OTULIN Restricts Met1-Linked Ubiquitination to Control Innate Immune Signaling

Abstract

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.

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…RIP2 was later uncovered to be the prime substrate of LUBAC in NOD2 signaling through quantitative proteomics analysis of the components obtained from M1-Ub-affinity purification. 21 Although the UBD of TAB2/3, specifically the NZF domain, preferentially binds to K63-type chains, the UBD in NEMO, referred to as UBAN (ubiquitin binding in ABIN and NEMO), binds significantly more strongly to M1 chains compared with K63 chains. Thus, the synchronous addition of K63-and M1-linked chains on RIP2 may provide an optimal platform to facilitate the TAK1-catalyzed activation of the IKK complex.…”

“…The linear ubiquitin chain-specific DUB OTULIN binds to the catalytic LUBAC subunit HOIP and selectively trims the M1-linked chains from RIP2, thereby attenuating NOD2 signaling. 21 In addition, HOIP also interacts with CYLD, which limits the extension of both K63-and M1-linked chains on RIP2 to restrict NOD2 signaling. 22 …”

A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production

“…RIPK2 plays a central role in the NOD signalling pathway and cells deficient in RIPK2 fail to mount a cytokine response upon NOD stimulation 14,15 . We and others have shown that upon NOD stimulation, RIPK2 is ubiquitylated by inhibitor of apoptosis (IAP) proteins, and this recruits the linear ubiquitination assembly complex (LUBAC), which is essential for downstream signalling [16][17][18] .…”

OTULIN deficiency causes auto-inflammatory syndrome

“…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.…”

“…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.…”