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Regulation of theTNFR1-signalling complex by LUBAC and associated deubiquitinases

Kupka, S; (2017) Regulation of theTNFR1-signalling complex by LUBAC and associated deubiquitinases. Doctoral thesis , UCL (University College London). Green open access

PhD Thesis Sebastian Kupka.pdf

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TNF is an inflammatory cytokine, vital for innate immune responses but also involved in pathological conditions including rheumatoid arthritis, psoriasis and inflammatory bowel disease. Assembly of the TNFR1-signalling complex (SC) is regulated and post-translational modifications play a crucial part in executing this regulation. The aim of this study was to characterise how LUBAC contributes to TNFR1-SC assembly, NF-B and MAPK pathway activation and prevention of cell death by influencing complex composition and ubiquitination. Using mass spectrometry OTULIN, CYLD and SPATA2 were identified as constitutive interaction partners of LUBAC. Interaction studies revealed that despite constitutive binding of OTULIN, SPATA2 and CYLD with the LUBAC component HOIP, only SPATA2 and CYLD are recruited to SCs. Strikingly, CYLD requires HOIP and SPATA2 for its recruitment to SCs, where CYLD counteracts LUBAC by cleaving ubiquitin chains. Consequently, CYLD enables TNF-induced cell death and supresses NF-B and MAPK activation in NOD2 signalling. Using a newly developed methodology TNFR1 and TRADD were identified as LUBAC substrates and absence of CYLD leads to increased ubiquitination of these proteins. In line with pro-survival functions of linear ubiquitination, depletion of either CYLD or SPATA2 protects cells from TNF-induced necroptosis. OTULIN, on the other hand, antagonises linear ubiquitination and regulates LUBAC in basal conditions by deubiquitinating its subunits and preventing aberrant linear ubiquitination. The protein A20 was found to require linear ubiquitin chains for its recruitment to the TNFR1-SC. A20, although negatively regulating NF-B signalling, is required to prevent TNF-induced cell death by stabilising linear ubiquitination of TNFR1-SC components. In summary, this study identified LUBAC to be central for CYLD and A20 recruitment to SCs and provides an explanation for the opposing role of CYLD and A20 in regulating TNF-mediated cell death despite their overlapping function in suppression of gene activatory pathways.

Type: Thesis (Doctoral)
Title: Regulation of theTNFR1-signalling complex by LUBAC and associated deubiquitinases
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
URI: http://discovery.ucl.ac.uk/id/eprint/1559749
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