LUBAC-Recruited CYLD and A20 Regulate Gene Activation and Cell Death by Exerting Opposing Effects on Linear Ubiquitin in Signaling Complexes

Abstract

SummaryUbiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1) ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC), at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC’s M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.

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

“…77 Whereas CYLD directly binds LUBAC and removes linear chains, thereby sensitizing cells to TNF-provoked apoptosis, A20 binding to the linear chains via its ZnF7 domain antagonizes their removal, consequently blocking cell death. 78 Dissimilarly, the RIP1-independent apoptotic branch is controlled by the level of cellular FLICE Inhibitory Protein (c-FLIPL), which effectively blocks caspase-8 activation by competitively binding to FADD. The E3 ligase Itch activated by JNK1 phosphorylation acts to provoke apoptosis by directing c-FLIPL for proteasomal destruction.…”

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.…”

“…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. In addition to OTULIN and CYLD, the DUB A20 has also been implicated in Met1-Ub biology.…”