LUBAC PUB domain interactions restrict Met1-linked ubiquitination to prevent murine embryonic lethality and immune pathology

Met1-linked ubiquitin assembled by LUBAC coordinates immune receptor signalling and inflammation. The PUB domain of the LUBAC subunit HOIP interacts with PUB-interacting motif-containing proteins, including the deubiquitinases OTULIN and SPATA2-CYLD. Yet, how these interactions regulate LUBAC remains unclear. Here, we investigate the consequences of ablating PUB-interacting motif interactions by generating mice carrying an N101D mutation in the HOIP PUB domain. The N101D mutation causes embryonic lethality at mid-gestation driven partly by TNF receptor 1 but not solely by the loss of interaction with OTULIN and SPATA2-CYLD. Mutation of the HOIP PUB domain causes Met1-linked autoubiquitination without inhibiting LUBAC activity, leading to superfluous Met1-linked ubiquitination of TNF receptor complex components after TNF, resulting in impaired gene activation and increased cell death. In adult mice, the N101D mutation causes cytokine storm, immune dysregulation, and weight loss. We report the HOIP PUB domain as a critical protein-interaction interface regulating LUBAC function in receptor signalling and immune homoeostasis. LUBAC-dependent Met1-ubiquitination critically modulates TNFR1 and other immune receptor signalling. Here, the function of the PUB domain of HOIP, a subunit of the LUBAC complex, is investigated by introducing an N101D point mutation. The authors report that the PUB domain interactions regulate LUBAC functions by controlling the fidelity of Met1-ubiquitin deposition within receptor complexes and that loss of PUB domain interactions alters TNFR1 signalling, contributing to embryonic lethality, immune pathology, and sensitivity to TNF-mediated cell death.