不依赖于E1和E2的新型泛素化修饰的分子机制和化学干预研究

Protein ubiquitination can control virtually all types of cellular events. The classical ubiquitination system relies on the E1-E2-E3 triple enzyme cascade and the cofactor ATP. However, the recently identified SidE family proteins from Legionella pneumophila have been found to be able to rely on a single protein to bind NAD as a cofactor and to ligase the phosphoribosylation ubiquitin to the serine residue of the substrate protein. Moreover, this novel ubiquitination modification can only be removed by the newly identified deubiquitinase SidJ, which is also derived from L. pneumophila. As a frontier issue of common concern in the fields of both pathogen infection and protein ubiquitination, the proposed study of this novel ubiquitination system will help to broaden the understanding of ubiquitination and to treat a variety of bacterial infectious diseases. Based on the preliminary data, a research program that combines approaches of biochemical, structural biology, cell biology and chemical biology to study the molecular mechanisms and the regulation network of this novel ubiquitination system. By using the high-throughput drug screening method, we are also planning to identify the small-molecule inhibitors for this novel system, so as to provide chemical intervention for both basic research and the therapies to treat infectious diseases.

蛋白质泛素化修饰几乎可调控所有的细胞活动。经典的泛素化修饰依赖于辅因子ATP和E1-E2-E3三酶级联反应。然而，最近鉴定的来源于嗜肺军团菌的SidE蛋白家族却能够依靠单一蛋白，以NAD作为辅因子，将磷酸核糖化的泛素连接到底物蛋白的丝氨酸上。而且，这种新型的泛素化修饰只能被同样来自于病原菌的新型去泛素化酶SidJ催化去除。作为感染免疫领域和泛素化修饰领域共同关注的前沿问题，对该新型修饰系统的研究，将既有助于拓宽人们对泛素化修饰的固有理解，还将对多种传染性疾病的治疗提供帮助。申请人在前期工作的基础上，拟结合生物化学、结构生物学、细胞生物学和化学生物学等多种手段研究此系统中还未解决的基本生物学问题，如催化反应的分子机制及修饰和识别的作用网络；并通过高通量药物筛选，鉴定针对此系统的小分子抑制剂，从而为相关基础研究和疾病治疗提供化学干预手段。

蛋白质泛素化修饰几乎可调控所有的细胞活动。经典的泛素化修饰依赖于辅因子ATP和E1-E2-E3三酶级联反应。然而，来源于高致病性嗜肺军团菌的两类效应蛋白（SidE和MavC），却能够完全不依靠经典泛素化修饰体系，以全新且完全不同的机制完成对宿主蛋白的泛素化修饰。在前期工作基础上，本项目拟结合生物化学、结构生物学、细胞生物学和化学生物学等多种手段研究此系统中还未解决的基本生物学问题，如催化反应的分子机制及修饰和识别的作用网络；并通过高通量药物筛选，鉴定针对此系统的小分子抑制剂，从而为相关基础研究和疾病治疗提供化学干预手段。本项目取得的重要进展包括：1）解析了SidE单独，及其与泛素、NAD、ADP-ribose复合物的结构，揭示了SidE催化新型泛素化修饰的机制；2）解析了MavC与UBE2N~Ub复合物、MavC与抑制蛋白Lpg2149复合物、以及MavC的同源蛋白MvcA与Lpg2149复合物等多个结构，揭示了MavC类型的新型泛素化修饰酶的催化和调控机制。