopharm (Chatswood, Australia). G. Kroemer is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; European Research Council Advanced Investigator Grand “ICD-Cancer”, Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council (ICD-Cancer), European Union Horizon 2020 Projects Oncobiome and Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM)
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- 2023
42. Determination of Cell Survival by RING-Mediated Regulation of Inhibitor of Apoptosis (IAP) Protein Abundance
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Silke, John, Ekert, Paul G., Day, Catherine L., Pakusch, Miha, and Vaux, David L.
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- 2005
43. DIABLO Promotes Apoptosis by Removing MIHA/XIAP from Processed Caspase 9
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Ekert, Paul G., Silke, John, Hawkins, Christine J., Verhagen, Anne M., and Vaux, David L.
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- 2001
44. Inhibitor of Apoptosis Proteins and Caspases
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Yu, Jai Y., Silke, John, Ekert, Paul G., and Srivastava, Rakesh, editor
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- 2007
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45. Caspase‐8‐driven apoptotic and pyroptotic crosstalk causes cell death and IL‐1β release in X‐linked inhibitor of apoptosis (XIAP) deficiency.
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Hughes, Sebastian A, Lin, Meng, Weir, Ashley, Huang, Bing, Xiong, Liya, Chua, Ngee Kiat, Pang, Jiyi, Santavanond, Jascinta P, Tixeira, Rochelle, Doerflinger, Marcel, Deng, Yexuan, Yu, Chien‐Hsiung, Silke, Natasha, Conos, Stephanie A, Frank, Daniel, Simpson, Daniel S, Murphy, James M, Lawlor, Kate E, Pearson, Jaclyn S, and Silke, John
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CELL death ,CROHN'S disease ,APOPTOSIS ,INFLAMMATORY bowel diseases ,MUCOUS membranes ,NLRP3 protein - Abstract
Genetic lesions in X‐linked inhibitor of apoptosis (XIAP) pre‐dispose humans to cell death–associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency–associated inflammatory bowel disease display increased inflammatory IL‐1β maturation as well as cell death–associated caspase‐8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase‐8‐driven cell death and bioactive IL‐1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase‐8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase‐1, ‐3, ‐7, ‐11 and BID), while caspase‐8 can still cause cell death in the absence of both GSDMD and GSDME when caspase‐3 and caspase‐7 are present. Neither caspase‐3 and caspase‐7‐mediated activation of the pannexin‐1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase‐1 and IL‐1β maturation downstream of XIAP inhibition and caspase‐8 activation, even though the pannexin‐1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co‐opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency. Synopsis: XIAP deficiency causes hemophagocytic lymphohistiocytosis and Crohn's disease. Analysis of XIAP‐deficient patients and cellular studies show that apoptotic caspases and GSDMD act redundantly downstream of caspase‐8 to cause excess cell death and IL‐1β release. XIAP deficiency increases cleaved caspase‐8 and GSDMD in mucosal tissue and cellsCrosstalk in apoptosis and pyroptosis sensitises cells to caspase‐8‐driven death and IL‐1β activation upon XIAP inhibitionPannexin‐1 and GSDMD are dispensable for NLRP3 inflammasome formation downstream of caspase‐8Pannexin‐1 cleavage by caspase‐3 and caspase‐7 is required for efficient NLRP3 activation following intrinsic apoptosis [ABSTRACT FROM AUTHOR]
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- 2023
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- View/download PDF
46. Divalent Metal Transporter 1 (DMT1) Regulation by Ndfip 1 Prevents Metal Toxicity in Human Neurons
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Howitt, Jason, Putz, Ulrich, Lackovic, Jenny, Doan, Anh, Dorstyn, Loretta, Cheng, Hong, Yang, Baoli, Chan-Ling, Tailoi, Silke, John, Kumar, Sharad, Tan, Seong-Seng, and Rouault, Tracey A.
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- 2009
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47. TWEAK-FN14 Signaling Induces Lysosomal Degradation of a cIAP1-TRAF2 Complex to Sensitize Tumor Cells to TNFα
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Vince, James E., Chau, Diep, Callus, Bernard, Wong, W. Wei-Lynn, Hawkins, Christine J., Schneider, Pascal, McKinlay, Mark, Benetatos, Christopher A., Condon, Stephen M., Chunduru, Srinivas K., Yeoh, George, Brink, Robert, Vaux, David L., and Silke, John
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- 2008
- Full Text
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48. Oligomerization‐driven MLKL ubiquitylation antagonizes necroptosis.
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Liu, Zikou, Dagley, Laura F, Shield‐Artin, Kristy, Young, Samuel N, Bankovacki, Aleksandra, Wang, Xiangyi, Tang, Michelle, Howitt, Jason, Stafford, Che A, Nachbur, Ueli, Fitzgibbon, Cheree, Garnish, Sarah E, Webb, Andrew I, Komander, David, Murphy, James M, Hildebrand, Joanne M, and Silke, John
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UBIQUITINATION ,CELL death ,APOPTOSIS ,CELL membranes ,BLOOD proteins ,MEMBRANE proteins - Abstract
Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death. SYNOPSIS: RIPK3 phosphorylates the necroptotic effector molecule MLKL leading to its oligomerization and translocation to the plasma membrane to kill cells. MLKL becomes multi mono‐ubiquitylated in an oligomerization dependent manner. Forced de‐ubiquitylation of MLKL increases MLKL's cytotoxic potential and confers RIPK3 and necroptotic stimulus independent activation and cell death. UbiCRest analysis shows that MLKL becomes multi mono‐ubiquitylated contemporaneously with activating phosphorylation and translocation to membranes.Analysis of gain and loss of function MLKL mutants indicates that MLKL oligomerization is required for necroptosis induced ubiquitylation.MLKL‐deubiquitylating enzyme (DUB) fusions kill cells more rapidly than MLKL‐catalytically dead DUB fusions and can induce necroptosis without a necroptotic stimulus, suggesting that ubiquitylation serves as a brake on MLKL's cytotoxic potential. [ABSTRACT FROM AUTHOR]
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- 2021
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49. The Immuno-Modulatory Effects of Inhibitor of Apoptosis Protein Antagonists in Cancer Immunotherapy.
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Michie, Jessica, Hawkins, Edwin D., Silke, John, and Oliaro, Jane
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PROGRAMMED cell death 1 receptors ,CHIMERIC antigen receptors ,DRUG resistance in cancer cells ,MITOCHONDRIAL proteins ,IMMUNOTHERAPY ,CELL death ,APOPTOSIS - Abstract
One of the hallmarks of cancer cells is their ability to evade cell death via apoptosis. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that act to promote cell survival. For this reason, upregulation of IAPs is associated with a number of cancer types as a mechanism of resistance to cell death and chemotherapy. As such, IAPs are considered a promising therapeutic target for cancer treatment, based on the role of IAPs in resistance to apoptosis, tumour progression and poor patient prognosis. The mitochondrial protein smac (second mitochondrial activator of caspases), is an endogenous inhibitor of IAPs, and several small molecule mimetics of smac (smac-mimetics) have been developed in order to antagonise IAPs in cancer cells and restore sensitivity to apoptotic stimuli. However, recent studies have revealed that smac-mimetics have broader effects than was first attributed. It is now understood that they are key regulators of innate immune signalling and have wide reaching immuno-modulatory properties. As such, they are ideal candidates for immunotherapy combinations. Pre-clinically, successful combination therapies incorporating smac-mimetics and oncolytic viruses, as with chimeric antigen receptor (CAR) T cell therapy, have been reported, and clinical trials incorporating smac-mimetics and immune checkpoint blockade are ongoing. Here, the potential of IAP antagonism to enhance immunotherapy strategies for the treatment of cancer will be discussed. [ABSTRACT FROM AUTHOR]
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- 2020
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50. Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2
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Annibaldi, Alessandro, John, Sidonie Wicky, Vanden Berghe, Tom, Swatek, Kirby N, Ruan, Jianbin, Liccardi, Gianmaria, Bianchi, Katiuscia, Elliott, Paul R, Choi, Sze Men, Van Coillie, Samya, Bertin, John, Wu, Hao, Komander, David, Vandenabeele, Peter, Silke, John, and Meier, Pascal
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DOMAINS ,RIPK1 ,NF-KAPPA-B ,TNF ,necroptosis ,Apoptosis ,Protein Serine-Threonine Kinases ,CHAIN ASSEMBLY COMPLEX ,Article ,caspase-8 ,Inhibitor of Apoptosis Proteins ,ACTIVATION ,Mice ,INFLAMMATION ,BINDING ,ubiquitin ,Medicine and Health Sciences ,Animals ,Humans ,Mice, Knockout ,Tumor Necrosis Factor-alpha ,Intracellular Signaling Peptides and Proteins ,NF-kappa B ,Ubiquitination ,Biology and Life Sciences ,MAP Kinase Kinase Kinases ,TNF-ALPHA ,CANCER ,Baculoviral IAP Repeat-Containing 3 Protein ,APOPTOSIS ,I-kappa B Kinase ,Mice, Inbred C57BL ,cell death ,HEK293 Cells ,CELL-DEATH ,inflammation ,Receptor-Interacting Protein Serine-Threonine Kinases ,cIAPs ,SURVIVAL ,Signal Transduction - Abstract
Summary Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1’s cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity., Graphical Abstract, Highlights • Ubiquitylation directly controls RIPK1 kinase activity in TNF signaling • UBA-dependent ubiquitylation of RIPK1 represses its kinase activity and cell death • The UBA contributes to optimal occupancy of ubiquitin-acceptor lysines in RIPK1 • UBA-dependent ubiquitylation of RIPK1 also targets it for proteasomal degradation, Annibaldi et al. show that cIAP-mediated ubiquitylation of RIPK1 kinase suppresses its auto-activation and, in addition, marks it for proteasomal degradation. These results reveal a direct role for ubiquitin in controlling RIPK1 kinase activity and suppressing TNF-mediated cytotoxicity.
- Published
- 2018
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