Roles And Mechanisms Of Tyrosine Kinase Src And E3 Ubiquitin Lingase Trim13 In The Regulation Of Innate Immune Response

Innate immunity system is the first line of defense that protects the host from infection by pathogens through recognition and elimination of invading pathogens.Host cells can recognize the pathogens associated molecular patterns(PAMPs)of pathogens via pattern recognition receptors(PRRs),and initiate signaling pathways that regulate the expression of type I interferons(IFN),interleukine 6(IL-6)and tumor necrosis factor α(TNFα),finally leading to resistance to pathogen infection.However,excessive innate immune response can cause inflammatory response of host,which may lead to pathological damages to tissues.In order to ensure the body’s resistance to invasion and protection from injury,the regulation of innate immunity becomes important.Phosphorylation and ubiquitination are both important types of post-translational modification(PTM).Previous studies suggest that both types of modification play crucial roles in the regulation of immune responses during each steps of immunity.Our group has focused on the studies of roles and mechanisms of PTM in the regulation of innate immunity.In recent three years,we have investigated the regulation of innate immune response by the phosphorylation of TANK-binding kinase 1(TBK1)and the E3 ubiquitin ligase Trim13.As a key kinase in antiviral innate immune response,TANK-binding kinase 1(TBK1)is always the focus of molecular immunology researches.However,it is less clear how the kinase activity of TBK1 is regulated.So in the first part of this thesis,we will aim to explore the activation-related molecules of TBK1.We set out to identify the phosphorylation sites of TBK1 during the process of antiviral innate immune response,test the effects of inactive mutant of TBK1 on antiviral innate immune response,and identify Tyr179 as another site of TBK1 phosphorylation in addition to the autophsophorylation site Ser172 in TBK1.By using Western blot and ELISA assays,we find that Tyr179 phosphorylation of TBK1 is essential for Ser172 phosphorylation and type I IFNs production.By using immunoprecipitations(IPs)plus mass spectrometry(MS),we detect the tyrosine kinase Src in the TBK1 complex.Src is a member of the Src family kinases(SFK),and is composed of the N-terminal SH4 domain,the unique domain,the SH3 domain,the SH2 domain and the C-terminal kinase domain.As a non-receptor tyrosine kinase,Src can mediate the tyrosine phosphorylation of various protein substrates and regulate multiple biological processes including proliferation,differentiation,survival,adhesion and motility.In order to explore the roles of Src in antiviral innate response,we use Src inhibitor and Src-deficient cells to inhibit the activity and abolish the expression of Src,respectively.We find that Src deficiency can inhibit the activation of TBK1-IRF3 signaling pathway and the production of type I IFN,but does not affect the activation of NF-kB and mitogen-activated protein kinase(MAPK)pathway.We confirm that Src can modulate the signaling pathways mediated by adaptors like TRIF,MAVS and STING but not MyD88 by using gene reporter assays.By using IPs and recombinant GST fusion proteins,we find that Src can interact with TRIF,MAVS and STING to indirectly regulate the phosphorylation of TBK1 on Tyr179,finally leading to activation of TBK1-IRF3 pathway,production of type I IFNs and innate antiviral response.In this part of research,we not only identify another phosphorylation site in TBK1(Tyr179)but discover the upstream kinase for TBK1(Src).Upon viral invasion,Src can interact with adaptors including TRIF,MAVS and STING and mediate Tyr179 phosphorylation of TBK1 and then autophosphorylation of TBK1 on Ser172,thus resulting in sequential activation of TBK1,phosphorylation and nuclear import of IRF3 and expression of type I IFNs.This study provides mechanistic insights into the activation of TBK1 and elucidates an important role of Src in modulating antiviral innate response.In addition to phosphorylation,ubiquitin modification also plays an important role in innate immune regulation.As a kind of key molecule in the process of ubiquitin modify,E3 ubiquitin ligases are key focuses in the study of innate immune regulation.In the second part of the thesis,we investigate the roles of E3 ligase Trim13 in the regulation of innate immune response.By searching the database-oriented literatures in combination with siRNA-based functional selection,we find that Trim13 may be involved in the regulation of innate immune response.Trim13 is an E3 ubiquitin ligase belonging to the RBCC type of E3 ligases.At present the roles and mechanisms of Trim13 in the regulation of innate immune response have not been clearly understood.To investigate the roles of Trim13 in the innate immunity-associated signaling pathways,we silenced the expression of Trim13 in RAW264.7 cells by using Trim13-specific siRNAs.We find that Trim13 knockdown inhibits the expression of IFN-β induced by VSV infection and promotes the expression of IL-6 and IFN-β induced by LPS treatments and HSV-1 infection.Comparably,overexpression of Trim13 promotes the expression of IFN-β induced by VSV infection and inhibits the expression of IL-6 and IFN-β induced by LPS treatments and HSV-1 infection.We then established the knockout mice for Trim13,and repeated the experiments using the above mentioned treatments in peritoneal macrophages and bone marrow-derived macrophages derived from wild type(Trim13-wt)and knockout mice(Trim13-ko).Similar results are obtained,as compared to the si RNA experiments.Our data indicate that Trim13 may play dual roles in the regulation of innate immune response upon encounter of different pathogens by macrophages.In vivo,4 hours after intraperitoneal injection of Listeria monocytogen,the serum levels of TNFα and IL-6 of Trim13-ko mice are significantly higher than that in Trim13-wt mice;12 hours after intraperitoneal injection of VSV,the serum levels of TNFα and IL-6 of Trim13-ko mice are significantly lower than that in Trim13-wt mice.So we propose that Trim13 dually regulates innate immune response: when Trim13 positively regulates innate immune response triggered by RNA virus infection,it may negatively regulate the innate immune response triggered by DNA viruses and TLR agonists.We went further to explore the signaling pathways potentially regulated by Trim13.In HEK293 T cells,overexpression of Trim13 can significantly inhibit the activation of IFN-β and NF-κB reporters elicited by TRIF and STING while Trim13 significantly inhibit the activation of IFN-β and NF-κB reporters elicited by CARD domain of RIG-I(RIG-I-CARD).But Trim13 overexpression does not affect the activation of IFN-β and NF-κB reporters elicited by MyD88.These data convincingly suggest that Trim13 is involved in the regulation of multiple innate signaling pathways.By using Western blot,we find that Trim13 overexpression decreases the protein levels of STING but not TRIF and MyD88,indicating that Trim13 may be involved in the polyubiquitination and degradation of STING.In this part of research,we find that Trim13 may play bidirectional roles in regulation of innate immune response.However,more studies are required to elucidate the mechanisms for Trim13-mediated regulation of innate immune response,e.g.the interacting molecules for Trim13,the protein substrates modified by Trim13 and ubiquitination type mediated by Trim13.