Supporting the latter scenario, HOIP oligomerizes via the N-terminal part of the protein (Elliott et?al., 2014), and CYLD has been shown to co-immunoprecipitate OTULIN in a LUBAC-dependent manner and vice versa (Takiuchi et?al., 2014). Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually MMV390048 required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation. Graphical Abstract Open in a separate window Introduction Ubiquitin (Ub) chains linked via the N-terminal methionine (Met1) of Ub (Met1-Ub, also termed linear Ub) and lysine 63 (Lys63-Ub) facilitate innate immune signaling initiated by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and nucleotide-oligomerization domain (NOD)-like receptors and cytokine?receptors such as tumor necrosis factor (TNF) receptor 1 (TNFR1) (Fiil and Gyrd-Hansen, 2014, Jiang and Chen, 2012). The linear Ub chain assembly complex (LUBAC), composed of HOIL-1, HOIP, and SHARPIN, is the only known Ub ligase to generate Met1-Ub (Gerlach et?al., 2011, Ikeda et?al., 2011, Kirisako et?al., 2006, Tokunaga et?al., 2011). LUBAC activity is counterbalanced by the Met1-specific deubiquitinase (DUB) OTULIN (Fiil et?al., 2013, Keusekotten et?al., 2013, Rivkin et?al., 2013), which binds to the catalytic subunit HOIP via interactions between the HOIP peptide:N-glycanase/UBA- or UBX-containing proteins (PUB) domain and a PUB-interacting motif (PIM) in OTULIN (Elliott et?al., 2014, Rabbit Polyclonal to ARHGEF5 Schaeffer et?al., 2014). The importance of Met1-Ub in immune signaling is underscored by identification of mutations within the LUBAC-encoding genes in human patients with immunological disease (Boisson et?al., 2012, Boisson et?al., 2015). Lys63-Ub can be generated by Ub ligases that interact with the dimeric E2 complex Ubc13/Uev1a, which exclusively conjugates this linkage (Deng et?al., 2000). Lys63-Ub is particularly important in MyD88-dependent MMV390048 immune-signaling pathways activated MMV390048 by TLRs and interleukin-1 receptors (IL-1R) whereas the role of Lys63-Ub in the NOD-containing protein 2 (NOD2) and TNFR1 pathways is not fully understood (Fiil and Gyrd-Hansen, 2014, Xu et?al., 2009). NOD2 is an intracellular bacteria-sensing PRR that recognizes MDP (muramyl dipeptide) constituents of bacterial peptidoglycan and plays a critical role in gastro-intestinal immunity (Philpott et?al., 2014). Upon stimulation, NOD2 binds receptor-interacting protein kinase 2 (RIPK2, also known as RIP2 or RICK), leading to recruitment of several Ub ligases including the inhibitor of apoptosis (IAP) proteins, cIAP1, cIAP2, and XIAP (Bertrand et?al., 2009, Damgaard et?al., 2012). XIAP is indispensable for NOD2 pathway functionality, where it ubiquitinates RIPK2 to facilitate recruitment of LUBAC (Bauler et?al., 2008, Damgaard MMV390048 et?al., 2012). In turn, LUBAC assembles Met1-Ub on RIPK2 to enable downstream signal transduction (Fiil et?al., 2013). Additionally, TRAF2, ITCH, cIAP1/2, TRAF6, and PELI3 are reported to contribute to the assembly of Lys63-Ub on RIPK2, but their individual contribution to this process and to NOD2 signaling is not fully resolved (Bertrand et?al., 2009, Hasegawa et?al., 2008, Tao et?al., 2009, Watanabe et?al., 2014, Yang et?al., 2013). A central regulatory point for productive innate immune signaling and transcription of nuclear factor-B (NF-B) target genes is the activation of the IKK (IB kinase) complex. IKK activation is dependent on phosphorylation by the TAB/TAK1 complex that interacts with Lys63-Ub and on the conjugation of Met1-Ub by LUBAC, which is bound by MMV390048 the IKK subunit NEMO (also known as IKK; Fiil and Gyrd-Hansen, 2014, Jiang and Chen, 2012). For appropriate and beneficial innate immune signaling, the assembly of Ub chains at receptor complexes must be carefully counterbalanced by DUBs. The linkage-selective DUBs OTULIN, CYLD, and A20 regulate various aspects of pro-inflammatory signaling (Fiil and Gyrd-Hansen, 2014, Harhaj and Dixit, 2012). The A20 gene (expression is not induced by stimulation of NF-B activity (Fiil et?al., 2013, Keusekotten et?al., 2013, Rivkin et?al., 2013). CYLD is a bona fide tumor suppressor and negatively regulates pro-inflammatory signaling (Bignell et?al., 2000, Harhaj and Dixit, 2012). CYLD belongs to the USP (Ub-specific protease) family of DUBs (Brummelkamp et?al., 2003, Kovalenko et?al., 2003, Trompouki et?al., 2003) and in?vitro cleaves Lys63-Ub and Met1-Ub with similar efficiency while displaying less activity toward Lys11-Ub and Lys48-Ub (Komander et?al., 2009, Ritorto et?al., 2014, Sato et?al., 2015). Unexpectedly, CYLD was recently reported to interact with HOIP, the catalytic subunit of LUBAC, and to inhibit LUBAC-dependent activation.