Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis

Abstract

The innate immune system is equipped with pattern-recognition receptors that recognize pathogen-associated molecular patterns 1 . Pattern-recognition receptors include transmembrane Toll-like receptors (TLRs) and cytosolic Nod-like receptors 2 . Nod1 and Nod2 recognize structures in bacterial peptidoglycan 3 . Loss-of-function mutants of Nod2 are associated with Crohn's disease 4-6 , whereas gain-offunction mutants result in predisposition to the development of earlyonset sarcoidosis and Blau syndrome 7,8 . Nod2 responds to muramyl dipeptide (MDP), a derivative of peptidoglycan 9,10 . Nod2 consists of two amino-terminal caspase-recruitment domains (CARDs), a central self-oligomerization NACHT region and multiple carboxyterminal leucine-rich repeats 11 . Engagement of the leucine-rich repeats by MDP promotes a conformational change that exposes the NACHT domain, which allows self-oligomerization of Nod2 and the binding of its CARDs to the CARD-containing kinase RIP2 (refs. 12,13). RIP2 interacts with the kinase TAK1, which leads to activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs) and induction of the expression of proinflammatory cytokines [14][15][16][17][18] . Ubiquitination of RIP2 is critical for Nod2 signaling pathways 15,16 .The attachment of polyubiquitin chains to RIP2 serves to recruit TAK1 via the adaptors TAB2 and TAB3 (ref. 19), and that facilitates TAK1-induced phosphorylation and activation of IκB kinases (IKKs) that induce phosphorylation of inhibitory IκB proteins 20 . Phosphorylated IκB proteins undergo proteasome-mediated degradation 21 that allows NF-κB to translocate to the nucleus and induce proinflammatory gene expression 22 . Studies have investigated the enzymes responsible for catalyzing the ubiquitination of RIP2. The Ubc13-Uev1a dimer acts as the E2 conjugating enzyme in the Lys63 (K63)-linked polyubiquitination of RIP2 (refs. 15,16), but the identity of the E3 ubiquitin ligase(s) that directly ubiquitinate(s) RIP2 to mediate Nod2-induced activation of NF-κB remains unclear. TRAF6 has been proposed as the main E3 ligase for RIP2 (ref. 15), but the ubiquitination of RIP2 is intact in TRAF6-deficient cells 16 and knockdown of TRAF6 does not affect RIP2-mediated activation of NF-κB 9,14 . Three members of the 'IAP' family of E3 ubiquitin ligases (XIAP, cIAP1 and cIAP2) have been proposed to regulate RIP2 ubiquitination 23,24 . Although the conclusions of the last two studies differ about the functional importance of cIAP1 and cIAP2 in mediating Nod2-induced ubiquitination of RIP2, one demonstrated that XIAP promotes the ubiquitination of RIP2 and recruitment of the linear ubiquitin chain-assembly complex (LUBAC) to Nod2 (ref. 23). However, the XIAP-mediated polyubiquitination of RIP2 is not K63 linked, a type of linkage associated with RIP2-induced activation of NF-κB. The E3 ligase Itch can also directly ubiquitinate RIP2 to negatively regulate Nod2-induced activation of NF-κB 25 . Thus, it remains unclear which E3 ubiquitin ligase directly cataly...

RIP kinases: key decision makers in cell death and innate immunity

“…Thus the ubiquitination of RIP2 is intact in TRAF6-deficient cells, 165 pharmacological depletion of cIAP1 and cIAP2 has no effect on RIP2 ubiquitination 171 and ITCHmediated ubiquitination of RIP2 is associated with negative regulation of RIP2-mediated NFkB signalling. 173 We have recently described a key role for the E3 ubiquitin ligase Pellino3 in directly ubiquitinating RIP2 and mediating NOD2 downstream signalling, including its activation of NFkB and protective effects in colitis 174,175 (Figure 4). We also showed that Pellino3 protein expression is greatly reduced in the colons of Crohn's disease subjects consistent with a protective role in human disease.…”

“…We also showed that Pellino3 protein expression is greatly reduced in the colons of Crohn's disease subjects consistent with a protective role in human disease. 174 We have proposed functional cooperation between Pellino3 and XIAP in that the former promotes the formation of polyubiquitin chains on RIP2 in which the isopeptide linkages between adjacent ubiquitin molecules are linked via lysine 63 of ubiquitin and XIAP facilitates linear ubiquitination of components of the RIP2 complex in which individual ubiquitin proteins are joined head to tail. This shows remarkable similarity to the RIP1-containing complex I in the TNFR-1 signalling pathway in which components of the complex are initially modified by lysine 63-linked chains followed by LUBAC-mediated linear ubiquitination that serves to stabilize the complex and further enhance downstream signalling pathways, such as NFkB and inflammatory gene expression.…”

Small Bowel Crohn's Disease: An Emerging Disease Phenotype?

“…In particular, SNP 13 appears to be associated with small bowel involvement and severe phenotype [17,18]. The discovery of NOD2/CARD15 has paved the way for the discovery of additional defective mechanisms [19]. In the small bowel, NOD2 receptors can be found in abundance in Paneth cells of the epithelial layer.…”

International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

“…Ubiquitination also plays a crucial role in NOD1 and NOD2 signaling. Studies of the adaptor RIP2 have revealed extensive ubiquitination by a range of E3 ligases including cellular inhibitor of apoptosis protein 1, cellular inhibitor of apoptosis protein 2, X-linked inhibitor of apoptosis protein, Pellino3, and itchy E3 ubiquitin protein ligase (Bertrand et al, 2009;Krieg et al, 2009;Tao et al, 2009;Yang et al, 2013b;TignoAranjuez et al, 2013). RIP2 ubiquitination enhances NFkB and JNK-mediated signaling, except when mediated by itchy E3 ubiquitin protein ligase, in which case the JNK and mitogen-activated protein kinase (MAPK) pathways are favored.…”