The binding of OTULIN restrains LUBAC activity to prevent TNF-driven immunopathology

Abstract Met1-linked ubiquitin chains (Met1-Ub), synthesised by the linear ubiquitin chain assembly complex (LUBAC) and disassembled by the deubiquitinase OTULIN, critically regulate inflammatory signalling. Although OTULIN’s activity is essential to prevent TNF-driven autoinflammatory pathology and embryonic lethality, the regulatory significance of its direct interaction with LUBAC remains unclear. Here, we reveal that mice harbouring a point mutation (OTULIN Y56A ) in the OTULIN PUB-interacting motif, which disrupts OTULIN-LUBAC interaction, are viable without spontaneous immunopathology. However, Otulin Y56A/Y56A mice exhibited hypersensitivity to TNF-induced toxicity, which was not prevented by inhibiting RIPK1 kinase-mediated cell death. Mechanistically, disruption of the OTULIN-LUBAC interaction led to Met1-linked autoubiquitination, which enhanced LUBAC’s activity and increased Met1-Ub accumulation at the TNF receptor signalling complex. This stabilised the signalling complex even after dissociation from TNF, increased NF-κB signalling and, contrary to loss of OTULIN or its activity, protected cells from TNF-induced apoptosis. During systemic Listeria monocytogenes infection, the increased response to TNF in Otulin Y56A/Y56A mice exaggerated pathology without affecting bacterial burden. Collectively, we identify the physical association of OTULIN to LUBAC as a critical brake that restricts LUBAC’s function and Met1-Ub-dependent inflammatory signalling, thereby preserving tissue integrity and promoting disease tolerance during acute immune activation.