MAVS通过TRAF3调控ASC介导的炎症信号通路的研究

发布时间：2018-06-27 17:21

本文选题：MAVS + TRAF3　；参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：先天性免疫系统是机体识别和清除入侵病原体的第一道防线，具有作用广泛、启动迅速的特点，在保护机体、抵抗感染的过程中具有极其重要的作用。先天性免疫应答的激活主要通过宿主的模式识别受体(PRRs)识别病原微生物的病原相关分子模式(PAMPs)，进而招募相应的接头蛋白，激活信号级联反应，诱导I型干扰素、促炎细胞因子、趋化因子的分泌和表达，从而杀伤和清除病原微生物。针对不同的病原相关分子模式，机体有不同的模式识别受体应答。目前发现的模式识别受体包括三种：TLRs、RLRs和NLRs。它们可以通过激活NF-B和IRF等转录因子促进I型干扰素等基因的转录，也可以通过起始胞浆内炎症小体复合物(inflammsome)的组装促进促炎细胞因子的成熟与释放。炎症小体由受体蛋白，接头蛋白ASC和效应分子pro-caspase-1构成。宿主炎症小体的功能状态与病原微生物的致病性密切相关，一方面炎症小体的适度激活具有杀灭病原体以及抑制感染的作用；另一方面，过度的炎症反应会导致多种组织和器官伤害，甚至危及生命。因此，炎症小体需要精准的调控，鉴定和筛选新的炎症小体调控分子对于抵抗病原微生物感染具有十分重要的意义。凋亡相关点状蛋白ASC作为炎症小体信号通路中关键的接头蛋白，在信号传递过程中必不可少。它通过PYRIN和CARD结构域连接受体蛋白和pro-caspase-1，使pro-caspase-1切割为有活性的caspase-1，最终促进IL-1和IL-18等成熟和分泌。鉴于ASC在炎症小体介导的细胞因子成熟过程中的重要作用，我们将其作为靶点筛选参与炎症小体激活的新调控分子，通过酵母双杂交实验发现线粒体抗病毒信号蛋白MAVS有可能和ASC相互作用，进一步研究表明： 1. MAVS与ASC发生相互作用：通过免疫共沉淀、GST-pulldown、线粒体分离、免疫荧光、免疫印迹等方法，发现MAVS与ASC在体内外形成复合物；通过构建截短突变体等方法，发现MAVS通过CARD和TM结构域与ASC的CARD结构域相互作用。 2. MAVS通过上调ASC的蛋白稳定性促进其介导的IL-1产生：ELISA结果表明，MAVS能促进ASC介导的IL-1产生，该现象依赖于MAVS的CARD结构域、TM结构域及其二聚化；免疫印迹结果表明，过表达的MAVS能上调ASC的蛋白水平，敲低细胞内源MAVS的表达导致ASC的蛋白水平降低；RT-PCR结果表明，MAVS不影响ASC的mRNA水平。 3. MAVS通过E3泛素连接酶TRAF3调控ASC：通过筛选6种与MAVS密切相关的E3泛素连接酶，发现TRAF3可以增强ASC的蛋白稳定性及其介导的IL-1水平；进一步研究表明，TRAF3能与ASC相互作用并使其泛素化，而丧失了E3连接酶功能的TRAF3(C53AC56A)和TRAF3(C68AH70A)突变体不能泛素化ASC；在TRAF3knockdown细胞中，MAVS不能明显上调ASC的蛋白稳定性。 4. TRAF3催化ASC K174位发生K63-连接的泛素化：通过构建一系列Ub的KR突变体，发现TRAF3催化ASC发生K63-连接的泛素化；通过构建一系列ASC的KR突变体，发现ASC第174位赖氨酸是可能的泛素化位点；与野生型ASC相比，ASC(K174R)突变体的蛋白稳定性及其介导的IL-1水平均不受MAVS与TRAF3的影响。 5.病毒感染后ASC的核质穿梭依赖于TRAF3对其泛素化修饰：VSV感染后，ASC出现明显的泛素化修饰与核质穿梭；与野生型细胞相比，在MAVS与TRAF3knockdown细胞中，，病毒感染引起的ASC的泛素化修饰水平降低，核质穿梭现象减弱。核质分离和免疫荧光实验表明，病毒感染后，TRAF3促进野生型ASC由细胞核向细胞质转移，而对ASC(K174R)突变体的细胞定位无明显影响。 6.病毒感染后MAVS与TRAF3促进炎症小体的激活：与野生型MEF细胞相比，在MAVS-/-与TRAF3-/-细胞中，SeV感染诱导的pro-caspase-1的切割减弱、IL-1水平下降；用VSV分别感染野生型与MAVS-/-小鼠的BMDC细胞后，MAVS-/-细胞中pro-caspase-1的切割程度与caspase-1的酶活性均低于野生型细胞。综上所述，本研究发现MAVS通过E3泛素连接酶TRAF3，上调ASC的蛋白稳定性，促进其介导的IL-1产生。TRAF3催化ASC发生K63-连接的泛素化修饰，修饰的可能位点为ASC第174位赖氨酸。进一步研究表明，ASC的泛素化能介导病毒感染诱导的核质穿梭，在此基础上促进炎症小体的激活。本研究不但鉴定了新的炎症小体调控因子，阐明了MAVS与TRAF3调控炎症小体的分子机制，同时也为揭示不同信号转导途径之间的交联机制提供了重要的理论依据。
[Abstract]:The present invention relates to a kind of pattern recognition receptor , which is composed of receptor protein , linker protein ASC and effector molecule pro - caspase - 1 . The present invention has the functions of activating NF - B and IRF and promoting the maturation and release of pro - inflammatory cytokines .
On the other hand , excessive inflammatory reaction can lead to injury of various tissues and organs , even life - threatening . Therefore , it is very important to regulate , identify and screen new inflammatory small - body regulatory molecules to resist pathogenic microorganism infection .

In view of the important role of ASC in the process of cytokine maturation mediated by inflammatory small body , we use it as a target to screen the active caspase - 1 and eventually promote the maturation and secretion of IL - 1 and IL - 18 . In view of the important role of ASC in the pathogenesis of inflammatory small body mediated cytokine maturation , we find that the mitochondrial antiviral signaling protein MAVS can interact with ASC by yeast two - hybrid experiment .

1 . MAVS interacting with ASC : MAVS and ASC were found to form complexes in vivo by means of immune codeposition , GST - transferase , mitochondrial separation , immunofluorescence , Western blotting , etc .
By constructing truncated mutants and the like , MAVS was found to interact with the CARD domain of ASC through CARD and TM domains .

2 . MAVS promotes its mediated IL - 1 production by up - regulation of the protein stability of ASC : ELISA results show that MAVS can promote the production of ASC - mediated IL - 1 , which depends on the CARD domain , TM domain and dimerization of MAVS ;
Western blot analysis showed that the overexpression of MAVS could up - regulate the protein level of ASC , and the expression of MAVS in low cell line could decrease the protein level of ASC .
RT - PCR showed that MAVS did not affect the mRNA level of ASC .

3 . MAVS regulated ASC by E3 ubiquitin ligase TRAF3 : by screening six E3 ubiquitin ligase closely related to MAVS , TRAF3 could enhance the protein stability of ASC and its mediated IL - 1 level ;
Further studies have shown that TRAF3 can interact with ASC and ubiquitination , while TRAF3 ( C53AC56 ) and TRAF3 ( C68AH70A ) mutants with loss of E3 ligase function do not ubiquitinated ASC ;
MAVS could not up - regulate the protein stability of ASC in mammalian cells .

4.TRAF3 catalyzes the ubiquitination of K63 - connections in ASC K174 sites : K63 - linked ubiquitination of the K63 - connection is found catalyzed by TRAF3 by constructing a series of KR mutants of Ub ;
By constructing a series of KR mutants of ASC , it was found that the 174 lysine in ASC was a possible ubiquitination site ;
Compared with wild - type ASC , the protein stability of ASC ( K174R ) mutant and its mediated IL - 1 level were not influenced by MAVS and TRAF3 .

5 . The nuclear shuttle of ASC after virus infection depends on TRAF3 ' s ubiquitination modification : after the infection , the ASC has obvious ubiquitination modification and nuclear shuttle ;
Compared with wild - type cells , the ubiquitination modification level of ASC induced by viral infection decreased and the nuclear shuttle phenomenon was weakened . After virus infection , TRAF3 promoted the transfer of wild - type ASC from nucleus to cytoplasm , and had no obvious effect on cell localization of ASC ( K174R ) mutant .

6 . MAVS and TRAF3 after viral infection promote the activation of inflammatory cells : in MAVS - / - and TRAF3 - / - cells , the cleavage of pro - caspase - 1 induced by SeV infection decreased and the level of IL - 1 was decreased compared with wild type MEFs .
The cleavage degree of pro - caspase - 1 and the activity of caspase - 1 in MAVS - / - cells were lower than that of wild - type cells after infected with BMDC cells of wild - type and MAVS - / - mice , respectively .

In conclusion , the study found that MAVS increased the protein stability of ASC by E3 ubiquitin ligase TRAF3 , and promoted its mediated IL - 1 production . TRAF3 catalyzed ASC to produce K63 - linked ubiquitination modification , and the possible site of modification was ASC 174 lysine .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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