精氨酸N-乙酰葡萄糖胺化修饰在病原细菌感染中结构机理及功能的研究

During infection processes,pathogens can inject effector proteins into host cells by the type III secretion system (T3SS) to subvert multiple host cell functions. Investigation of the pathogen and host interaction relationship can not only help us understand the pathogenic mechanism, but also broaden out insights into host cell signalling pathways. In previous study, we reported that death domains (DDs) in several DD-containing proteins, including TRADD, FADD, RIPK1and TNFR1 were directly inactivated through a novel post-translational modification, arginine GlcNAcylation, by NleB family, a series of type III secretion system effectors produced by enteropathogenic Escherichia coli (EPEC) and related pathogens. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification. To further understand this novel argnine GlcNAc modification, we propose this project with two major questions. At first, Our preliminary data show that the overall structure of NleB is a GT-A family glycosyltransferase, so it is intriguing that why the NleB family catalyzed GlcNAcylation occured on arginine residue but not serine/threonine residues. Determination of crystal structures of NleB and its complex with the death domain substrate will uncover this mystery. Structural-based biochemical and enzymatic analysis will allow us to propose a general molecular mechanism of the novel enzyme activity and the specific recognition with host targets. Furthermore, the significance of the enzymatic activity of NleB will be verified by bacterial colonization in the mouse model of EPEC infection. Secondly, we are curious about the existence and significance of the endogenous arginine GlcNAcylation in host cells, since the O-GlcNAcylation occurred on serine/threonine has been developed into a leading research topic. We propose to begin with preparing sensitive and specific rabbit monoclonal antibody of arginine GlcNAcylation, and then with the tool of mass spectrometry to explore the host cell signalling pathways.

许多革兰氏阴性致病菌在侵染过程中，通过三型分泌系统向宿主注入效应蛋白，来操控宿主细胞的信号转导过程。我们已报道三型效应蛋白NleB家族，以一种全新的精氨酸N-乙酰葡萄糖胺化修饰作用于宿主细胞的死亡结构域，从而抑制宿主死亡受体所介导的细胞死亡（Li et al.,Nature,2013)。在原有工作的基础上，本项目初期研究解析了NleB单体及NleB/GlcNAc复合体的结构，将进一步通过解析NleB与其底物死亡结构域蛋白所形成的复合体的晶体结构，揭示这种全新酶活催化和识别底物的分子机制，并阐明其在多种病原菌感染过程中的生理意义，帮助我们深入理解病原菌的致病机理。另外，本项目初期已成功合成含有精氨酸N-乙酰葡萄糖胺修饰的糖肽，将通过制备特异性兔源单抗，结合高精度质谱分析，探索真核宿主体內内源精氨酸N-乙酰葡萄糖胺修饰的存在性及意义，这将为我们利用病源细菌研究真核宿主信号通路提供新的方向。

对三型分泌系统效应分子的研究是我们研究病原微生物与宿主互作关系中重要的研究方向，效应蛋白经常以新颖的酶学活性作用于宿主细胞中的靶蛋白。研究并揭示其作用的分子机制，不仅使我们更加深入的理解病原细菌的致病机理，为发现诊断与治疗的新靶点，控制病原菌感染和提高病原菌感染相关疾病的治愈率奠定基础，同时也对我们探索真核细胞内新的酶学反应过程和信号转导通路有重要的指导意义。我们已报道三型效应蛋白NleB家族，以一种全新的精氨酸 N-乙酰葡萄糖胺化修饰作用于宿主细胞的死亡结构域，从而抑制宿主死亡受体所介导的细胞死亡(Li et al.,Nature,2013)。本项目在自然界首次合成含有精氨酸N-乙酰葡萄糖胺修饰的糖肽，成功制备识别修饰的抗体，因多肽合成的新颖性，抗体极高的特异性和灵敏度，论文发表在《德国应用化学》（Pan & Li，et al.,Angew Chemi Int Ed, 2014），与Abcam公司合作开发的抗体已经商业化，取得了良好的经济效益，利用该抗体，学界已经在Molecular cell, Plos pathogen, Infection and Immunity等期刊发表高水平学术论文多篇。修饰特异性抗体的制备，为研究此全新的蛋白翻译后修饰开辟了一条道路。为了进一步研究精氨酸N-乙酰葡萄糖胺化修饰的酶学机理，本项目解析NleB单体及 NleB/GlcNAc 复合体的结构，鉴定其为GT-A家族的糖基转移酶。解析了NleB与底物死亡结构域蛋白复合物的结构，鉴定了精氨酸N-乙酰葡萄糖胺转移酶NleB中与金属离子和糖基配体结合结合的关键位点，并在动物感染模型中鉴定了NleB关键位点突变体在病源菌感染动物的模型中的毒力，揭示了其催化全新的蛋白质翻译后修饰的酶活反应和底物特异性识别的分子机理。该工作已审回至《Molecular Cell》。另依托该项目发现来自细菌的毒素表位疫苗对幽门螺旋杆菌侵染有保护作用（Pan et al., Frontiers in Immunology），报道李斯特菌在湖北十堰地区监测情况（吕均等，中国卫生检验杂志，2017），受邀撰写综述三篇 (Luo et al., Apoptosis, 2015；朱平等，生物技术通报，2018；朱平等，微生物学通报，2018)。