ISGylation By HERCs Facilitates STING Activation

Research backgroundInnate immunity is the first line of defense against the invasion of pathogenic microorganisms,and it is also the prerequisite for activating adaptive immunity.Exogenous pathogenic DNA from viral and bacterial infections and endogenous unstable ectopic DNA caused by tumors and other diseases can be recognized by DNA receptors in the cytoplasm,and the host produces an inflammatory response and induces the production of type I IFNs.cGAS activates the STING after recognizing double-stranded DNA,altering the STING conformation and further activating TBK1 and IRF3 to induce the production of type I IFNs.STING is the hub for cGAS-mediated cascade reactions.Abnormal activation of STING leads to abnormal interferon and inflammatory responses mediated,and ectopic DNA cannot be effectively cleared,resulting in autoinflammatory diseases and cancer.Therefore,precise regulation of STING activation is essential for maintaining immune homeostasis.PTMs regulate protein structure and function.PTMs such as phosphorylation,ubiquitination,and SUMO of STING precisely regulate the activation of STING by changing the structure,positioning and stability of STING.Related studies have found that the carbonylation of STING inhibits its activation by inhibiting the translocation of STING from the endoplasmic reticulum to the Golgi apparatus.The ubiquitination of K63 positions catalyzed by RNF115 and TRIM56 promotes translocation and dimerization of STING,respectively.The ubiquitination of K48 positions mediated by RNF5,RNF90,TRIM30 and TRIM29 enhances the degradation of STING and inhibits STING activation.Secreted type Ⅰ IFNs can be recognized by interferon receptors on cell membranes,inducing the expression of a large number of interferon stimulates genes(ISGs),including ISG15.ISG15 not only promotes the secretion of pro-inflammatory and chemokines in its free form,but also connects to target proteins to cause ISGylation under the action of a variety of enzymes.ISGylation is a tertiary enzyme-linked reaction:ISG15 binds to the E1 enzyme UBE1L,is transferred to the E2 enzyme UBCH8,and subsequently binds to the target protein by E3 ligase.HERC5(human)and HERC6(mouse)are the most dominant E3 ligases that mediate ISGylation,catalyzing the binding of most substrates to ISG15.ISGylation can alter protein stability,localization,and function.ISGylation of MDA5 is necessary for its activation and restriction of viral replication;ISGylation of RIG-I inhibits its mediated pathway to prevent overactivation.However,whether STING can undergo ISGylation modification,and whether isgylation plays a role in the cgas-sting pathway is unknown.Purpose of the study1)To study the regulatory role of HERCs in the activation of the cGAS-STING pathway and elucidate the molecular mechanism of their occurrence.2)Elucidate the interaction between STING ISGylation and ubiquitination modification during STING activation,and further reveal the activation regulation mechanism of cGASSTING pathway.3)To explore the role of HERC5(human)and HERC6(mouse)in the activation of the cGAS-STING pathway.Research methods1)Using Herc6 gene knockout mice,mouse peritoneal macrophages transfected with Herc6 siRNA,human myeloid leukemia mononuclear cells(THP-1)transfected with HERC5 siRNA,Herc6 gene knockout,knockdown,or decreased transcription and release of IFN-βinduced after activation of the cGAS-STING pathway after knockdown of the HERC5 gene.Then,Western blot studies have found that HERCs enhance phosphorylation of downstream molecules(TBK1,IRF3,STAT1)and ISGs such as IFIT1,IFIT2,CXCL10,Max1,etc.These results suggest that HERCs promote cGAS-STING signaling pathway activation.2)Using a variety of mouse disease models(DNA virus HSV-1 infection model and DMXAA-induced STING activation model)and a variety of infection modes(intraperitoneal injection and intraventricular injection)through ELISA,RT-PCR,western blotting,immunohistochemical staining experiments,it was found that the secretion of IFN-β,TNF-αand IL-6 in Herc6-/-mice was significantly lower than that of Herc6+/+ mice in the control group.Next,we found that HERCs can bind to STING but not to cGAS through coimmunoprecipitation,immunofluorescence and other experiments.Then,immunoblotting studies found that HERCs enhanced phosphorylation of downstream molecules(TBK1,IRF3,STAT1)and enhanced transcription of ISGs(IFIT1,IFIT2,CXCL10,Max1).Inhibitors such as protein synthesis inhibitor CHX and ubiquitin-proteasome inhibitor MG 132 were used to act on Herc6-/-mice,mouse intraperitoneal macrophages and HEK293T cells transfected with Herc6 siRNA.The results confirmed that HERCs can stabilize their expression by inhibiting the pathway of proteasome degradation of STING.3)Transfection of relevant plasmids into HEK293T cells,through co-immunoprecipitation and western blotting analysis,it was found that STING can undergo ISGylation before transposition,and HERCs can enhance ISGylation of STING and inhibit ubiquitination.At the same time,the E3 ligase inactivation site mutant plasmid HERC5-C994A of HERC5 was transfected in HEK293T cells,and it was found that the mutant lost the ability to enhance STING ISGylation and weaken STING ubiquitination through co-immunoprecipitation and immunoblottingResearch results1)This study clarifies the role of E3 ISGylationHERCs in the activation of cGAS-STING signaling pathway,and reveals the molecular mechanism of HERCs to antagonize the ubiquitination degradation pathway in the physiological state of STING by catalyzing the ISGylation modification of STING,thereby positively regulating the expression level of STING.2)This study reveals the specific mechanism by which ISGylation of STING regulates the expression of STING protein.At the same time,the interaction between STING ubiquitination and ISGation was found.Protein expression of STING is associated with activation of the cGAS-STING pathway.Provide new targets for the treatment of related immune disorders.