【作者】 王敏；

【导师】 郑学星； 赵伟；

【作者基本信息】 山东大学 ， 公共卫生（专业学位）， 2023， 硕士

【摘要】 研究背景病毒、细菌感染产生的外源性致病DNA以及肿瘤等疾病导致的内源性不稳定异位DNA能够被胞质内的DNA感受器(DNA receptors)识别后,宿主产生炎症反应并且诱导Ⅰ型干扰素(Interferon,IFNs)生成。环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase,cGAS)作为胞质内DNA感受器,识别双链DNA后激活干扰素基因刺激因子(stimulator of interferon genes,STING),STING激活后转移到高尔基体,招募并磷酸化TANK结合激酶 1(TANK-bindingkinase 1,TBK1)和干扰素调节因子 3(Interferon regulatory Factor 3,IRF3),诱导Ⅰ型IFNs的产生。STING是cGAS介导的级联反应的枢纽。STING的活化异常导致其所介导的干扰素和炎症反应异常,异位DNA不能被有效清除致使自身炎性疾病和癌症等疾病发生。因此,精准调节STING的活化对于维持免疫稳态至关重要。蛋白翻译后修饰(Post-translational modification of proteins,PTMs)能调节蛋白质结构和功能。STING 的磷酸化(Phosphorylation)、泛素化(Ubiquitination)、SUMO 化等 PTMs 通过改变 STING 的结构、定位、稳定性来精准调节STING的活化。相关研究发现STING的羰基化通过抑制STING从内质网到高尔基体的转位而抑制其活化。RNF115和TRIM56催化的K63位的泛素化,分别促进STING的转位和二聚化。而RNF5、RNF90、TRIM30和TRIM29介导的K48位的泛素化增强STING的降解,抑制STING活化。分泌的1型IFNs可以被细胞膜上的干扰素受体识别,诱导包括ISG15在内的大量的干扰素刺激基因(Interferon stimulates genes,ISGs)的表达。ISG15不仅以游离的形式促进促炎因子和趋化因子的释放,还可以在多种酶的作用下连接到靶蛋白上使其发生ISG化。ISG化是一种三级酶联反应:ISG15与E1酶UBE1L结合后被转移到E2酶UBCH8上,随后通过E3连接酶与靶蛋白结合。HERC5(人类)和HERC6(小鼠)是介导ISG化最主要的E3连接酶,催化大多数底物与ISG15的结合。ISG化可以改变蛋白的稳定性、定位和功能。MDA5的ISG化是其激活和限制病毒复制所必需的;而RIG-Ⅰ的ISG化抑制其介导的通路,以防过度活化。然而,STING是否可以发生ISG化修饰,ISG化在cGAS-STING通路中是否发挥作用是未知的。研究目的1)研究HERCs在cGAS-STING通路活化中的调控作用,并阐明其发生的分子机制。2)阐明STING活化过程中STING ISG化与泛素化修饰的相互作用,进一步揭示cGAS-STING通路的活化调控机制。3)探究HERC5(人)和HERC6(小鼠)在cGAS-STING通路活化过程中的作用。研究方法1)利用Herc6基因敲除小鼠、转染Herc6siRNA的小鼠腹腔巨噬细胞、转染HERC5 siRNA 的人单核巨噬细胞(Human myeloid leukemia mononuclear cells,THP-1),通过ELISA及RT-PCR实验研究发现Herc6基因敲除、敲低、或HERC5基因敲低后cGAS-STING通路活化后诱导的IFN-β的转录和释放减少。然后,通过免疫印迹研究发现HERCs增强STING下游分子(TBK1、IRF3、信号传导及转录激活蛋白1(Signaltransducer andactivatoroftranscription 1,STAT1))的磷酸化以及ISGs 比如干扰素诱导蛋白与四肽重复 1(Interferon induced protein with tetratricopeptiderepeats 1 Gene,IFIT1)、干扰素诱导蛋白与四肽重复 2(Interferon induced protein with tetratricopeptide repeats 2 Gene,IFIT2)、人 C-X-C 基序趋化因子配体 10(Small inducible cytokine subfamily B(Cys-X-Cys)member 10,CXCL10)、黏病毒耐药蛋白 1(Myxovirus resistantprotein 1,Max1)等的转录。这些结果说明HERCs促进cGAS-STING信号通路活化。2)利用多种小鼠疾病模型(DNA病毒HSV-1感染模型和DMXAA诱导STING激活模型)和多种感染方式(腹腔注射和脑室内注射)通过ELISA、RT-PCR、免疫印迹、免疫组织化学染色实验,发现Herc6-/小鼠体内IFN-β、TNF-α和IL-6分泌显著低于对照组Herc6+/+小鼠。接下来我们通过免疫共沉淀、免疫荧光等试验,发现HERCs可以与STING结合而不与cGAS结合。然后,通过免疫印迹研究发现HERCs增强STING下游分子(TBK1、IRF3、STAT1)的磷酸化以及增强 ISGs(IFIT1、IFIT2、CXCL10、Max1)的转录。再利用蛋白质合成抑制剂CHX、泛素-蛋白酶体抑制剂MG132等抑制剂作用于Herc6-/-小鼠、转染Herc6 siRNA的小鼠腹腔巨噬细胞和HEK293T细胞。结果证实HERCs可通过抑制STING的蛋白酶体降解的途径来稳定其表达。3)转染相关质粒到HEK293T细胞,通过免疫共沉淀和免疫印迹分析发现了 STING可以在转位之前发生ISG化,并且HERCs可以增强的STING的ISG化而抑制泛素化。同时在HEK293T细胞中转染HERC5的E3连接酶酶失活位点突变质粒HERC5-C994A,通过免疫共沉淀和免疫印迹发现了该突变体失去了增强STING ISG化和减弱STING泛素化的能力。研究结果1)本研究明确了 E3 ISG连接酶HERCs在cGAS-STING信号通路活化中的作用,并揭示了 HERCs通过催化STING发生ISG化修饰来拮抗STING生理状态下的泛素化降解途径,从而正向调控STING表达水平的分子机制。2)本研究揭示了 STING的ISG化调节STING蛋白表达的具体机制。同时发现STING泛素化和ISG化之间的相互作用关系。STING的蛋白表达与cGAS-STING通路的活化相关。为治疗相关免疫异常疾病提供新的靶点。更多还原

【Abstract】 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.更多还原