RNF122负向调控抗病毒天然免疫反应及作用机制研究

发布时间：2018-04-29 11:03

本文选题：天然免疫 + Ⅰ型干扰素　；参考：《北京协和医学院》2016年博士论文

【摘要】：当病毒入侵机体时,宿主细胞可以识别病毒的核酸,通过产生Ⅰ型干扰素(Type I interferon, IFN-I)和促炎因子来触发抗病毒天然免疫反应。作为一种重要的天然免疫受体,RIG-I (Retinoic acid-inducible gene 1)能够识别病毒RNA,通过其C末端解螺旋酶结构域结合RNA,随后发生构象改变并募集至线粒体附近,通过与线粒体上的MAVS (Mitochondrial antiviral signaling)相互作用而激活下游信号转导通路。MAVS的下游信号转导最终激活转录因子IRF3 (Interferon regulatory factor 3)和NF-κB,活化的IRF3和NF-κB入核刺激Ⅰ型干扰素和促炎因子转录,帮助宿主抵御病毒入侵。这一过程中,RIG-I的活性需要受到严密的调控,使机体可以有效清除病原体,同时又不至于产生过强的炎症反应而对机体造成免疫损伤。那么,如何及时终止RIG-I信号,避免产生过量的Ⅰ型干扰素以及造成自身免疫性疾病是一个需要深入研究的课题。蛋白质的翻译后修饰(post-translational modifications,PTMs),尤其是泛素化修饰(ubiquitination),在RIG-I活性的调控中发挥重要的作用。目前己报道有多种E3泛素连接酶参与调节RIG-I信号通路。为了全面深入地认识RIG-I信号通路及其调控机制,需要继续发现新的目前未知的负向调控因子。在本课题的研究中,我们在病毒感染的细胞中免疫沉淀RIG-I,通过质谱鉴定RIG-I相互作用蛋白时,发现了一个新的E3泛素连接酶RNF122 (Ring finger protein 122)。RNF122含有N端的穿膜结构域和C端的RING (Really interesting new gene)结构域,RING结构域提示RNF122可能具有E3泛素连接酶活性。序列比对发现RNF122高度保守,鼠源RNF122与人源RNF122有97%的同源性。RNF122在小鼠各组织脏器中广泛表达,并高表达于免疫器官和免疫细胞中。我们的研究显示,VSV (Vesicular stomatitis virus)刺激前后RNF122与RIG-I在腹腔巨噬细胞中均共定位于胞浆。进一步的研究发现,RNF122通过其穿膜结构域与RIG-I的CARD (Caspase activation and recruitment domain)结构域结合,并通过RING结构域介导了CARD上第115位和第146位赖氨酸的泛素化,通过K48位连接的泛素化修饰促进RIG-I降解,从而显著降低RIG-I下游信号通路的活化水平。RNF122表达缺失或敲低的巨噬细胞在感染VSV、SeV (Sendai Virus)或转染poly(I:C)时,IFN-p的mRNA表达水平显著高于对照细胞。RNF122-/-小鼠的腹腔巨噬细胞在RNA病毒感染而非DNA病毒感染时分泌更多的Ⅰ型干扰素(IFN-α和IFN-β)、炎症因子TNF-α和IL-6。免疫印迹结果也显示RNF122表达缺失或敲低的巨噬细胞在VSV感染时IRF3和p65的活化水平显著高于对照组细胞。这些结果显示RNF122是RIG-I信号通路特异性的负向调控因子,能够显著抑制RNA病毒感染引发的抗病毒天然免疫反应。RNF122表达缺失的小鼠具有正常的骨髓细胞发育和巨噬细胞分化能力。当使用致死剂量的VSV感染小鼠时,RNF122-/-小鼠血清中Ⅰ型干扰素和促炎因子水平明显高于对照组小鼠,肺脏中炎性细胞浸润较少,生存时间也明显延长, 表明RNF122对于RNA病毒感染所触发的Ⅰ干扰素产生与炎症发生具有抑制作用。此外,RNF122可发生自身泛素化,其蛋白表达量在病毒感染的细胞和小鼠中均明显升高,这种升高对于其反馈限制Ⅰ干扰素的过度产生、发挥免疫抑制功能具有重要意义。综上所述,我们发现了RIG-I相互作用蛋白RNF122可以通过K48位连接的泛素化修饰增加RIG-I的降解,从而抑制RNA病毒感染所触发的Ⅰ干扰素的产生。RNF122的这种负向调控作用为我们理解宿主细胞抗病毒天然免疫反应与炎症的调控机制提供了新的视角。
[Abstract]:When a virus invades the body, the host cell can identify the virus's nucleic acid and trigger an antiviral natural immune response by producing Type I interferon (IFN-I) and pro-inflammatory factors. As an important natural immune receptor, RIG-I (Retinoic acid-inducible gene 1) can identify virus RNA and solve spiral enzyme through its C end. The structure domain combined with RNA, followed by conformation changes and recruitment to the mitochondria, activating downstream signal transduction pathway.MAVS downstream signal transduction pathway, IRF3 (Interferon regulatory factor 3) and NF- kappa B by interacting with MAVS (Mitochondrial antiviral signaling) on the mitochondria. Nuclear stimulation of type I interferon and pro-inflammatory factor transcription helps the host to resist the invasion of the virus. In this process, the activity of RIG-I needs to be closely regulated, so that the organism can effectively remove the pathogen and not produce an excessive inflammatory response to the body. Then, how to stop the RIG-I signal and avoid production in time. Excessive production of type I interferon and the cause of autoimmune diseases are a subject that needs further study. Post-translational modifications (PTMs), especially ubiquitination (ubiquitination), plays an important role in the regulation of RIG-I activity. There have been a variety of E3 ubiquitin ligase. With the regulation of the RIG-I signaling pathway, in order to fully understand the RIG-I signaling pathway and its regulatory mechanism, we need to continue to discover new and unknown negative regulatory factors. In this study, we immunized RIG-I in the virus infected cells and found a new E3 ubiquitin in the identification of RIG-I interacting proteins by mass spectrometry. The ligase RNF122 (Ring finger protein 122).RNF122 contains the membrane domain of the N terminal and the RING (Really interesting new gene) domain of the C end. It is widely expressed in organs and highly expressed in immune organs and immune cells. Our study showed that RNF122 and RIG-I were Co located in the cytoplasm of peritoneal macrophages before and after the stimulation of VSV (Vesicular stomatitis virus). Further studies found that RNF122 through its membrane domain and RIG-I's CARD (Caspase activation and) T domain) combines the domain of the domain and mediates the ubiquitination of 115th and 146th lysine on the CARD domain through the RING domain, and promotes the degradation of RIG-I through the ubiquitination of K48 bit connection, thus significantly reducing the activation level of the downstream signal pathway of the RIG-I and the macrophages in the infection VSV, SeV (Sendai Virus) or transfection. At Poly (I:C), the expression level of mRNA in IFN-p was significantly higher than that of the peritoneal macrophages in the.RNF122-/- mice of the control cells in the RNA virus infection but not in the DNA virus infection. The inflammatory factor TNF- alpha and IL-6. immunoblotting results also showed that the macrophages with the absence of the RNF122 expression or the low number of knockout were infected. The activation level of RF3 and p65 was significantly higher than that of the control group. These results showed that RNF122 was a specific negative regulator of RIG-I signaling pathway, and could significantly inhibit the normal immune response to.RNF122 expression induced by RNA virus infection in mice with normal bone marrow cell development and macrophage differentiation. When the dead dose of VSV infected mice, the level of interferon I and pro-inflammatory factors in the serum of RNF122-/- mice was significantly higher than that of the control mice. The infiltration of inflammatory cells in the lungs was less and the survival time was prolonged obviously. It showed that the RNF122 interferon I caused by the RNA virus infection was inhibited by the inflammation. In addition, RNF122 could be found. The increase in the protein expression in the virus infected cells and mice is significantly higher than that in the virus infected cells and mice. This increase is of great significance to its feedback restriction overproduction of interferon I and play an immunosuppressive function. In summary, we have found that the RIG-I interaction protein RNF122 can increase R through the ubiquitination of K48 connection. The degradation of IG-I, thus inhibiting the negative regulation of.RNF122 produced by interferon I triggered by RNA virus infection, provides a new perspective for our understanding of the regulatory mechanism of the host cell's antiviral natural immune response and inflammation.

【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】：2016
【分类号】：R392

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