本文選題：天然免疫 + Ⅰ型干擾素　； 參考：《北京協(xié)和醫(yī)學院》2016年博士論文

【摘要】：當病毒入侵機體時,宿主細胞可以識別病毒的核酸,通過產生Ⅰ型干擾素(Type I interferon, IFN-I)和促炎因子來觸發(fā)抗病毒天然免疫反應。作為一種重要的天然免疫受體,RIG-I (Retinoic acid-inducible gene 1)能夠識別病毒RNA,通過其C末端解螺旋酶結構域結合RNA,隨后發(fā)生構象改變并募集至線粒體附近,通過與線粒體上的MAVS (Mitochondrial antiviral signaling)相互作用而激活下游信號轉導通路。MAVS的下游信號轉導最終激活轉錄因子IRF3 (Interferon regulatory factor 3)和NF-κB,活化的IRF3和NF-κB入核刺激Ⅰ型干擾素和促炎因子轉錄,幫助宿主抵御病毒入侵。這一過程中,RIG-I的活性需要受到嚴密的調控,使機體可以有效清除病原體,同時又不至于產生過強的炎癥反應而對機體造成免疫損傷。那么,如何及時終止RIG-I信號,避免產生過量的Ⅰ型干擾素以及造成自身免疫性疾病是一個需要深入研究的課題。蛋白質的翻譯后修飾(post-translational modifications,PTMs),尤其是泛素化修飾(ubiquitination),在RIG-I活性的調控中發(fā)揮重要的作用。目前己報道有多種E3泛素連接酶參與調節(jié)RIG-I信號通路。為了全面深入地認識RIG-I信號通路及其調控機制,需要繼續(xù)發(fā)現(xiàn)新的目前未知的負向調控因子。在本課題的研究中,我們在病毒感染的細胞中免疫沉淀RIG-I,通過質譜鑒定RIG-I相互作用蛋白時,發(fā)現(xiàn)了一個新的E3泛素連接酶RNF122 (Ring finger protein 122)。RNF122含有N端的穿膜結構域和C端的RING (Really interesting new gene)結構域,RING結構域提示RNF122可能具有E3泛素連接酶活性。序列比對發(fā)現(xiàn)RNF122高度保守,鼠源RNF122與人源RNF122有97%的同源性。RNF122在小鼠各組織臟器中廣泛表達,并高表達于免疫器官和免疫細胞中。我們的研究顯示,VSV (Vesicular stomatitis virus)刺激前后RNF122與RIG-I在腹腔巨噬細胞中均共定位于胞漿。進一步的研究發(fā)現(xiàn),RNF122通過其穿膜結構域與RIG-I的CARD (Caspase activation and recruitment domain)結構域結合,并通過RING結構域介導了CARD上第115位和第146位賴氨酸的泛素化,通過K48位連接的泛素化修飾促進RIG-I降解,從而顯著降低RIG-I下游信號通路的活化水平。RNF122表達缺失或敲低的巨噬細胞在感染VSV、SeV (Sendai Virus)或轉染poly(I:C)時,IFN-p的mRNA表達水平顯著高于對照細胞。RNF122-/-小鼠的腹腔巨噬細胞在RNA病毒感染而非DNA病毒感染時分泌更多的Ⅰ型干擾素(IFN-α和IFN-β)、炎癥因子TNF-α和IL-6。免疫印跡結果也顯示RNF122表達缺失或敲低的巨噬細胞在VSV感染時IRF3和p65的活化水平顯著高于對照組細胞。這些結果顯示RNF122是RIG-I信號通路特異性的負向調控因子,能夠顯著抑制RNA病毒感染引發(fā)的抗病毒天然免疫反應。RNF122表達缺失的小鼠具有正常的骨髓細胞發(fā)育和巨噬細胞分化能力。當使用致死劑量的VSV感染小鼠時,RNF122-/-小鼠血清中Ⅰ型干擾素和促炎因子水平明顯高于對照組小鼠,肺臟中炎性細胞浸潤較少,生存時間也明顯延長, 表明RNF122對于RNA病毒感染所觸發(fā)的Ⅰ干擾素產生與炎癥發(fā)生具有抑制作用。此外,RNF122可發(fā)生自身泛素化,其蛋白表達量在病毒感染的細胞和小鼠中均明顯升高,這種升高對于其反饋限制Ⅰ干擾素的過度產生、發(fā)揮免疫抑制功能具有重要意義。綜上所述,我們發(fā)現(xiàn)了RIG-I相互作用蛋白RNF122可以通過K48位連接的泛素化修飾增加RIG-I的降解,從而抑制RNA病毒感染所觸發(fā)的Ⅰ干擾素的產生。RNF122的這種負向調控作用為我們理解宿主細胞抗病毒天然免疫反應與炎癥的調控機制提供了新的視角。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392

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