OTULIN Antagonizes LUBAC Signaling by Specifically Hydrolyzing Met1-Linked Polyubiquitin

Abstract

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.

Biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease

“…S4 B and C). OTULIN cleaves Met1-linked linear polyubiquitin chains from target substrates, such as NEMO (IKKγ), RIPK1, ASC, and TNFR1 to restrict signaling activation and propagation (7,14,15). To investigate the effect of OTULIN mutations on its deubiquitinase function, we cotransfected WT and mutant OTULIN plasmids into HEK293 cells along with plasmids encoding the LUBAC subunits, mono specific-ubiquitin plasmid, and each of the OTULIN substrates NEMO (Fig.…”

“…OTULIN and CYLD are deubiquitinases (DUBs) that cleave Met1-linked chains (6). Although OTULIN functions exclusively as a Met1 deubiquitinase (7,8), CYLD may also hydrolyze Lys63-linked ubiquitin (9). OTULIN is an evolutionarily highly conserved protein, and in mice complete deficiency is embryonically lethal (8).…”

“…Recently, we reported patients with heterozygous germline mutations in TNFAIP3/ A20 (10), which has DUB activity for K63-linked polyubiquitin chains. Both OTULIN and A20 are important gatekeepers of innate immunity (7,11).…”

OTULIN deficiency causes auto-inflammatory syndrome

“…Previous studies have revealed that OTULIN exclusively hydrolyzes Met1-Ub, prevents accumulation of Met1-Ub on LUBAC components under basal conditions, and restricts ubiquitination of LUBAC substrates after receptor stimulation [2][3][4]. However, the contribution of OTULIN to regulation of physiological immune responses had not been investigated.…”

“…Met1-Ub chains are assembled by the linear ubiquitin chain assembly complex (LU-BAC), composed of HOIP, HOIL-1, and SHARPIN [1]. LUBAC function is regulated by the deubiquitinases (DUBs) OTULIN [2-4] and CYLD [5][6][7], which both associate with the catalytic subunit HOIP [5][6][7][8].Previous studies have revealed that OTULIN exclusively hydrolyzes Met1-Ub, prevents accumulation of Met1-Ub on LUBAC components under basal conditions, and restricts ubiquitination of LUBAC substrates after receptor stimulation [2][3][4]. However, the contribution of OTULIN to regulation of physiological immune responses had not been investigated.…”