The Ubiquitin E3 Ligase TRIM10 Promotes STING Aggregation And Activation In The Golgi Apparatus

STING is a transmembrane protein located in the endoplasmic reticulum,which plays an important role in antiviral innate immunity.After the invasion of pathogenic microorganisms,DNA viruses,as pathogen-related molecular patterns,are recognized by the pattern recognition receptor cGAS and produce the second messenger cGAMP.cGAMP binds to STING and mediates the transfer of STING from endoplasmic reticulum to Golgi apparatus.STING phosphorylated the downstream kinase TBK1,activated TBK1 and further activated IRF3,which enabled IRF3 to enter the nucleus and bind to interferon genes,inducing the expression of type I interferon,and thus inhibiting viral replication.Therefore,STING is a very important connector molecule in interferon signaling pathway.However,the exact mechanisms of STING activation and translocation remain largely unknown.TRIM 10 is a member of the TRIM family of E3 ubiquitin ligases that have previously been reported to be involved in oncogenesis,neurodegenerative diseases,and antiviral immune responses.In this paper,we found that TRIM 10 can positively regulate STING activation and aggregation on the Golgi apparatus.We screened some members of the TRIM family by immunofluorescence and found that TRIM 10 was localized to the Golgi apparatus after the stimulation of ISD.Macrophages with TRIM10 knockout produced significantly less type I interferon and ISGs when stimulated by dsDNA or cGAMP compared to the wild type.The specific mechanism is based on the ubiquitination of K27 and K29 by TRIM 10 on the lysine of the 289th and 370th positions of STING.This modification facilitated the transport of STING from the endoplasmic reticulum to the Golgi apparatus,the formation of STING aggregates,and the recruitment of TBK1 to STING,ultimately promoting the STING-dependent type I interferon response.Our study suggests that TRIM 10 is a key regulator of cGAS-STING mediated antiviral and antitumor immunity.Methods and Results:1.TRIM10 was translocated to the Golgi apparatus after ISD stimulation.HeLa cells were transfected with TRIMs overexpressed vectors for 24h and then infected with DNA ligand ISD for 4h.We observed that TRIM 10 was localized in the Golgi apparatus after ISD stimulation.We extracted the Golgi apparatus-enriched component from HeLa cells stimulated with ISD.Western blot results showed that the level of STING and TRIM 10 in the Golgi apparatus increased after ISD stimulation.These results suggest that the translocation of TRIM 10 to the Golgi apparatus after ISD stimulation may be related to STING.2.TRIM10 positively regulates the expression of IFN-β mediated by cGASSTING.We stimulated peritoneal macrophages with RLR ligands(5’ppp RNA,Poly(I:C)(HMW),Poly(I:C)(LMW))and TLR4 ligand LPS or infected with SeV to check whether TRIM10 regulates RLR and TLR4 signaling.Interestingly,we found that the expression of Ifnb1 was comparable between Trim10-/-and Trim10+/+ peritoneal macrophages after RLR and LPS stimulation.According to the results of qRT-PCR,the deletion of TRIM 10 did not affect the expression of Ifnb1 mediated by TLR and RLR pathways.But after the infection of HSV-1,the expression of Ifnb1 in the TRIM10 knockout group was significantly lower than that in the wild-type group.These results suggest that TRIM 10 positively regulates the expression of DNA virusmediated IFN-β,but has no effect on RLR and TLR pathways.3.TRIM10 interacts with STINGCo-immunoprecipitation experiments showed that TRIM 10 interacts with STING in vitro.Next,we verified this hypothesis again through endogenous binding experiment.We prepared primary peritoneal macrophages from Trim10+/+ and Trim10-/-mice,and the peritoneal macrophages were infected with HSV-1.AntiSTING antibody was used for co-immunoprecipitation.Western blot revealed that endogenous TRIM 10 was bound to STING,and with the increase of infection time,the interaction is also growing.According to the above experimental results,we confirmed the specific binding of TRIM10 to STING.4.TRIM10 regulates ubiquitination of STINGTo study the types of STING polyubiquitination mediated by TRIM 10,we transfected various ubiquitin mutants with only one intact lysine residue into HEK293T cells together with TRIM10 and STING.We observed that TRIM10 increases STING polyubiquitination only in the cells transfected with HA-ubiquitin WT,K27,and K29.We also observed increased STING ubiquitination in the presence of ubiquitin WT,K27,and K29,but not ubiquitin K48 and K63 with the in vitro ubiquitination assay.Since TRIM 10 interacts with the CBD domain of STING,we hypothesize that TRIM 10 might modulate the ubiquitination of lysine residues in the CBD domain or near the CBD domain of STING.We mutated the lysine residues in the CBD domain and the nearby region into arginine and transfected them into HEK293T cells together with TRIM10.Co-immunoprecipitation showed that TRIM10 could not promote the ubiquitination of STING mutants K289R and K370R,in which the lysine residues at 289 and 370 were mutated to arginine,indicating K289 and K370 are the main ubiquitin acceptor residue in STING mediated by TRIM10.5.TRIM10 promotes the aggregation of STING on the GolgiAccording to the previous experimental results,we know that TRIM10 regulates STING ubiquitination.To directly verify the effect of TRIM10 on STING activation,we prepared Trim10+/+ and Trim10-/-peritoneal macrophages and measured the formation of endogenous STING oligomers after HSV-1 infection.Native polyacrylamide gel electrophoresis(Native PAGE)showed that HSV-1 infection induced STING aggregation in Trim10+/+ macrophages.However,STING aggregation induced by HSV-1 infection in Trim10-/-macrophages was decreased compared to that in Trim10+/+ macrophages.STING-associated vasculopathy with onset in infancy(SAVI)is an autoimmune disease caused by gain-of-function mutations in the STING-encoding gene TMEM173.These mutations do not localize in the ER and can lead to STING hyperactivation in a ligand-independent manner.We showed that STING V147L forms aggregation without cGAMP stimulation,while STING WT only forms aggregate after cGAMP stimulation.Notably,we found TRIM10 could promote the aggregation of STING(V147L)similarly to STING WT.These results indicate that TRIM 10 can promote the aggregation of STING in the Golgi apparatus.Conclusion1.TRIM 10 facilitates cGAS-STING-mediated type Ⅰ IFN production.2.TRIM10 promotes K27-/K29-linked polyubiquitination of STING at K289 and K370,which facilitates STING aggregation and activation in Golgi apparatus.3.After DNA stimulation,TRIM 10 was translocated to the Golgi apparatus in a STING-dependent manner,and promoted the aggregation of STING through ubiquitination,which further affected the recruitment and activation of TBK1 by STING.Innovation1.The research on STING translocation and aggregation mechanism was still unclear.Our research demonstrated that TRIM 10 promotes K27/K29 linked polyubiquitination of STING at K289 and K370,which facilitates STING aggregation and TBK1 recruitment.2.In this study,we found STING first interacts with TRIM 10 and then co-translocates to the Golgi apparatus after dsDNA stimulation.Knockout of TRIM 10 decreased translocation of STING to the Golgi apparatus and STING aggregation.The reduced translocation and aggregation of STING are restored with the reintroduction of TRIM 10 WT,but not the E3 ligase dead mutant TRIM 10 ΔRING.These findings suggest that TRIM10 can regulate STING ER-to-Golgi translocation and aggregation on the Golgi through its E3 ubiquitin ligase activity.