SummaryThe linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling.

SummaryThe linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked “linear” Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain specifically. Analysis of LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.