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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-? (TNF?)-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 TNF?, 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 TNF?, BV6 and zVAD-fmk. c-IAP1 deficiency likely decreases necroptotic cell death through the activation of the noncanonical NF-?B 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 TNF?-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.

SUBMITTER: de Almagro MC

PROVIDER: S-EPMC4669837 | biostudies-other | 2015 Jun

REPOSITORIES: biostudies-other

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Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.

de Almagro M C MC Goncharov T T Newton K K Vucic D D

Cell death & disease 20150625

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 co ...[more]

PMID: 26111062

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Project description:Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a deubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.Abbreviations: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CUL1-FBXL20: cullin 1-F-box and leucine rich repeat protein 20; CUL3-KLHL20: cullin 3-kelch like family member 20; CUL4-AMBRA1: cullin 4-autophagy and beclin 1 regulator 1; CYLD: CYLD lysine 63 deubiquitinase; DAPI: 4',6-diamidino-2-phenylindole; DUB: deubiquitinating enzyme; EBSS: Earle's Balanced Salt Solution; GFP: green fluorescent protein; GST: glutathione S-transferase; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; LUBAC: linear ubiquitin chain assembly complex; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3B; MIM: MIT-interacting motif; mRFP: monomeric red fluorescent protein; NEDD4: NEDD4 E3 ubiquitin protein ligase; NFKB: NF-kappaB complex; OPTN: optineurin; OTULIN: OTU deubiquitinase with linear linkage specificity; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns: phosphatidylinositol; PtdIns3K: class III phosphatidylinositol 3-kinase complex; PtdIns3P: phosphatidylinositol 3-phosphate; RBCK1/HOIL1: RANBP2-type and C3HC4-type zinc finger containing 1; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RIPK1: receptor interacting serine/threonine kinase 1; RNF216: ring finger protein 216; RNF31/HOIP: ring finger protein 31; RT-PCR: reverse transcriptase polymerase chain reaction; S. Typhimurium: Salmonella enterica serovar Typhimurium; SHARPIN: SHANK associated RH domain interactor; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING: stimulator of interferon response cGAMP interactor 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TNF/TNF-alpha: tumor necrosis factor; TNFAIP3/A20: TNF alpha induced protein 3; TRAF6: TNF receptor associated factor 6; TRIM32: tripartite motif containing 32; UBAN: ubiquitin binding in TNIP/ABIN and IKBKG/NEMO proteins; ULK1/2: unc-51 like autophagy activating kinase 1/2; USP: ubiquitin specific peptidase; UVRAG: UV radiation resistance associated; VCPIP1: valosin containing protein interacting protein 1; WIPI2: WD repeat domain, phosphoinositide interacting protein 2; ZBTB16-CUL3-RBX1: zinc finger and BTB domain containing protein 16-cullin 3-ring-box 1; ZRANB1: zinc finger RANBP2-type containing 1.

Dataset Information

Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.

Publications

Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.

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