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Linear ubiquitination prevents inflammation and regulates immune signalling

Abstract

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpincpdm/cpdm) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

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Figure 1: SHARPIN is recruited to the native CD40- and TNF-RSCs.
Figure 2: SHARPIN forms a tripartite complex with HOIP and HOIL-1 that is capable of forming linear ubiquitin chains.
Figure 3: NEMO and RIP1 are linearly ubiquitinated in the native TNF-RSC.
Figure 4: SHARPIN is required for full TNF-, CD40L- and IL-1β-induced activation of NF-κB and JNK and to prevent TNF-induced cell death.
Figure 5: Genetic ablation of TNF rescues the skin phenotype and reduces inflammation in cpdm mice but does not revert the immunological distortion.

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Acknowledgements

We thank M. Miasari for developing the cIAP2 antibody, M. Leverkus for advice on skin histology, J. Lokan for advice on liver histology, C. Rappl, N. Barboza, S. Kupka, D. Heinze, J. Zipprich, J. Corbin, S. Wiegelmann and A. Bankovacki for excellent technical assistance, M. Bolognesi and P. Seneci for SM-164, H. Koerner for Tnf/− mice, and all past and present members of the Walczak and Silke laboratories for continuous stimulating scientific discussions and support. Research in the Walczak lab is supported by grants from Cancer Research UK, AICR, BBSRC (ERASysBio PLUS), Ovarian Cancer Action and the EU Marie Curie Research Training Network ApopTRAIN. Work in the Silke lab is supported by NHMRC grants 541901, 541902 and 602516. Work in the Purcell lab is supported by the NHMRC (Senior Research Fellowships for A.W.P. and a C. J. Martin Overseas Biomedical Fellowship to A.I.W.), and by grants from the NHMRC and ARC. U.W. is funded by HGF/SBCancer. E.R. and T.L.H. are ApopTRAIN fellows and U.N. is supported by the Schweizer Nationalfonds (SNF).

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Contributions

T.L.H. and H.W. conceived the moTAP procedure. B.G., E.R., H.W. and U.W., and T.L.H. and U.W. determined the composition of the CD40- and TNF-RSC, respectively. B.G. performed all other experiments involving CD40L. A.C.S. and C.H.E. cloned and purified all recombinant proteins, determined the molecular interaction between the LUBAC components and performed the in vitro ubiquitination assays. B.G., S.M.C., A.C.S., C.H.E. and E.R. provided moTAP-purified TNF-RSCs for 2D-MRM analysis which was conceived by A.I.W. and H.W., performed by A.I.W., and analysed by A.I.W. and A.W.P.; J.S. and H.W. planned and J.A.R., H.A., U.N., W.W.-L.W., L.G., B.G., S.M.C. and E.R. performed the analyses of cells, tissues and blood samples obtained from all mouse strains used in this study. H.W. and J.S. wrote the manuscript assisted by B.G., S.M.C., A.C.S., C.H.E., E.R. and A.I.W.

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Correspondence to Henning Walczak.

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Competing interests

H.W. is founder, shareholder and scientific advisor of Apogenix GmbH, Heidelberg, Germany. J.S. is a consultant for TetraLogic Pharmaceuticals, Malvern, Pennsylvania, USA.

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Gerlach, B., Cordier, S., Schmukle, A. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011). https://doi.org/10.1038/nature09816

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