E3 ligase WWP2 negatively regulates TLR3-mediated innate immune response by targeting TRIF for ubiquitination and degradation

Abstract

Recognition of viral double-stranded RNA by Toll-like receptor 3 (TLR3) triggers activation of the transcription factors NF-κB and interferon regulated factor 3, leading to induction of type I interferons and proinflammatory cytokines. TIR-domain-containing adapter-inducing interferon-β (TRIF) is an adapter protein required for TLR3-mediated signaling. Here we identified the E3 ubiquitin ligase WW domaincontaining protein 2 (WWP2) as a TRIF-associated protein by biochemical purification. WWP2 mediated K48-linked ubiquitination and degradation of TRIF upon TLR3 activation. Overexpression of WWP2 inhibited TLR3-mediated NF-κB and interferon regulated factor 3 activation, whereas knockdown of WWP2 had opposite effects. We generated Wwp2-deficient mice to further investigate the roles of Wwp2 in innate immune responses. Consistently, production of IFN-β, CCL5, TNFα, and IL-6 in response to the TLR3 ligand poly(I:C) was elevated in Wwp2 −/− macrophages and Wwp2-deficient mice exhibited increased susceptibility to poly(I:C)-induced death than the control littermates. Our findings suggest that WWP2 negatively regulates TLR3-mediated innate immune and inflammatory responses by targeting TRIF for ubiquitination and degradation.T oll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors (PRRs) that are critically involved in host defense from plants to humans. So far, 13 TLRs (named TLR1 to TLR13) have been identified in humans and mice, each of which recognizes a distinct set of pathogen-associated molecular patterns (PAMPs) (1). TLRs contain an extracellular domain consisting of leucine rich repeats which is responsible for PAMP recognition, a transmembrane domain, and a conserved cytoplasmic toll/IL-1 receptor (TIR) domain which is able to mediate homotypic protein-protein interactions (2). The TIR domains of TLRs are responsible for their homo-or hetero-dimerization, and upon ligand stimulation, they also act as platforms to recruit downstream TIR domain-containing adaptor proteins and signaling molecules, leading to the activation of transcription factors such as NF-κB and interferon regulated factor 3 (IRF3) (1, 3, 4). These transcription factors act alone or in collaboration to induce transcription of proinflammatory cytokines and/or type I interferons (IFNs).Among the TLRs, TLR3 has been reported to recognize viral dsRNA as well as its analog poly(I:C). Recognition of these ligands by TLR3 activates signaling pathways leading to the activation of NF-κB and IRF3 and subsequent production of type I IFNs and proinflammatory cytokines (5). TLR3-mediated signaling critically depends on the TIR domain-containing adapter TIRdomain-containing adapter-inducing interferon-β (TRIF; also called TICAM-1) (6). It has been shown that Trif −/− lung fibroblasts are defective in poly(I:C)-induced activation of NF-κB and IRF3 as well as production of type I IFNs, demonstrating that TRIF is indispensable for TLR3-mediated signaling (7). In addition, the TLR4 ligand LPS has been shown to signal through TRIF-...

TRIM38 Negatively Regulates TLR3/4-Mediated Innate Immune and Inflammatory Responses by Two Sequential and Distinct Mechanisms

“…Finally, mouse gene-knockout studies demonstrated that Nap1 is not required for TLR3/4-mediated innate immune responses (32). Previously, we reported that another E3 ubiquitin ligase, WWP2, also targets TRIF for K48-linked polyubiquitination and proteasomal degradation (16). However, WWP2 functions differently with TRIM38.…”

“…Mouse anti-TRIF, mouse anti-IkBa, mouse anti-TAB2, and rabbit anti-TAB3 were described previously (11,(16)(17)(18).…”

Long noncoding RNAs in viral infections

“…Several sensors and mediators of IFN synthesis and signaling modify their stability or functionality by ubiquitination. These include MyD88, TRIF or nuclear IRF3, degraded after the action of several E3 ligases, whose increase reduces IFN synthesis (Xue et al, 2012; Yang et al, 2013a; Wang et al, 2015). In contrast, USP13 (Ubiquitin Specific Peptidase 13) is a DUB that deubiquitinates STAT1 to avoid degradation and potentiate IFN signaling (Yeh et al, 2013).…”

Regulation of cellular innate antiviral signaling by ubiquitin modification

“…Pellino1 promotes K63-linked polyubiquitination of RIP1 and thus selectively facilitates TLR3-mediated activation of NF-κB and induction of pro-inflammatory cytokines [33]. In contrast, the E3 ligases WWP2, TRIM38, and Nrdp1 catalyze K48-linked ubiquitination and induce the proteasome-dependent degradation of TRIF and MyD88, respectively, turning down TLR-mediated signaling [34][35][36].…”