title: Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.
abstract: Necroptosis is a caspase-independent regulated type of cell death that relies on receptor-interacting protein kinases RIP1 (receptor-interacting protein kinases 1) and RIP3. Tumor necrosis factor-α (TNFα)-stimulated assembly of the TNFR1 (TNF receptor 1)-associated signaling complex leads to the recruitment of RIP1, whose ubiquitination is mediated by the cellular inhibitors of apoptosis (c-IAPs). Translocation of RIP1 to the cytoplasm and association of RIP1 with the necrosome is believed to correlate with deubiquitination of RIP1. However, we found that RIP1 is ubiquitinated with K63 and linear polyubiquitin chains during TNFα, IAP antagonist BV6 and caspase inhibitor zVAD-fmk-induced necroptotic signaling. Furthermore, ubiquitinated RIP1 is associated with the necrosome, and RIP1 ubiquitination in the necrosome coincides with RIP3 phosphorylation. Both cellular IAPs and LUBAC (linear ubiquitin chain assembly complex) modulate RIP1 ubiquitination in IAP antagonist-treated necrotic cells, but they use different mechanisms. c-IAP1 regulates RIP1 recruitment to the necrosome without directly affecting RIP1 ubiquitination, whereas HOIP and HOIL1 mediate linear ubiquitination of RIP1 in the necrosome, but are not essential for necrosome formation. Knockdown of the E3 ligase c-IAP1 decreased RIP1 ubiquitination, necrosome assembly and necroptosis induced by TNFα, BV6 and zVAD-fmk. c-IAP1 deficiency likely decreases necroptotic cell death through the activation of the noncanonical NF-κB pathway and consequent c-IAP2 upregulation. The ability to upregulate c-IAP2 could determine whether c-IAP1 absence will have a positive or negative impact on TNFα-induced necroptotic cell death and necrosome formation. Collectively, these results reveal unexpected complexity of the roles of IAP proteins, IAP antagonists and LUBAC in the regulation of necrosome assembly.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: de Almagro
authors.affiliation: Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: T
authors.initials: T
authors.lastname: Goncharov
authors.affiliation: Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: K
authors.initials: K
authors.lastname: Newton
authors.affiliation: Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: D
authors.initials: D
authors.lastname: Vucic
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D061945:Caspase Inhibitors / Q000494:pharmacology; D045744:Cell Line, Tumor; D019073:HT29 Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007739:L Cells; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009336:Necrosis / Q000473:pathology*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C119724:AGFG1 protein, human; D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D016328:NF-kappa B; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; D047888:Receptors, Tumor Necrosis Factor, Type I; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C494833:BIRC2 protein, human; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; C504507:RBCK1 protein, human; C558338:RNF31 protein, human; D044767:Ubiquitin-Protein Ligases; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases