马TRIM5α激活AP-1信号通路的分子机制研究

发布时间：2017-12-26 18:29

本文关键词：马TRIM5α激活AP-1信号通路的分子机制研究　出处：《中国农业科学院》2016年硕士论文　论文类型：学位论文

【摘要】：TRIM5α是TRIM家族的一个成员,具有典型的RBCC结构(RING、B-box和Coil-Coiled)和一个C端SPRY结构域。TRIM5α是存在于多种宿主细胞内的抗逆转录病毒天然免疫限制因子,可通过SPRY结构靶向降解逆转录病毒衣壳蛋白(CA)和激活天然免疫来发挥抗病毒功能。我们前期研究证明,相对于其它动物而言,马TRIM5α具有独特的分子特征,在进化过程中缺失了重要的C末端结构域而失去降解EIAV的CA蛋白功能,但过表达后仍明显限制马传染性贫血病病毒(EIAV)的感染。为了进一步确定这个结果,本论文利用慢病毒表达载体建立了稳定表达马TRIM5α和恒河猴TRIM5α的HEK293细胞系,并进行了抗病毒实验。结果显示马TRIM5α表现出明显的限制EIAV感染作用。同时,利用AP-1和NF-κB荧光素酶报告系统检测马TRIM5α激活天然免疫的活性,结果证明了马TRIM5α可以很好地激活AP-1和NF-κB信号通路。上述结果说明由于马TRIM5α没有完整的SPRY结构导致其不能靶向降解EIAV的Gag蛋白,但是马TRIM5α仍可以发挥抗病毒功能并激活天然免疫信号,因此这提示我们马TRIM5α可能是通过激活天然免疫来抵抗病毒感染。灵长类TRIM5α发挥天然免疫激活功能依赖完整的SPRY结构域,而马TRIM5α蛋白仅包含五分之一SPRY结构,仍能显著激活天然免疫信号通路。这提示不同种属TRIM5α发挥激活天然免疫功能的分子基础与分子机制不同。为了确定马TRIM5α激活AP-1信号通路的关键氨基酸,本研究对马TRIM5α从C端开始进行截短表达,构建了截短表达突变体(eqTRIM5-333、eqTRIM5-328、eqTRIM5-324、eqTRIM5-318、eqTRIM5-310、eqTRIM5-303、eqTRIM5-282),利用双荧光素酶报告系统初步确定了马TRIM5α激活AP-1的关键氨基酸在第303位至第310位氨基酸这7个氨基酸之间。因此在eqTRIM5-310的基础上对这7个氨基酸逐一进行了点突变,构建了重组质粒(eqTRIM5-310-303、eqTRIM5-310-304、eqTRIM5-310-305、eqTRIM5-310-306、eqTRIM5-310-307、eqTRIM5-310-308、eqTRIM5-310-309),确定了马TRIM5α激活AP-1的关键氨基酸是第303位苏氨酸和第304位亮氨酸,并且这两个氨基酸对其激活NF-κB也是重要的。为进一步研究马TRIM5α激活AP-1信号通路的分子机制,我们将马TRIM5α及其突变体分别和TAB2、TAK1共转293T细胞,结果表明马TRIM5α及其突变体可以降解TAB2,但是失去激活作用的突变体均失去了与TAK1的相互作用。通过将马TRIM5α及其突变体、TAK1、泛素(Ub)共转293T细胞,结果表明马TRIM5α可能是通过泛素化TAK1进而激活下游的AP-1和NF-κB信号通路。本研究揭示了马TRIM5α激活AP-1信号通路的分子机制,提示马TRIM5α可能是通过激活天然免疫从而限制病毒感染,这为进一步理解宿主细胞天然免疫分子进化及与病毒相互适应的机理提供了研究基础,并且马TRIM5α可作为潜在的广谱抗病毒新型分子,在开发新型抗病毒制剂和药物等方面具有应用前景。
[Abstract]:TRIM5 alpha is a member of the TRIM family and has a typical RBCC structure (RING, B-box and Coil-Coiled) and a C end SPRY domain. TRIM5 alpha is an antiretroviral natural immune limiting factor existing in various host cells. It can play the antiviral function by targeting the degradation of retroviral capsid protein (CA) by SPRY structure and activating innate immunity. Our previous studies have demonstrated that compared with other animal, horse TRIM5 alpha has unique molecular characteristic, in the evolutionary process of the deletion of C terminal domain important without degradation of EIAV CA protein function, but still significantly restricted expression of equine infectious anemia virus (EIAV) infection. In order to further confirm this result, a HEK293 cell line stably expressing horse TRIM5 alpha and Ganges RIver monkey TRIM5 alpha was established by Lentivirus Expression Vector, and antiviral experiments were carried out. The results showed that the TRIM5 alpha showed a significant effect on EIAV infection. Meanwhile, AP-1 and NF- kappa B luciferase reporter system were used to detect the activity of natural immunization activated by horse TRIM5 TRIM5, which proved that horse TRIM5 alpha can activate AP-1 and NF- kappa B signaling pathway. The results show that because the horse TRIM5 alpha no complete structure of the SPRY it can not lead to targeted degradation of EIAV Gag protein, but the horse TRIM5 alpha can still play antiviral function and activate innate immune signal, so this suggests that Ma TRIM5 alpha may be through the activation of innate immunity against viral infection. Primate TRIM5 alpha exerts natural immune activation function and relies on a complete SPRY domain, while the horse TRIM5 alpha protein contains only 1/5 SPRY structure, which still can significantly activate the innate immune signaling pathway. This suggests that different species of TRIM5 alpha play a different molecular basis and molecular mechanism to activate the natural immune function. In order to determine the key amino acid horse TRIM5 alpha activation of the AP-1 pathway, the study of Ma TRIM5 alpha began expression of truncated from the C, constructed the expression of truncated mutants (eqTRIM5-333, eqTRIM5-328, eqTRIM5-324, eqTRIM5-318, eqTRIM5-310, eqTRIM5-303, eqTRIM5-282), using the dual luciferase reporter system initially identified the key amino acid horse TRIM5 alpha activation AP-1 between 303rd to 310th amino acid of the 7 amino acids. Therefore, on the basis of the eqTRIM5-310 of these 7 amino acids by point mutations, to construct recombinant plasmid (eqTRIM5-310-303, eqTRIM5-310-304, eqTRIM5-310-305, eqTRIM5-310-306, eqTRIM5-310-307, eqTRIM5-310-308, eqTRIM5-310-309), to determine the key amino acids of TRIM5 alpha horse activation AP-1 is 303rd bit and 304th bit leucine and threonine, these two amino acids of the the activation of NF- K B is important. The molecular mechanism of activation of AP-1 signaling pathway for the further study of horse TRIM5 alpha, we will ma TRIM5 alpha and TAB2, TAK1 and its mutants were co transfected 293T cells. The results showed that the horse TRIM5 alpha and its mutants can degrade TAB2, but lost activation mutants lost the interaction with TAK1. Through the transformation of horse TRIM5 alpha and its mutant, TAK1 and ubiquitin (Ub) into 293T cells, the results indicate that TRIM5 TRIM5 may activate downstream AP-1 and NF- kappa B signaling pathway through ubiquitination. This study reveals the molecular mechanism of TRIM5 alpha activation of the AP-1 pathway, suggesting that TRIM5 may be through the activation of alpha horse natural immunity to limit virus infection, it may provide a basis for the further understanding of the host innate immune cells and molecular evolution mechanism to adapt to each other with the virus, and the horse can be used as a broad-spectrum antiviral TRIM5 a new molecular potential and has the application prospect in the development of new antiviral agents and drugs etc..
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S821

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