【作者】 尚桂军；

【导师】 谷立川；

【作者基本信息】 山东大学 ， 生物物理学， 2014， 博士

【摘要】 细菌六型分泌系统(T6SS)利用自身注射器样的分泌装置,以直接接触的方式注射多种效应蛋白到与其比邻的同种或者异种的细胞中,导致异种受体细胞死亡或生长停滞。同时为了使自身细胞免受这些效应分子的毒害,T6SS+细菌同时表达与各种效应分子对应的同宗免疫蛋白(cognate immunity protein)而使其失活。这种效应蛋白和免疫蛋白对(E-I pair)使拥有T6SS的细菌在与其它细菌竞争中保持了优势。通过分泌组学和质谱的方法,在铜绿假单胞菌中鉴定出三种受H1-T6SS控制的效应蛋白Tse1、Tse2和Tse3。其中Tsel和Tse3分别降解敌对细菌中细胞壁成分的肽侧链和糖主链部分。本文利用X-射线晶体学技术研究了效应因子Tse3和Tse3-Tsi3复合物的三维结构,揭示了Tse3除含有一个C端的溶菌酶结构域以外,N端还含有一个annexin repeat-like结构域；来源于N端结构域的loop12和C端结构域的螺旋α9在钙离子存在下把Tse3和Tse3-Tsi3锚定到膜上；膜锚定对于Tse3的活性是必需的；Tse3可以利用Tsel的产物作为底物,首次发现了T6SS效应蛋白之间的协同效应；发现在Tse3催化中心和底物结合位点分别含有一个钙结合基序,它们对催化活性也是必需的。另外,Tse3-Tsi3复合物的结构揭示了Tsi3通过三个功能性loop结合到Tse3的活性中心而使其失去结合底物的能力,并发现Tsi3对Yse3的抑制也是钙依赖的,即Tsi3是一种新颖的G-型溶菌酶抑制剂。Tse3和Tsi3结构与功能的研究丰富了我们对T6SS效应蛋白的认识,并为控制病原菌感染提供了必要的结构基础。在高等生物天然免疫系统中,STING蛋白是细胞内对双链DNA (dsDNA)免疫应答的重要接头分子。细胞内的DNA感受器能把dsDNA信号传递给STING蛋白,STING招募下游的激酶分子TBK1,使其磷酸化转录因子IRF3,磷酸化的IRF3二聚化而进入核内,使INF-β表达上调,继而激活病原菌入侵应答。最近发现的CDNs信号通路使我们对STING介导的dsDNA信号通路的认识更加深入。我们借助X-射线晶体学的方法解析了人源STING蛋白的C端结构域(CTD)及其与细菌第二信使c-di-GMP复合物的三维空间结构。我们的晶体学结构显示h-STINGCTD以二体发挥功能,c-di-GMP的结合并没有使h-STINGCTD构象变化,而人细胞内第二信使2’5’-cGAMP则可引起h-STINGCTD构象从open到close的变化,位于β2-β3的loop变为了反平行的β片层盖子结构把CDNs封闭在二体形成的口袋内；同时体内实验显示h-STING并不对c-di-GMP的刺激产生免疫应答,鉴于I型干扰素可能有利于细菌的侵染,推测细菌很有可能通过模拟真核细胞的第二信使来劫持宿主的天然免疫系统。人的STING蛋白则通过对c-di-GMP不响应,成功地逃避了细菌的劫持,这在进化上对人类是有利的。而小鼠的STING蛋白则对所有的CDNs应答,并且其构象始终保持close的状态,这提示了STING蛋白在天然免疫中的物种间的差异性。总之,STING结构的解析,为我们治疗病原侵染和自身免疫疾病提供了重要的结构基础。更多还原

【Abstract】 Gram negative bacteria using a syringe-like tool of Type VI secretion system (T6SS) inject the effector proteins into the rival cells in a cell-contact manner leading to their death. Concurrently, bacteria employ cognate immunity proteins neutralizing these effector proteins to prevent self-intoxition. This effector-immunity pairs (E-I pairs) of T6SS endow the bacteria with great advantage in the fierce niche competiton. Three effector proteins (Tsel, Tse2and Tse3) controlled by H1-T6SS from Pseudomonas aeruginosa have been identified by Secretome and Mass spectrum analysis. Tsel and Tse3were demonstrated to be the amidase and muramidase degrading the peptide and glycan moiety of peptidoglycan (PG) respectively. In this thesis, X-ray crystallography method was performed to elucidate the mechanism used by Tse3and Tsi3in details. Tse3has an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. Interestingly, one loop (loop12) at the N-terminal domain, together with one helix (a9) from the C-terminal domain, anchors Tse3or the Tse3-Tsi3complex to membrane in a calcium-dependent manner in vitro. Moreover, the membrane-binding ability is shown to be essential for Tse3’s activity. In the C-terminal domain, a Y-shaped groove occurs on the surface probably serving as the PG-binding site. Two calcium-binding motifs are also observed in the groove and they are necessary for Tse3’s activity. In the Tse3-Tsi3structure, three loops of Tsi3insert into the substrate-binding groove of Tse3. Three calcium ions found in the interface of the complex are demonstrated to be indispensable for the formation of Tse3-Tsi3complex. These findings further our perspective on the T6SS effector proteins and provide the structural basis for eliminating and combating the pathogenic infection.STING protein plays an essential role in the double strands DNA sensing pathway in the cytosol for innate immunity. The DNA sensors relayed the signal to STING which recruited the kinase TBK1and transcript factor IRF3. Then, IRF3was phosphated by TBK1, which leaded to its dimerization. Dimerized IRF3was transferred to the nucleus and promote the transcription of type I INF which initialized the anti-pathogen process by innate immunity. Recently, the cyclic dinucleotides (CDNs) pathway was discovered making adaptor protein STING more fascinating. In this thesis, we solved the crystal structures of h-STINGCTD domain and its complex with c-di-GMP. The h-STINGCTD and c-di-GMP-h-STINGCTD complex behaves as a dimmer in the solution and crystal indicating it may function as a dimmer. The c-di-GMP was found in the dimmer interface of two STING protomer with a moderate binding affinity. Comparing to the apo form structure, c-di-GMP binding did not induce significant conformation change occurring in h-STINGCTD. However, the metazoan second messenger2’5’-cGAMP binded to h-STINGCTD resulting in drastic structural changes:The open state h-STINGCTD became close state; Loops between β2and β3became anti-parallel β sheet capping the CDNs in the pocket formed by two STING protomers. At the same time, the c-di-GMP did not induce significant immune response mediated by h-STING in vivo. Considering the complicated results of type I INF for the infection of bacteria, they may hijack the CDNs pathway for their purpose by mimcing the metazoan second messenger and human STING protein may evade this bacterial strategy in the evolution process. As for the mouse STING protein, it always adopts the close form and strongly responses to stimulates of CDNs, which indicates the functional differences of STING protein among species. All in all, our structural information of h-STINGCTD provides the molecular basis for the therapy of infection and autoimmune diseases such as systemic lupus erythematosus, SLE.更多还原