TRIM38调节天然免疫的研究

【作者】 刘新蕾；

【导师】 洪涛； 王健伟；

【作者基本信息】 北京协和医学院 ， 病原生物学， 2010， 博士

【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。更多还原

【Abstract】 The innate immune response is the first line of defense against viruses and is the foundation of adaptive immune systems. Initially, host sensor molecules detect specific pathogen-associated molecular patterns (PAMPs) and activate the innate immune system through pathogen recognition receptors (PRRs). Among those PRRs, Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) can detect viral RNA in cytoplasm and induce interferons and proinflammatory cytokines, thus playing a pivotal role in host defense. Specificly, RLRs selectively detect the non-self-RNA, and induce ATP-dependent conformational change to form a dimmer or oligomer, which allows its interaction with the downstream adapter protein IPS-1 (IFN-βpromoter stimulator 1). IPS-1 resides on the outer membrane of mitochondria, and recruitment of RIG-I to IPS-1 leads to the activation the two IKK-related kinases, TANK(TRAF family member-associated NF-κB activator) binding kinase 1(TBK1) and inducible inhibitor-KB kinase (IKKi), both of which phosphorylate interferon refulatory factor 3/7, in turn, induce the expression of type I interferon. In regards to the importance of innate immune signaling regulation, extensive study is required to identify novel immune regulating molecules.The tripartite motif (TRIM) protein family was originally described as the RBCC family because members belonging to this family contain a RING finger, one or two B-boxes, followed by a coiled-coil domain. There are now more than 70 TRIM proteins known in humans. Members of the TRIM protein family are involved in various cellular processes, including cell proliferation, differentiation, oncogenesis and apoptosis. Importantly, some TRIM proteins involve in innate immune signaling regulation and/or display antiviral properties. However, only few TRIM proteins were fully elucidated.TRIM38 located in the chromosome 6p21.3. In comparison of the TRIM38 sequence with other TRIM proteins, we found that TRIM38 contains several evolutionary conserved molecular domains, including a RING finger, two B-boxes, and one pry/spry domain. Previous studies have demonstrated that the activity of most E3 ligases is specified by a RIGN domain. Accordingly, our observations reveal that forced expression of TRIM38 protein results in a remarkable increase in the level of ubiquitin-protein conjugates. Further studies demostrated that TRIM38 was conjugated with K48-linked poly-ubiquitin chains, which suggests that TRIM38 targets itself for proteasomal degradation through the K48-linked poly-ubiquitylation, and which indicates that TRIM38 may function as an E3 ubiquitin ligase.To explore the role of TRIM38 in the regulation of innate immune signaling, the interferon-βpromoter activity assays were conducted. Results showed that overexpression of TRIM38 inhibits Sendai virus(SeV) triggered type I interferon induction. To investigate the mechanism by which TRIM38 inhibits the innate immune signaling,293T cells were transfected with plasmids encoding TRIM38 together with RIG-1, MDA5, IPS1, TBK1, IKKi, and interferon-βreporter plasmids. Results showed that TRIM38 inhibits RIG-I-mediated IFN-P promoter activity in a dose dependent manner, wheares MDA5, IPS1, TBK1 or IKKi mediated activation of IFN-P promoter remain unattenuated. Immunoprecipitation studies suggest that TRIM38 associates with RIG-I, and TRIM38 could disrupt the interaction between RIG-I and IPSl/TBKl.These findings showed that TRIM38 may function as an E3 ubiquitin ligase and is a negative regulator of RIG-I-mediated innate antiviral response. The investigations on the function and mechanism of TRIM38 will facilitate the development of new strategies to control immune injury, and also will contribute to the better understanding of immune recognition and immune regulation.更多还原