GLTSCR2负调控Ⅰ型干扰素及抗病毒应答的分子机制

发布时间：2018-01-07 06:02

本文关键词：GLTSCR2负调控Ⅰ型干扰素及抗病毒应答的分子机制　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：胶质瘤抑制候选基因 2(Glioma tumour-suppressor candidate region gene 2,GLTSCR2)基因位于人类肿瘤抑制功能区域19号染色体上,编码分子量为60kDa的细胞蛋白。在肿瘤学中有广泛的研究,但在病毒学领域中的功能尚不清楚。本研究探索GLTSCR2调控Ⅰ型干扰素的产生及细胞抗病毒应答的分子机制,以揭示GLTSCR2在病毒复制中的作用。本研究首先采用基因沉默或过表达细胞中的GLTSCR2基因,通过荧光定量PCR、病毒效价测定以及免疫印迹试验,发现GLTSCR2蛋白与病毒的复制直接相关。进一步的GLTSCR2截短体、突变体试验表明GLTSCR2羧基末端330-432位氨基酸,特别是出核位点氨基酸对病毒的复制促进作用至关重要。病毒感染或poly(I:C)刺激可引起GLTSCR2的细胞定位发生改变:从细胞核移位到细胞质中,而出核位点缺失的GLTSCR2则不能迁移出核,无论是否有病毒感染。为了探索GLTSCR2在促进病毒复制的机理,我们采用双荧光素酶报告系统试验,发现GLTSCR2显著抑制IFN-β和NF-κB的活化,进一步运用免疫共沉淀和荧光素酶试验,结果表明GLTSCR2,特别是羧基末端GLTSCR2靶向RIG-Ⅰ,并非MAVS、TBK-1,因此负调控IFN-β的活化,而出核位点缺失的GLTSCR2则不能显著促进IFN-β的活化。为了揭示GLTSCR2负调控IFN-β产生的分子机制,进行了 GLTSCR2对RIG-Ⅰ磷酸化或泛素化突变体试验,结果表明GLTSCR2并非通过磷酸化RIG-Ⅰ负调控IFN-β的产生,而是通过特异性移除RIG-Ⅰ上K63偶联的泛素化链抑制RIG-Ⅰ的活化,从而负调控RIG-Ⅰ信号通路。进一步采用泛素化试验,结果表明GLTSCR2能通过USP15,并非USP3、USP21或TRIM25,进而去除RIG-Ⅰ上K63偶联的泛素化链,从而负调控RIG-Ⅰ活化和IFN-β的产生。最后,我们将GLTSCR2的截短体片段串联穿透肽,构建G4-TAT基因,进行原核表达,细胞试验结果表明,重组蛋白G4-TAT可以穿透细胞膜进入细胞,抑制Ⅰ型干扰素信号通路,负调控细胞抗病毒应答,因而利于病毒复制。综上所述,GLTSCR2可以促进病毒的复制,病毒的感染导致GLTSCR2从细胞核移位到细胞质中,并通过招募USP15去泛素化RIG-Ⅰ的K63位泛素化链负调RIG-Ⅰ的活化,从而负调控Ⅰ型干扰素的抗病毒应答。而GLTSCR2羧基末端原核表达蛋白亦能负调控Ⅰ型干扰素的产生从而促进病毒的复制。
[Abstract]:Glioma 2 candidate genes (Glioma tumour-suppressor candidate region gene 2, GLTSCR2) gene in human tumor suppressor function region on chromosome 19, encoding a molecular weight of 60kDa cell protein. There has been extensive research in oncology, but in the field of Virology in function is unclear. This study explores the molecular mechanism and cell antiviral response GLTSCR2 regulation of type I IFN, in order to reveal the role of GLTSCR2 in viral replication. In this study we used the gene silencing or overexpression of GLTSCR2 gene in the cells by fluorescence quantitative PCR, virus titer determination and Western blot test, found GLTSCR2 protein and the replication of the virus directly related. The truncated GLTSCR2 further. Experiments show that the GLTSCR2 mutant 330-432 carboxy terminal amino acids, especially nuclear sites of virus replication amino acids promote the key to The virus infection or poly (I:C) stimulation can induce the cellular localization of GLTSCR2 is changed from nuclear translocation to the cytoplasm and nuclear sites of deletion of GLTSCR2 is not a nuclear migration, regardless of whether there is a virus infection. In order to explore the mechanism of GLTSCR2 in promoting viral replication, we used dual luciferase reporter system test found the activation of GLTSCR2 significantly inhibited IFN- and NF- Beta Kappa B, further by immunoprecipitation and luciferase test, the results show that GLTSCR2, especially the carboxyl terminus of GLTSCR2 targeting RIG- I, TBK-1 is not MAVS, therefore, the negative regulation of IFN- beta activation, and nuclear gene deletion of GLTSCR2 does not significantly promote the activation of IFN- beta. In order to reveal the molecular mechanism of GLTSCR2 negative regulation of IFN- beta, the GLTSCR2 of RIG- I phosphorylation or ubiquitination of mutant test results show that the GLTSCR2 is not through the phosphorylation of RIG- of the negative regulation of IFN- beta Production, but through the ubiquitin chain specific RIG- 1 K63 remove coupling to inhibit activation of RIG- I, RIG- I and negatively regulates the signal pathway. The test results show that ubiquitination, GLTSCR2 through USP15, not USP3, USP21 or TRIM25, and then remove the ubiquitin chain K63 coupling RIG- 1, which the negative regulation of RIG- activation and IFN- beta 1. Finally, we will be truncated fragments of GLTSCR2 tandem penetrating peptides, construct G4-TAT gene, prokaryotic expression, cell test results showed that the recombinant protein G4-TAT can penetrate the cell membrane into the cell, inhibition of type I interferon signaling pathway negatively regulates antiviral response, which is conducive to the virus copy. In conclusion, GLTSCR2 can promote the replication of the virus, the virus infection from GLTSCR2 nuclear translocation to the cytoplasm, and through the recruitment of USP15 to RIG- I K63 a ubiquitin ubiquitin chain negative The activation of RIG- I negatively regulates the antiviral response of type I interferon, while the prokaryotic expression protein of GLTSCR2 carboxy terminal can also negatively regulate the production of type I interferon and promote the replication of virus.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q939.91;Q78

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