MAVS Regulates ASC-mediated Inflammatory Signaling Pathway Via TRAF3

The innate immune system, which represents the first line in recognizing andeliminating invading microbes, has broad antimicrobial functions that are activatedrapidly after microbes are encountered. It plays a crucial role in protecting organismsfrom infection. The activation of innate immune response relies on germ-line encodedpattern recognition receptors (PRRs) from host that recognize essential pathogenmolecules, so-called pathogen associated molecular patterns (PAMPs). Followingligand binding, the host receptors recruit adaptor proteins, activate signaling cascades,induce the production of various antimicrobial molecules such as type I IFNs,pro-inflammatory cytokines and chemokines, thereby lead to the killing and clearanceof pathogenic microorganisms. For different PAMPs, the host responses with specificPRRs. So far, three classes of viral PRRs have been identified: the Toll-like receptors(TLRs), the RIG-like receptors (RLRs) and the NOD-like receptors (NLRs). Theseproteins could promote transcriptional activation of IFN-I expression through theNF-B and IRF-transcriptional activator pathways, or initiate the assembly of acytoplasmic signaling complex known as inflammasome, leading to the release ofmature pro-inflammatory cytokines. A typical inflammasome consists of an activatedreceptor, the adapter protein ASC and the effector molecular pro-caspase-1. Thefunctional state of host inflammasome can be a double-edged sword, which tightlycorresponded to the pathogenesis of microbes. On one hand, appropriate activation ofinflammasome is essential for killing pathogens and preventing infection; on the otherhand, excessive inflammatory response might lead to the damage of various tissuesand organs, even threaten the hosts life. Thus, inflammatory reactions in response topathogen infection are highly coordinated, identifying and screening new regulatorymolecular have great significance in combating microbial infection.As the key adaptor protein in inflammasome signaling pathway, ASC, which iscalled apoptosis-associated speck-like protein containing a CARD, is indispensablein this signal transduction. It links the receptor and pro-caspase-1throughCARD-CARD and PYRIN-PYRIN domain interactions, which leads to the proteolytic cleavage of pro-caspase-1into the active form. The activation of caspase-1is essential for the processing of pro-IL-1and pro-IL-18and secretion of theirmature biologically active forms. Whereas ASC has a pivotal role in the maturation ofcytokines mediated by inflammasome, the potential new regulators of inflammasomestargeting ASC are under investigation. Via the yeast two-hybrid experiment, we foundmitochondrial antiviral signaling protein MAVS might interact with ASC. Furtherexperiments showed that:1. MAVS interacts with ASC in vivo and in vitro: By using immunoprecipitation,GST-pulldown, mitochondrial separation, immunofluorescence microscopy andimmunoblot, MAVS was found to form specific complex with ASC in vivo and invitro. By constructing various truncated mutants, we identified that N-terminal CARDdomain and C-terminal TM domain of MAVS are required for MAVS associating withASC CARD domain.2. MAVS modulates ASC-mediated IL-1secretion by stabilizing ASC: ELISAanalysis indicated that MAVS could increase ASC-mediated IL-1secretion, whilethe CARD deletion mutant, TM deletion mutant and dimerization mutant failed to doso. Overexpression of MAVS led to considerable increase of ASC protein abundance,while specific suppression of MAVS decreased endogenous ASC expression. RT-PCRdata showed that ASC mRNA abundance was not altered with MAVS expression.3. MAVS regulates ASC via E3ligase TRAF3: By screening6E3ligasesinvolved in MAVS signaling pathway, we found TRAF3could increase ASC proteinstabilization and IL-1secretion. Further study revealed that TRAF3associated withASC and promoted its ubiquitylation. The TRAF3C53AC56A or C68AH70A mutant,which losses E3ligase activity, had compromised activity on ASC ubiquitylation.After specific suppression of TRAF3by transfecting siRNA into the cells, MAVS wasfound to enhance ASC stabilization unsuccessfully.4. TRAF3catalyzes K63-linked polyubiquitylation of ASC at lysine174: Byconstructing a series of Ub KR mutants and ASC KR mutants, we identifiedK63-linked polyubiquitylation of ASC and at lysine174might be the ubiquitylationsite by TRAF3. Further investigation suggested that MAVS and TRAF3had littleeffect on the cellular protein abundance and ubiquitylation levels of ASC (K174R)mutant.5. TRAF3-mediated ubiquitylation is indispensable for ASC redistribution uponviral infection: The infection of VSV induced ASC ubiquitylation and nucleus to cytosol redistribution, this phenomenon was reduced in MAVS or TRAF3knockdowncells in contrast with WT cells. Cell fractionation and immunoflurecence experimentsshowed that TRAF3promoted WT ASC transferring from the nucleus to the cytosol,but not ASC (K174R) mutant.6. MAVS and TRAF3promote virus-induced inflammasome activation:Compared with WT MEFs, MAVS-/-and TRAF3-/-MEFs had impaired IL-1production and decreased proteolytic cleavage of pro-caspase-1in response to SeV. Inconcert with this finding, MAVS-/-BMDCs also showed decreased activation ofcaspase-1upon VSV stimulation.In summary, our research showed that MAVS incresed ASC stabilization andIL-1secretion via E3ligase TRAF3, which catalyzed K63-linked polyubiquitylationof ASC at lysine174. In addition, TRAF3-mediated ubiquitylation faciliated ASCredistribution upon viral infection and promoted the activation of inflammasome. Wehave identified new regulators of inflammasome, clarified the molecular mechanismof MAVS and TRAF3in regulating this signaling pathway, suggested bettertheoretical basis for the crosstalk of different transduction pathways.