Suppression of LUBAC‐mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN

Abstract

Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC) play an important role in NF-jB activation. However, the regulation of linear ubiquitin chain generation by LUBAC is not well characterized. Here, we identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear linkage specificity (OTULIN/Gumby/ FAM105B) and cylindromatosis (CYLD) that can cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP interacts with both CYLD and OTULIN even in unstimulated cells. The interaction of CYLD and OTULIN with HOIP synergistically suppresses LUBACmediated linear polyubiquitination and NF-jB activation. Moreover, introduction of a HOIP mutant unable to bind either deubiquitinase into HOIP-null cells augments the activation of NF-jB by TNF-a stimulation. Thus, the interactions between these two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-a-induced NF-jB activation in cells by fine-tuning the generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling pathway activation by LUBAC. Our observations provide molecular insights into the roles of ligase-deubiquitinase interactions in regulating molecular events resulting from linear ubiquitin conjugation.

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase

“…In contrast, USP10 was able to disassemble the Lys-63-linked chain by cleaving the individual ubiquitin moiety (21). Another well characterized DUB, CYLD, was also found to cleave both the Lys-63 chain and linear chain in vitro (43) and form a complex with OTULIN to suppress linear ubiquitination in cells (44). We found that overexpression of CYLD or OTULIN diminished NEMO linear ubiquiti- nation by genotoxic stress, whereas deletion of TANK had little impact on CYLD/OTULIN-dependent deubiquitination of NEMO (data not shown).…”

Control of p97 function by cofactor binding

“…Three PUB-domain containing proteins have been described so far in humans and all interact with the p97 C-terminal tail: PNGase (peptide N-glycanase) [85,86], which is involved in the deglycosylation of misfolded glycoproteins [87], the UBX protein UBXD1 [68,85,88], and HOIP (HOIL-1-interacting protein) [89][90][91], the catalytic subunit of the E3 ubiquitin ligase LUBAC, which catalyzes the assembly of linear ubiquitin chains [92]. Molecular insights into the interaction of the PUB domain with p97 were revealed by crystal structures of the PNGase and HOIP PUB domains in complex with peptides comprising the five and four, respectively, C-terminal residues of p97 called PIM (PUB Interacting Motif) [86,91] (Fig.…”

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.…”

“…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].…”

“…The observation that Met1-Ub accumulated in OTULIN-deficient cells rather than being disassembled by other cellular DUBs such as CYLD is intriguing because CYLD, like OTULIN, cleaves Met1-Ub and associates with HOIP [5][6][7]. However, OTULIN and CYLD interact with distinct LUBAC complexes in a mutually exclusive manner [7], thus a possible explanation is that CYLD only regulates ubiquitin chains conjugated by a subset of LUBAC complexes.…”