Promotion Of Anti-viral Innate Immune Responses By E3 Ubiquitin-ligating Enzyme UFL1 And Its Mechanisms

The innate immune system is the first line of host defense against invading pathogens.Activation of the innate immune system relies on pattern recognition receptors(PPRs)to recognize pathogen-associated molecular patterns(PAMPs)or danger associated molecular patterns(DAMPs)and then activate downstream signaling pathways to resist pathogens invasion.cGAS-STING signaling pathway plays a key role in the anti-viral innate immune response.Cytoplasmic DNA senser cyclic guanylate-adenylate synthase(c GAS)can recognize pathogen DNA,abnormal DNA or cyclic dinucleotides(CDNs),and dimerizes to catalyze GMP and AMP to form c GAMP.Then,c GAMP acts as a second messenger to bind and activate the stimulator of interferon genes(STING),the dimerization of STING can activate downstream TANK binding kinase(TBK1)and Ik B kinase(I-kappa B kinase,IKK)to trigger the activation of IRF3 and NF-κB,which induces the production of interferon(IFN)and inflammatory factors.The functional regulation of STING proteins has received extensive attention as it is a key regulator of effective innate immune response.At present,most researches focus on post-translation modification.Post-translational modification(PTM)means the covalent or enzymatic modification of a protein during or after synthesis.Up to date,more than 200 types of PTMs have been reported.Among them,ubiquitination is one of the most common PTMs,next to phosphorylation and glycosylation.Ubiquitin protein covalently binds to the target proteins to perform functions under the catalysis of a complex enzymatic reaction chain,which includes:an ubiquitin-activating enzyme E1,an ubiquitin-conjugating enzyme E2,and an ubiquitin-ligating enzyme E3.In addition to the classic ubiquitin proteins,there are a series of ubiquitin-like proteins(Ub-like proteins,UBLs)whose tertiary structure is similar to ubiquitin proteins.These UBLs also bind to the target proteins through the enzymatic reaction chain to exercise its biological functions.Ubiquitin fold modifier1(UFM1)is one of the newly identified UBLs which can mediate Ufmyylation modification.It’s specific enzymatic reaction chain includes the UFM1-activating enzyme UBA5(ubiquitin-like modifier activating enzyme5),the UFM1 conjugating enzyme 1(UFC1),the UFM1 specific ligase 1(UFL1)and the UFM1-specific proteases(Uf SPs).Preliminary experimental results showed that the m RNA and protein expression of UFL1 were significantly decreased in the primary macrophages with virus infected,so we speculated that UFL might be involved in the regulation of anti-viral innate immune responseand carried out following research.Through the small interfering RNA(si RNA)to knock down the expression of UFL1 in primary peritoneal macrophages and bone marrow-derived macrophages,our study found that decreased expression of UFL1 could inhibit the secretion of interferon β(IFN-β)and interleukin-6(IL-6)induced by viruses or exogenous nucleic acid stimulation.But it only weaken resistance of cells to DNA viruses and promote intracellular viral replication.Similarly,the expression of IFN-β and IL-6 induced by double-stranded nucleic acid stimulation was also significantly reduced in the Ufl1 knockout L929 cells.These results indicated that UFL1 could promote anti-viral innate immune response.To investigate the mechanism of UFL1 promoting the anti-viral innate immune response,our study used luciferase reporter gene experiments and found UFL1 could effectively enhance the activation of IFN-β gene expression induced by c GAS plus STING.Next,we detected the activation of IRF3 and NF-κB signaling pathways in macrophages infected with HSV and found that after knocking down UFL1 expression,phosphorylation levels of IKKβ,P65,TBK1,and IRF3 were significantly decreased.These results suggest that the decreased expression of UFL1 suppresses the anti-viral innate immune response and it acts on c GAS or STING.Our study used exogenous and endogenous co-immunoprecipitation experiments in order to determine the target,and found that UFL1 interacts with STING.Fluorescence confocal experiments also proved that UFL1 and STING had co-localization in macrophages regardless of HSV infection.The above experimental results suggested that UFL1 regulates the activation of c GAS-STING signaling pathway mainly by targeting STING.In order to explore whether the interaction between UFL1 and STING can regulate the innate immune response induced by RNA virus,we knocked down the expression of UFL1 in Sting knockout L929 cells and infected with VSV.The results showed that the decreased expression of UFL1 could not inhibit the expression of IFN-β and IL-6,which mesns UFL1 regulates anti-RNA virus innate immune response by targeting STING as well.In order to further explore the effect of UFL1,our study examined the changes in m RNA and protein levels of STING after knocking down UFL1,and found there is no change in m RNA level,but the protein of STING was significantly reduced.Therefore,we gradient-transfected UFL1 in MEF cells and found that the protein of STING gradually increased with the ramp-up expression of UFL1.Given the dynamic balance between synthesis and degradation of proteins,our study co-transfected UFL1 and STING in MEF cells and treated them with protein synthesis inhibitor CHX,protein degradation inhibitor MG132 and CQ.The result showed that only MG132 could reverse the phenomenon of UFL1 promoting STING expression.At the same time,the above three inhibitors were added to peritoneal macrophages which were knocked down of UFL1 expression.Unsurprisingly only MG132 could reverse the degradation rate of STING.The above experiments showed that UFL1 could inhibit the proteasome degradation of STING.To further investigate how UFL1 regulates the proteasome degradation of STING,our study examined the ubiquitination level of STING and found UFL1 could reduce the K48-linked ubiquitination of STING,and Lys338,Lys347,Lys370 are key sites.Subsequent experimental results showed that UFL1 and TRIM29 are in competitive combination with STING,thereby reduce the K48-linked ubiquitination and the proteasome degradation of it.In conclusion,Our study completed the regulatory network of STING and anti-viral innate immunity,and provided new strategies and ideas for the treatment of related diseases.