【作者】 王晓芳；

【导师】 郑慧；

【作者基本信息】 苏州大学 ， 免疫学， 2017， 硕士

【摘要】 研究背景及目的:细胞因子信号抑制子SOCS1(Suppressor of cytokine signaling 1)是1997年发现的JAK-STAT通路中特异性负调控因子。研究发现,SOCS1主要通过三种途径对IFNs信号负向调节:与JAKs结合,抑制其激酶活性,抑制STAT1磷酸化,最终抑制JAK-STAT信号通路;通过SH2结构域与活化的JAKs,STATs结合,随之被蛋白酶体降解,从而发挥负向调节作用;SOCS1蛋白可以直接与IFNs受体结合,从而竞争结合受体酪氨酸磷酸化,抑制STATs磷酸化。目前,主要研究集中在SOCS1蛋白通过影响其他信号分子的活性而发挥其信号抑制的作用,而对其自身泛素化的调控及其抗病毒功能机制影响的研究尚未见报道。泛素化修饰是由泛素蛋白酶体系统(Ubiqutin-Proteasomesystem,UPS)精密调控的,其中去泛素化酶在UPS途径中起关键作用,成为目前国际泛素研究领域的前沿和热点。鉴定与疾病相关的重要蛋白的去泛素化酶,并寻找其小分子抑制剂,可能为以后临床靶向治疗相关疾病提供一定的科学依据。SOCS1作为一个IFNs负向调控因子,其泛素化调控和功能的研究尚浅。本课题通过探讨JOSD1调节IFNs信号中SOCS1的泛素化,旨在为临床靶向治疗病毒感染提供新的实验依据。研究方法:在HEK293T细胞中过表达Myc-SOCS1,利用免疫沉淀探究SOCS1的泛素化修饰及蛋白降解。利用去泛素化酶文库初步筛选SOCS1的去泛素化酶JOSD1,进一步利用Western blot技术和Real-time qPCR的技术从蛋白水平、相互作用、去泛素化水平、稳定性以及相关性方面鉴定SOCS1的去泛素化酶JOSD1。最后用免疫印迹和免疫荧光技术分析去泛素化酶JOSD1通过调控SOCS1蛋白水平对IFN介导的信号通路的影响。研究结果:1.SOCS1自身发生泛素化,并且主要发生K48位泛素化。2.利用DUB文库筛选出去泛素化酶JOSD1增加SOCS1蛋白水平。3.SOCS1与JOSD1相互作用。4.JOSD1调控SOCS1的泛素化水平。5.SOCS1的稳定性能够被JOSD1调节。6.在病毒刺激下,JOSD1与SOCS1蛋白水平具有相关性。7.SOCS1的去泛素化酶JOSD1抑制IFNα信号通路,从而负向调控其抗病毒功能。研究结论:JOSD1作为SOCS1的去泛素化酶,能够去SOCS1的泛素化水平稳定SOCS1的蛋白水平,从而抑制Ⅰ型IFN的信号通路,进一步抑制Ⅰ型IFN介导的抗病毒功能。更多还原

【Abstract】 Background and purpose:In 1997,SOCS1 was found as a negative regulator of the JAK-STAT pathway.Thereare three means by which SOCS1 negatively regulates IFNs signaling pathway.Firstly,SOCS1 can interact with JAKs and inhibits JAK kinase activity,thereby restricting JAK-STAT signaling pathway.Secondly,SH2 domain of SOCS1 binds to activated JAKs and STATs,and then results in their degradation by the protesome pathway.Thirdly,SOCS1 directly combines with IFNs receptor,thus competitively inhibits the phosphorylation of tyrosine and phosphorylation of STATs.The current studies clearly demonstrate that SOCS1 protein is a negative regulator in IFN signaling pathway.The regulation of SOCS1 ubiquitination and its antiviral response have not yet been reported.Ubiquitination is thr precise regulation by the ubiquitin proteasome system.Deubiquitases play crucial roles in the UPS,which become more and more popular.Identifying the important proteins involved in the diseases may provide a scientific basis for clinical targeted therapies.SOCS1 is a negative regulator of IFNs signaling,and the regulation and function of its ubiquitination are still unknown.This study aims to investigate the role of JOSD1 in regulating SOCS1 ubiquitination in IFN signaling,in order to provide a potential target for IFN-mediated antiviral therapy.Methods:Myc-SOCS1 was transfected into HEK293T cells.Ubiquitination and degradation were analyzed by Western blot.Deubiquitinase of SOCS1 protein were identified by using deubiquitinase library.Analyses of the DUB of SOCS1 with Western blot and Real-time qPCR in several respects,including protein level,interaction,deubiquitination,stability and correlation.The effect of JOSD1 via on stabilizing SOCS1 in IFN I signaling pathway and its antiviral function were explored by Western blot and immunofluorescence technique.Results:1.SOCS1 can form polyubiquitination modification and the lysine 48(K48)-linked polyUbiquitination is the major form of SOCS1 ubiquitination.2.Deubiquitinase JOSD1 up-regulates the level of SOCS1 protein.3.JOSD1 physically interacts with SOCS1.4.JOSD1 deubiquitinates SOCS1.5.JOSD1 stabilizes SOCS1.6.The levels of JOSD1 and SOCS1 undergo down-regulation during the early stage of viral infections.7.JOSD1 negatively regulates IFN-I-mediated signaling pathway and restricted its antiviral defense.Conclusions:As the deubiquitase of SOCS1,JOSD1 upregulates SOCS1 protein levels and deubiqutinates SOCS1,thereby enhancing SOCS1 protein stability.Importantly,JOSD1 inhibits IFN-I-induced signaling pathway and restricts IFN-I-mediated antviral response during viral infections.更多还原