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

Met1-linked ubiquitin (Met1-Ub) assembled by LUBAC crucially regulates immune receptor signalling. However, the regulation of LUBAC’s function remains widely debated. Here, we reveal that the interaction between LUBAC and PUB-interacting motif (PIM)-containing proteins, including deubiquitinases OTULIN and SPATA2-CYLD, safeguards the fidelity of Met1-Ub deposition to prevent embryonic lethality and immune pathology. Mutation of the PUB domain in the LUBAC subunit HOIP ablated PIM-interactions and led to excessive Met1-Ub on signalling components after immune receptor stimulation, yet impaired productive signalling and sensitised to TNF-induced cell death. In vivo, ablation of HOIP PIM-interactions caused embryonic lethality at midgestation and led to acute cytokine storm, immune dysregulation, and weight loss when induced in adult mice. Moreover, heterozygous ablation of HOIP PUB PIM-interactions, which did not cause spontaneous pathology, rendered mice remarkably sensitive to TNF-challenge. Thus, the PUB domain of HOIP is a critical protein-interaction interface regulating LUBAC’s function in receptor signalling and immune homeostasis.