GPATCH3调控RIG-I样受体介导的天然免疫反应的分子机制

发布时间：2018-03-31 08:58

本文选题：天然免疫反应　切入点：GPATCH3　出处：《中国科学院大学(中国科学院武汉病毒研究所)》2017年博士论文

【摘要】：天然免疫是机体在长期的进化过程中逐渐形成的一种免疫防御机制,是机体抵抗病毒感染的第一道防线。病毒感染宿主细胞后,细胞内的模式识别受体(pathogen recognition receptors,PRRs)能够识别病毒的病原相关分子模式(pathogen-associated molecular patterns,PAMPs),起始一系列复杂的信号级联反应,诱导I型干扰素及炎症基因的表达。随后,I型干扰素通过JAK-STAT信号通路诱导大量的抗病毒蛋白基因——干扰素刺激基因(interferon stimulated genes,ISGs)的转录。这些抗病毒蛋白及细胞炎症因子共同抑制病毒的感染及复制,并促进适应性免疫反应的发生。研究表明,RIG-I样受体(RIG-I like receprors,RLRs)成员RIG-I(retinoic acid-inducible gene-1)和MDA5(melanoma differentiation-associated gene 5)能识别病毒在感染与复制过程中释放在胞浆中的RNA。识别并结合病毒RNA的RIG-I及MDA5发生构象变化,招募并激活位于线粒体上的重要接头蛋白VISA(也称作MAVS、IPS-1或Cardif)。活化的VISA发生朊病毒样聚集并招募TRAF蛋白(TRAF2、TRAF5及TRAF6)、激酶TBK1及IKK,形成VISA复合物。随后,激酶TBK1及IKK,分别激活转录因子IRF3及NF-?B,进而诱导I型干扰素及炎症因子的产生。然而,过低或过高的I型干扰素产生都会引起多种疾病的发生,严重威胁机体健康。因此,病毒诱导I型干扰素产生的各个环节均受到机体严格的调控。在本研究中,为了进一步揭示抗病毒天然免疫信号转导的调控机制,我们通过表达克隆筛选的方式,筛选到一个能够显著抑制I型干扰素诱导表达的蛋白——GPATCH3(G-patch domain-containing protein 3)。GPATCH3广泛表达于多个组织中,但该分子的生物学功能目前还未被报道。我们的研究发现,过量表达GPATCH3能剂量依赖性地抑制RNA病毒SeV(Sendai virus)诱导的IFN-?启动子、ISRE及NF-?B报告基因的激活。降低细胞中GPATCH3的表达能够增强SeV诱导的IFNB1、IFNB1下游基因ISGs以及炎症基因的转录,但对DNA病毒HCMV及双链DNA(dsDNA)HSV-120诱导的IFNB1的转录没有影响。与此一致,降低GPATCH3的表达能够显著地抑制SeV和VSV(Vesicular stomatitis virus)两种RNA病毒的复制。我们进一步构建了GPATCH3基因敲除细胞,与野生型细胞相比,GPATCH3基因敲除细胞受到RNA病毒感染后,IFNB1的转录显著地增强。在作用机制研究方面,我们发现,VISA是GPATCH3的作用靶标分子。病毒感染能够增强GPATCH3与VISA的相互作用。进一步研究发现,GPATCH3能够减弱VISA-TBK1及VISA-TRAF6的相互作用,通过抑制VISA复合物的组装从而负调控RLRs介导的天然免疫反应。综上,我们的研究发现了一个参与调控RLRs信号通路的蛋白GPATCH3,并揭示了其作用机制。由于VISA是模式识别受体RIG-I及MDA5下游的关键接头蛋白,且VISA复合物的形成是RLRs介导的信号通路中极其重要的一步。因此,我们的研究对于深入认识RLRs介导的抗病毒天然免疫反应的调节机制具有重要意义。另外,本研究首次阐明了GPATCH3的生物学功能,为研究该分子的其他生物学功能奠定了基础。
[Abstract]:Natural immunity is an immune defense mechanism of the body gradually formed in the long process of evolution, is the first line of the body against infection. The virus infection of host cells, intracellular pattern recognition receptors (pathogen recognition receptors, PRRs) to identify pathogen associated molecular patterns virus (pathogen-associated molecular patterns, PAMPs), starting a series of complex signaling cascade, induce the expression of type I interferon and inflammatory gene. Subsequently, interferon type I through JAK-STAT signaling pathway induced by antiviral protein radicals by - interferon stimulated genes (interferon stimulated, genes, ISGs) infection and replication transcription. These antiviral protein and cell inflammatory factors together to suppress the virus, and promote the adaptive immune response occurred. The study shows that the RIG-I like receptor (RIG-I like receprors RLRs RIG-I (RET) members Inoic acid-inducible gene-1 (melanoma) and MDA5 differentiation-associated gene 5 RIG-I and MDA5) to identify the virus infection and replication process release RNA. recognition in the cytoplasm and combined with viral RNA conformational change to recruit and activate in important adaptor protein VISA on mitochondria (also known as MAVS, IPS-1 or Cardif) activation. VISA prion like aggregation and recruitment of TRAF protein (TRAF2, TRAF5 and TRAF6), TBK1 kinase and IKK, the formation of the VISA complex. Subsequently, TBK1 kinase and IKK, respectively. The activation of the transcription factor IRF3 and NF-? B, and the induction of type I interferon and inflammatory cytokines. However, type I interferon is too low or too high which will cause a variety of diseases, serious threat to human health. Therefore, all aspects of virus induction of type I interferon production are subject to strict regulation of the body. In this study, in order to further reveal the resistance The regulation mechanism of toxin innate immune signal transduction, we cloned by screening the expression and screening to a GPATCH3 protein significantly inhibited the induction of type I interferon expression (G-patch domain-containing protein 3).GPATCH3 is widely expressed in multiple tissues, but the biological function of this molecule has not yet been reported. Our study found overexpression of GPATCH3, dose dependently inhibited the RNA virus SeV (Sendai virus) induced IFN- promoter, and ISRE? NF-? B activation of reporter genes. Decreased expression of GPATCH3 in cells can enhance SeV induced IFNB1 transcription of IFNB1 downstream gene ISGs and inflammatory genes, but the DNA virus HCMV and double chain DNA (dsDNA) did not affect the transcription of HSV-120 induced by IFNB1. Consistent with this, reduce the expression of GPATCH3 can significantly inhibit SeV and VSV (Vesicular stomatitis virus) two RNA virus complex System. We further constructed the GPATCH3 gene knockout cells compared with wild-type cells, GPATCH3 gene knockout cells infected with RNA, the transcription of IFNB1 significantly enhanced. In the mechanism study, we found that VISA is the target molecule of GPATCH3. Virus infection can enhance the interaction between GPATCH3 and VISA further study found that the interaction of GPATCH3 can decrease VISA-TBK1 and VISA-TRAF6, through the assembly of VISA complexes to inhibit the negative regulation of RLRs mediated innate immune response. In summary, our study found that one participates in the regulation of RLRs signaling pathway protein GPATCH3, and reveal its mechanism. Because VISA is a key downstream joint pattern recognition receptors RIG-I and MDA5 protein, and the formation of the VISA complex is an important signal pathway mediated by RLRs in one step. Therefore, our study for deep understanding RLRs mediated the regulation mechanism of antiviral innate immune response. In addition, this study first elucidated the biological function of GPATCH3, which laid the foundation for studying other biological functions of the molecule.

【学位授予单位】：中国科学院大学(中国科学院武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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