新型病原菌效应蛋白SidE及IpaJ的结构与功能研究

Pathogen-host interaction are research highlights in recent years. One important pattern is that bacterial pathogens translocate effector proteins into host cells through their unique secretion systems to interfere with and escape from host immune system. SidE family from Legionella pneumophila can convert ubiquitin into phosphoribosylated ubiquitin. This atypical kind of ubiquitin competes with unmodified ubiquitin, thus affecting host ubiquitin signal pathway extensively. In the presence of substrates, SidE can conjugate phosphoribosylated ubiquitin to serine residues of substrates through a phosphodiester bond, which is different from the classic E1-E2-E3 cascade reaction system. Taken together, SidE defines a novel ubiquitin-modification mechanism and its molecular basis remains unclear, which is the key point in this research project. Shigella flexneri IpaJ can cleavage the N-myristoyl modifications of ARF1, thus blocking STING translocation from ER to ER-Golgi intermediate compartments and helping the infection of Shigella flexneri. In vitro, IpaJ can cleavage more broad N-myristoylated glycine of proteins. The illustration of IpaJ catalytic mechanism can provide a prompt for developing new anti-infectious drugs, and IpaJ shows the potential to be reprogrammed to cleavage novel substrates in vivo and study the biological roles of myristoyl modifications.

病原菌与宿主的相互作用是近年来研究的热点。一种方式为病原菌通过自身的分泌系统将效应蛋白注入到宿主细胞内，借此干扰和逃逸宿主的免疫系统。来源于嗜肺军团菌的SidE家族能够以NAD作为辅因子，将泛素修饰为磷酸核糖化的泛素。这种非典型的泛素分子同未修饰的泛素相互竞争，广泛的抑制宿主的泛素信号通路。在底物存在时，SidE能够将这种泛素连接到底物的丝氨酸上，这不同于经典的E1-E2-E3级联反应系统。可以说，SidE是一种新型的泛素修饰系统，其分子机制尚不清楚，亦即本项目的研究内容。来源于福氏志贺菌的IpaJ能够切除宿主ARF1蛋白的N端豆蔻酰化修饰，进而影响免疫信号蛋白STING在内质网和高尔基体间的转运能力，帮助福氏志贺菌的侵染。在体外，IpaJ能够更广泛的切割N端为豆蔻酰化甘氨酸的蛋白。IpaJ催化机制的揭示能够为开发新的抗感染药物提供思路，且有潜力成为研究豆蔻酰化修饰功能的工具。

经典泛素化修饰依赖于E1-E2-E3级联反应。然而，来源于嗜肺军团菌的蛋白SidE和MavC却以全新的方式进行泛素化修饰。SidE以NAD为辅因子，将磷酸核糖化的泛素连接到底物丝氨酸上。MavC则通过对泛素40位的谷氨酰胺脱氨，将其连接到E2结合酶UBE2N的92位或94位的赖氨酸上；同时，Lpg2149可抑制MavC的酶活。本项目通过结构生物学并结合生物化学和细胞生物学等手段阐明了SidE和MavC催化底物修饰及动态调控的分子基础。SidE的apo蛋白结构、mART结构域-泛素-NAD复合物结构及PDE结构域-泛素-ADPr复合物结构，分别揭示了SidE催化反应前、催化第一步反应及催化第二步反应的分子基础。同时，本研究还发现SidE家族蛋白对底物的识别不依赖于底物蛋白的特定三维结构。MavC与UBE2N~Ub复合物的晶体结构直接揭示了MavC与UBE2N以及Ub的互作界面，也解释了为何MvcA为何不能催化UBE2N的泛素化修饰。MavC-Lpg2149及MvcA-Lpg2149的晶体结构表明Lpg2149和Ub占据相似的结合位置，直接干扰Ub与MavC/MvcA的相互作用。同时，MavC和MvcA还具备去泛素化酶的活性，可以特异性去除MavC介导的UBE2N泛素化修饰。这些研究较为完整的阐明了SidE及Mavc介导的新型泛素化修饰的催化和调控机制，不仅拓展了人们对于泛素化修饰的认知，还增加了人们对于病原与宿主相互博弈多样性机制的理解。