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

Met1-linked ubiquitin chains (Met1-Ub), synthesized by the linear ubiquitin chain assembly complex (LUBAC) and disassembled by the deubiquitinase OTULIN, regulate inflammatory signaling and cell death. OTULIN activity prevents tumor necrosis factor (TNF)-driven autoinflammatory pathology and embryonic lethality, but the role of the direct OTULIN-LUBAC interaction remains unclear. Here, we show that mice harboring a point mutation in the OTULIN peptide:N-glycanase/UBA- or UBX-containing proteins (PUB)-interacting motif (OTULIN^Y56A), which disrupts LUBAC binding, are viable without spontaneous pathology but are highly sensitive to TNF-induced toxicity and TNF-mediated immunopathology during Listeria monocytogenes infection. Mechanistically, the OTULIN-LUBAC interaction prevents Met1-linked autoubiquitination, thereby limiting LUBAC activity and Met1-Ub conjugation on non-LUBAC substrates. Disrupting the OTULIN-LUBAC interaction increased Met1-Ub accumulation at the TNF receptor signaling complex, which stabilized the signaling complex, increased nuclear factor-kappa B (NF-κB) signaling, and, unlike loss of OTULIN or its activity, protected from TNF-induced apoptosis. Collectively, we identify the OTULIN-LUBAC interaction as a critical brake restricting LUBAC function and Met1-Ub-dependent signaling.