嗜肺军团菌OTU家族效应蛋白生化及生物学功能研究

Legionella pneumophila translocates more than 300 effector proteins into host cytosol via its Dot/Icm type IV secretion system, which interfere with multiple host signaling pathways to establish a niche permissive for its intracellular survival and replication. Studies aimed to unveil the biochemical and biological function of effector proteins will greatly improve our understandings on the pathogenesis of L. pneumophila. Ubiquitination is an enzymatic post-translational modification in which an ubiquitin protein is attached to a substrate protein. Ubiquitination regulates almost every aspect of cellular function and many cell signaling pathways in eukaryotes. Ubiquitin modification can be reversed by deubiquitinases, a family of diverse proteases that remove ubiquitin from the modified proteins. Previous studies have shown that L. pneumophila is capable of co-opting host ubiquitination machinery by multiple effector proteins to facilitate its intracellular survival and propagation. Bioinformatics analysis reveals that L. pneumophila encodes 4 effector proteins (Lpg0227, Lpg1621, Lpg2248 and Lpg2529) which contain conserved OTU (ovarian-tumour-domain-containing proteases) domain. OTU domain-containing proteins are cysteine protease which potentially possesses deubiquitinase activity. The aim of the proposed study is to characterize the biochemical function of L. pneumophila OTU family proteins, and further clarify their biological activities during bacterial infection, which would allow us gain more insight on the regulation of host ubiquitination machinery by L. pneumophila.

嗜肺军团菌通过其Dot/Icm四型分泌系统输送超过300个效应蛋白到宿主细胞内，干扰宿主的多种信号传导通路，以构建适于其定殖和生长的最适微环境，因此揭示效应蛋白的生化功能及其在感染中的生物相关性是阐明军团菌致病分子机制的关键。泛素化是由三种酶介导的对底物蛋白分子进行泛素修饰的过程，参与几乎一切细胞生命活动的调控；去泛素酶通过水解底物蛋白与泛素链之间的连接，发挥去泛素化作用，从而逆转泛素化对蛋白功能的影响。军团菌利用多种效应蛋白劫持宿主的泛素化信号通路以实现其在宿主内的有效增殖；军团菌编码4个含OTU结构域的效应蛋白，目前其生化和生物学功能仍不清楚。OTU家族蛋白属于半胱氨酸蛋白酶家族，具有潜在的去泛素化酶活性。本项目拟以军团菌OTU家族效应蛋白为研究对象，阐明其生化功能及其在军团菌感染过程中对泛素化通路的影响。该研究将有助于进一步认识军团菌对宿主泛素信号网络的调控机制。

嗜肺军团菌是导致军团菌肺炎和庞蒂亚克热的主要病原。军团菌属于兼性胞内寄生菌，通过其编码的四型分泌系统（T4SS）向宿主细胞内转运的超过330个效应蛋白，劫持多种宿主信号传导以构建其在胞内的最适生长微环境（LCV）。因此，解析T4SS效应蛋白的生化功能、揭示其宿主内靶标和对宿主细胞信号通路的干扰，是阐明军团菌致病分子机制的关键。蛋白质的泛素化是真核生物最重要的蛋白质翻译后修饰之一，参与细胞一切生命活动的调控。基于其在宿主免疫反应中的重要性，泛素化信号通路成为许多胞内寄生菌效应蛋白的非常重要的靶标之一。通过对已报道的军团菌效应蛋白进行生物信息学分析，发现了4个具有真核细胞OTU结构域的效应蛋白（Lpg0227、Lpg1621、Lpg2248和Lpg2529）。本项目以军团菌 OTU 家族蛋白为研究对象，阐明其生化功能及其在军团菌感染中的生物学作用。研究表明，效应蛋白Lpg1621（Ceg23）是一种可特异性切割K63连接多聚泛素链的去泛素化酶；Ceg23转运至宿主细胞内后定位于LCV表面，参与LCV泛素化的负调控；解析了Ceg23的晶体结构（2.8Å），结果显示，虽然与OTU蛋白的氨基酸序列差异较大，但Ceg23具有典型的OTU折叠。与Ceg23不同的是，Lpg2529（Lem27）主要对K6、K11和K48连接的多聚泛素链表现出强大的切割作用，Lem27定位于LCV表面并与泛素连接酶SidC和SdcA共同调控LCV的泛素化；Lem27与去泛素化酶自杀抑制剂Ub-PA的复合物晶体结构（2.43 Å）揭示Lem27具有类似于OTUB1的Cys-His催化二联体，并通过六个接触位点以多个氢键识别泛素分子。上述研究成果有助于我们进一步认识军团菌对宿主泛素信号网络的调控机制。