The Regulation And Mechanism Of E3 Ubiquitin Ligase Complex SCF-FBXO11 In Innate Immune Response

The innate immune system,as the first line of defense against pathogenic microorganisms,recognizes pathogen associated molecular patterns(PAMPs)of pathogenic microorganisms or the host derived damage associated molecular patterns(DAMPs)via a series of pattern-recognition receptor(PRR).Known innate immune receptor mainly includes Toll-like receptors,RIG-I like receptors,NLR receptors,the CLR receptors and DNA recognition receptors.A variety of innate immune recognition receptors released into the intracellular pathogen nucleic acids(DNA and RNA),which involved in recognition of DNA receptor mainly including TLR9,c GAS,AIM2,IFI16 and so on.By mediating DNA identification,pattern recognition receptor will activate the innate immune response signaling pathways and ultimately induce the secretion type-? interferons and inflammatory cytokines,to eliminate the invading pathogens.c GAS is the well accepted DNA sensor till now.After recognize intracellular ds DNA,c GAS is activated and synthesize c GAMP.c GAMP then moves to the ER to activate STING.Activated STING can promote TBK1 and IRF3 phosphorylation and induce the transcription and expression of type-? interferons.In the cgas-sting signaling pathway,STING is at the center of anti-DNA innate immune responses.There are a variety of ubiquitination ligases or deubiquitination enzymes that regulate the ubiquitination modification of STING,leading to the degradation of STING or affecting its activity,regulating the activation of downstream signaling pathway and maintaining the immune balance of the body.Ubiquitin is one of the key important regulation of protein function regulation,which is the antagonistic regulation between the ubiquitination enzyme system,including E1 ubiquitin activating enzyme,E2 ubiquitin binding enzyme and E3 ubiquitin ligase,and the deubiquitination enzyme system.Ubiquitination and deubiquitination play an important role in the functional regulation of proteins and play an important role in many immune processes.Ubiquitin is a small molecular peptide consisting of 76 amino acids residues with a monomer molecular weight of 8.6 k Da that is covalently linked to lysine or other residues of target proteins to modify their stability or activity.Ubiquitin binding to substrates usually occurs on lysine residues.Ubiquitin contains seven lysine residues(K6,K11,K27,K29,K33,K48,K63)and produce seven different types of polyubiquitin chains,among which K48 ubiquitin modification and K63 ubiquitin modification are the most studied.At present,some reports on the ubiquitination regulation of STING still need to be further explored and improved.Based on our previous study in STING signal pathways,we find the new E3 ubiquitin ligase which can interact with STING by the IP-MS experiment.By the analysis of mass spectrum data,we screen the E3 ubiquitin ligase which has not been reported to regulate STING and find E3 ubiquitin ligase complex components FBXO11 that has significant regulation function against DNA virus,HSV–1.FBXO11 plays an important role in regulating cell cycle,tumorigenesis and metastasis,but little is known about its role in antiviral immunity.In this study,we identified FBXO11 as a new regulator which influence STING ubquitination and regulates innate anti-DNA virus immune responses.The knockdown of FBXO11 significantly inhibited the expression levels of IFN-?,IFN-?4 and inflammatory cytokines induced by DNA virus hsv-1,while had no significant regulatory effect on the secretion of type-? interferons and inflammatory cytokines induced by RNA virus VSV in primary macrophages by real-time quantitative PCR.Dual luciferase reporter assay also found that FBXO11 could effectively enhance activity of IFN-? luciferase reporter induced by c GAS-STING,but the expression of IFN-? cannot be induced by RIG-I and MAVS in RNA recognition pathways and TBK1 and IRF3.Thus,FBXO11 selectively regulates the antiviral innate immune response induced by DNA viruses.In the mechanism study,analysis of the signal pathway showed that knockdown of FBXO11 inhibited the phosphorylation of TBK1,IRF3 and p65 in the c GAS-STING pathway,suggesting that FBXO11 targets STING or its upstream molecules.By immunoprecipitation experiment we found that FBXO11 interacted with STING.By constructing the truncation of FBXO11 and STING,it was found that the interaction between FBXO11 and STING depended on the Pb H domain of FBXO11 and the C-terminal domain of STING.In the following experiments,we elucidated that FBXO11 inhibited the proteasomal degradation of STING by impairing the K48-linked ubiquitination of STING,and promoting the dimerization of STING.By constructing the Lys-Arg single site mutant plasmid of STING,it was found that the K224 site of STING was the key site for FBXO11 to inhibit the ubiquitination of STING.After the mutation of this site,the promoting effect of FBXO11 on c GAS-STING induced IFN-? luciferase reporter gene disappeared,and the inhibiting effect on the degradation of STING also disappeared.However,the specific molecular mechanism by which FBXO11 impairs STING ubiquitination still needs further study.As a component of E3 ubiquitin ligase,FBXO11 does not promote the ubiquitination of STING,but inhibits its ubiquitination.We propose there may be two possible mechanisms:(1)FBXO11 can inhibit the K48 ubiquitination of STING mediated by another E3 ubiquitin ligase.FBXO11 indirectly inhibited the degradation of STING by preventing the E3 binding to STING and weakening its K48 ubiquitination.(2)FBXO11 promotes the deubiquitination of STING by promoting a DUB binding to STING,and indirectly inhibits the degradation of STING.We will carry out further research in the follow-up work.In summary,this study found that E3 ubiquitin ligase FBXO11 can positively regulate innate immune response,and verified the key role of STING in antiviral innate immune signaling pathway.FBXO11 can remove the K48 ubiquitination of STING by targeting the K224 site and maintain the stability of STING,thus to promote the antiviral innate immune response of the body.Our study provides a new way to further understand the mechanism of FBXO11 in innate anti-viral immunity and a potential target for the treatment of viral infection,or even cancer.