TRIM65在MDA-5抗病毒信号活化过程中的关键作用及其机制探究

发布时间：2018-05-10 21:14

本文选题：天然免疫抗病毒信号 + RIG-I样模式识别受体　；参考：《中国科学技术大学》2017年博士论文

【摘要】：天然免疫抗病毒信号活化是机体免疫系统抵抗外界病毒感染的第一道防线,而RIG-I样模式识别受体所调控的抗病毒信号活化是天然免疫抗病毒系统中非常重要的一个组成部分。RIG-I样模式识别受体能够特异性地感知RNA病毒的入侵,活化下游信号通路,诱导Ⅰ型干扰素及其他细胞因子的产生,诱发机体对病毒实施有效的抵抗。RIG-I样模式识别受体家族主要有三个成员,分别是RIG-I、MDA-5以及LGP2。其中RIG-I和MDA-5分另能够特异性地识别不同种类的RNA病毒,而LGP2主要是针对RIG-I和MDA-5的活化过程起到调控作用。RIG-I或MDA-5功能的缺失会使得机体不能够有效抵抗RNA病毒的感染,导致机体出现多种病症甚至死亡。而二者功能的过度活化同样会导致机体稳态的失衡,出现一系列自身免疫性疾病,如红斑狼疮。可见R1G-I和MDA-5功能的正常发挥对于机体抵抗RNA病毒以及稳态的维持是不可或缺的。目前,对于RIG-I信号活化机制的探究相对较为清楚,但是对于MDA-5是如何被活化的目前的研究还知之甚少。目前已知MDA-5与RIG-I相似也会被PP1介导发生磷酸化修饰,而其他修饰类型是否在其中也起着重要的作用目前仍是未知的。尤其是在RIG-I的活化过程中起着关键作用的泛素化修饰,是否在MDA-5活化过程中也起着至关重要的作用,以及有哪些泛素连接酶参与到其中目前还不清楚。基于以上原因,本文以前人的研究为基础,针对RNA活化MDA-5的分子机制展开了一系列探讨。在本文的研究中,我们利用免疫共沉淀及质谱分析的方法筛选到与MDA-5可能存在相互作用的蛋白。在众多筛选到的蛋白中,我们发现,一种E3泛素连接酶—TR1M65,与MDA-5存在着非常强的相互作用。后续的一系列实验也证实了这一相互作用的存在。为了探究二者的相互作用对于MDA-5信号活化的作用,我们构建了Trim65缺陷小鼠,并利用MDA-5信号特异性活化剂处理缺陷鼠以及缺陷鼠来源的细胞。结果发现,TRIM65表达缺失后,MDA-5抗病毒信号通路的活化受到了极大程度的抑制。在人源细胞中我们也得到了类似的结果。机制方面,我们发现TRIM65能够利用其自身的E3泛素连接酶的活性诱导MDA-5的743位赖氨酸发生K63连接方式的泛素化。进一步我们利用SDD-AGE以及激光共聚焦技术,发现了 TRIM65对MDA-5泛素化的诱导能够显著促进MDA-5寡聚体的形成,而MDA-5寡聚体的形成是MDA-5抗病毒信号活化的关键步骤。通过以上研究,我们不仅发现了 TRIM65是MDA-5信号活化过程中的一个关键组分,同时还证实了泛素化调控对于MDA-5寡聚化以及活化都是至关重要的,并且还为MDA-5相关疾病的治疗找到了新的关键靶点。
[Abstract]:The activation of innate antiviral signals is the first line of defense of the body's immune system against external virus infection. The activation of antiviral signal regulated by RIG-I like pattern recognition receptor is a very important part of innate immune antiviral system. RIG-I pattern recognition receptor can specifically perceive the invasion of RNA virus and activate downstream signal pathway. There are three main members of the RIG-I-like pattern recognition receptor family: RIG-ImDA-5 and LGP22, which induce the production of interferon type I and other cytokines. Among them, RIG-I and MDA-5 can specifically identify different RNA viruses, while LGP2 mainly regulates the activation process of RIG-I and MDA-5. The absence of RIG-I or MDA-5 makes the body unable to resist the infection of RNA virus effectively. Causes the body to appear many kinds of sickness even dies. The over-activation of both functions also leads to homeostasis and a series of autoimmune diseases such as lupus erythematosus. It can be seen that the normal function of R1G-I and MDA-5 is indispensable for the body to resist the RNA virus and maintain the homeostasis. At present, the mechanism of RIG-I signal activation is relatively clear, but little is known about how MDA-5 is activated. It is known that the phosphorylation of MDA-5 and RIG-I is mediated by PP1, but it is still unknown whether other modification types play an important role in it. Especially the ubiquitin modification which plays a key role in the activation of RIG-I and whether it also plays an important role in the activation of MDA-5 and which ubiquitin ligase is involved in it is not clear. Based on the previous studies, a series of studies on the molecular mechanism of MDA-5 activation by RNA have been carried out. In this study, we used immunoprecipitation and mass spectrometry to screen proteins that might interact with MDA-5. Among the selected proteins, we found that an E3 ubiquitin ligase TR1M65 interacts very strongly with MDA-5. A series of subsequent experiments have confirmed the existence of this interaction. In order to investigate the effect of the two interactions on the activation of MDA-5 signals, we constructed Trim65 deficient mice, and used MDA-5 signal specific activators to treat the cells from deficient mice and deficient mice. The results showed that the activation of MDA-5 antiviral signaling pathway was greatly inhibited after the deletion of TRIM65 expression. We have similar results in human cells. In terms of mechanism, we found that TRIM65 can induce the ubiquification of MDA-5 's 743 lysine ligand by using its own E3 ubiquitin ligase activity. Furthermore, by using SDD-AGE and laser confocal technique, we found that the induction of MDA-5 ubiquitin by TRIM65 can significantly promote the formation of MDA-5 oligomers, and the formation of MDA-5 oligomers is a key step in the activation of MDA-5 antiviral signals. Through the above studies, we not only found that TRIM65 is a key component in the process of MDA-5 signal activation, but also confirmed that the regulation of ubiquitin is very important for the oligopolymerization and activation of MDA-5. It also provides a new key target for the treatment of MDA-5 related diseases.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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