TRIM38调节天然免疫的研究

发布时间：2018-05-04 15:31

本文选题：天然免疫 + TRIM38　；参考：《北京协和医学院》2010年博士论文

【摘要】：天然免疫是宿主抵抗病毒感染的第一道防线,也是激活适应性免疫的基础,在宿主清除病毒的免疫反应中具有关键作用,因此成为当前免疫学研究的热点。天然免疫应答是通过模式识别受体识别病原体相关分子模式来启动的。其中维甲酸诱导基因Ⅰ样受体(Retinoic acid-inducible gene-I like receptors, RLRs)是细胞内识别病毒RNA,诱导天然免疫反应的重要模式识别受体之一。RLRs主要存在于胞浆中,主要包括两个分子：维甲酸诱导基因Ⅰ(Retinoic acid-inducible gene-I,RIG-Ⅰ)和黑色素瘤分化相关基因5(Melanoma differentiation-associated gene-5),它们与病毒RNA结合之后,构象发生改变,从而募集下游效应分子IFN-8启动子激活因子1(IFN-βpromoter stimulator 1, IPS-1),引起TBK1/IKKi的激活,进一步引起干扰素调控因子(Interferon transcription factor, IRF) 3/7的磷酸化,最终诱导Ⅰ型干扰素(Type I interferon, I-IFN)、促炎症细胞因子等一系列抗病毒因子的产生。有关RLRs触发的天然免疫应答反应和信号传导过程的调节机制尚不十分清楚,成为近几年研究热点。 TRIM (Tripartite motif protein)家族是一类存在于胞浆中的,含有保守结构域的蛋白质,目前已经发现70多个成员,它们参与细胞增殖、分化、致癌和程序性死亡等很多细胞活动。近年的研究表明,部分TRIM蛋白具有调节免疫信号通路和抗病毒功能,但是目前仅有几个TRIM蛋白的功能得到阐述。为了进一步阐明TRIM蛋白在先天免疫信号调节中的功能,深入了解机体抗病毒的免疫机制,我们开展了对TRIM蛋白的研究。前期实验表明TRIM38可以抑制仙台病毒(Sendai virus, SeV)刺激的Ⅰ型干扰素的产生,我们推测TRIM38可能具有调节天然免疫的功能。目前有关TRIM38的基本功能及其调节天然免疫的机制还未见报道。本研究拟对TRIM38调控天然免疫的机制进行探讨。首先,我们分析了TRIM38的基本功能。细胞定位实验表明TRIM38主要在细胞质中表达。TRIM38具有TRIM蛋白家族典型的结构域特征,含有E3泛素连接酶典型的特征RING结构域。通过将TRIM38与泛素化质粒共转染,免疫共沉淀结果表明TRIM38可以增加细胞内的泛素化水平,并可自身泛素化,与TRIM22具有相似的特性。蛋白翻译后的泛素化修饰有两种,一种是通过48位赖氨酸(K)连接的多泛素链修饰,诱导蛋白降解；另外一种是通过63位赖氨酸(K)63连接的多泛素链修饰,可以激活蛋白降解以外的其它功能。我们进一步的研究表明TRIM38可以催化自身的K48连接的泛素化,提示TRIM38有可能通过该途径降解。随后,我们对TRIM38调节天然免疫反应的机制进行了探讨。首先,通过体外荧光素酶报告基因实验表明TRIM38可以抑制RIG-Ⅰ诱导的IFNβ、ISG56和ISRE启动子的激活,但是对于MDA5、IPS-1、TBK1和IKKi诱导的IFNβ启动子的激活没有抑制作用。结果提示TRIM38可能通过作用于RIG-Ⅰ抑制Ⅰ-IFN信号通路。免疫共沉淀结果表明RIG-Ⅰ与TRIM38存在于同一复合物中,且TRIM38可以与RIG-Ⅰ的N端和C端相互作用。进一步研究表明TRIM38与RIG-Ⅰ的相互作用抑制了其与下游效应分子IPS-1以及TBK1的结合,进而抑制IFNβ的产生。通过构建TRIM38各个结构域的突变体,经荧光素酶和免疫共沉淀实验证明TRIM38的RING结构域对于其泛素化和对RIG-Ⅰ信号通路的负反馈调节起着重要的作用。综上所述,TRIM38是一个具有典型TRIM蛋白家族保守结构域的蛋白,主要存在于细胞质,可自身泛素化,并可增加细胞内的泛素化水平。TRIM38可以通过与IPS1和TBK1竞争性结合RIG-Ⅰ,对RIG-Ⅰ介导的Ⅰ型干扰素通路进行负向调节。本研究发现了一个新的负向调节先天免疫信号通路的蛋白——TRIM38,并对其基本功能及负向调节RIG-Ⅰ介导的Ⅰ-IFN信号转导通路的机制进行了初步研究,该研究有助于进一步阐明机体的先天免疫调节机制,并为免疫调节药物靶点的设计提供依据。
[Abstract]:Natural immunity is the first line of defense against virus infection and the basis for activating adaptive immunity, which plays a key role in the immune response of the host. Therefore, it has become a hot spot in the current immunology research. Acid induced gene type I receptor (Retinoic acid-inducible gene-I like receptors, RLRs) is one of the important pattern recognition receptors for the identification of virus RNA in cells, which mainly exists in the cytoplasm, mainly including two molecules: retinoic acid inducible gene I (Retinoic acid-inducible gene-I, RIG- I) and melanin Tumor differentiation related gene 5 (Melanoma differentiation-associated gene-5), after combining with viral RNA, conformation changes, thus raising the downstream effector IFN-8 promoter Activator 1 (IFN- beta promoter stimulator 1, IPS-1), causing TBK1/IKKi activation, further causing interferon regulatory factors (Interferon transcription) The phosphorylation of actor, IRF) 3/7 eventually induces the production of a series of anti-virus factors such as Type I interferon (I-IFN) and inflammatory cytokines. The regulatory mechanism of natural immune response and signal transduction triggered by RLRs is still not very clear, which has become a hot research topic in recent years.
The TRIM (Tripartite motif protein) family is a class of proteins that exist in the cytoplasm and contains a conservative domain. At present, more than 70 members have been found. They are involved in cell proliferation, differentiation, carcinogenesis and programmed death. Recent studies have shown that the partial TRIM protein has the regulation of the immune signal pathway and the antiviral function. But only a few TRIM proteins have been described at present. In order to further clarify the function of TRIM protein in the regulation of innate immune signals and to understand the immune mechanism of the organism, we have carried out a study on the TRIM protein. Earlier experiments showed that TRIM38 could inhibit the type I interference stimulated by Sendai virus (Sendai virus, SeV). We speculate that TRIM38 may have the function of regulating natural immunity. The basic functions of TRIM38 and the mechanism of regulating natural immunity have not yet been reported. This study is to discuss the mechanism of the regulation of natural immunity by TRIM38.
First, we analyzed the basic functions of TRIM38. Cell localization experiments showed that TRIM38 mainly expressed.TRIM38 in the cytoplasm with typical domain characteristics of the TRIM protein family, containing the typical characteristic RING domain of E3 ubiquitin ligase. By CO transfection of TRIM38 with ubiquitination plasmids, the results of immunoprecipitation showed that TRIM38 could increase fine. The ubiquitination level in the cell, and its own ubiquitination, is similar to that of TRIM22. There are two kinds of ubiquitination modification after protein translation, one is modified by the multi ubiquitin chain modified by 48 lysine (K), and the other is modified by the multi ubiquitin chain linked by 63 lysine (K) 63, which can activate protein degradation Further studies have shown that TRIM38 can catalyze ubiquitination of its K48 connections, suggesting that TRIM38 may be degraded through this pathway.
Then, we explored the mechanism of TRIM38 regulating the natural immune response. First, through the in vitro luciferase reporter gene experiment, TRIM38 can inhibit the activation of IFN beta, ISG56 and ISRE promoters induced by RIG- I, but there is no inhibitory effect on the activation of IFN beta promoter induced by MDA5, IPS-1, TBK1 and IKKi. The results suggest TRIM3. 8 may inhibit I -IFN signal pathway by acting on RIG- I. The result of immunoprecipitation indicates that RIG- I and TRIM38 exist in the same complex, and TRIM38 can interact with the N end and C end of RIG- I. Further studies show that the interaction of TRIM38 and RIG- I inhibits the binding of its downstream effector molecule IPS-1 and TBK1. The production of IFN beta. Through the construction of TRIM38 mutants in each domain, the RING domain of TRIM38 plays an important role in its ubiquitination and negative feedback regulation on the RIG- I signaling pathway by the luciferase and co immunoprecipitation experiments.
To sum up, TRIM38 is a protein with typical TRIM protein family conserved domains, which mainly exist in cytoplasm, can be ubiquitin and can increase the level of ubiquitination in cells,.TRIM38 can be negatively regulated by RIG- I mediated interferon pathway mediated by RIG- I mediated by RIG- I with IPS1 and TBK1. A new negative regulation of the protein of the innate immune signal pathway, TRIM38, has been preliminarily studied on its basic function and the mechanism of the negative regulation of RIG- I mediated I -IFN signal transduction pathway. This study is helpful to further elucidate the body's innate immune regulation mechanism and provide the basis for the design of immunoregulatory targets.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1

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