锌在梅毒螺旋体Tp0751结构与功能中的作用研究

项目名称： 锌在梅毒螺旋体Tp0751结构与功能中的作用研究

项目编号： No.81201331

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 医学四处

项目作者： 刘双全

作者单位： 南华大学

项目金额： 23万元

中文摘要： Tp0751是Tp重要的致病因素，锌可能在维系Tp0751的蛋白水解活性中发挥重要的作用。但是，Tp0751是否为锌依赖金属蛋白尚需要利用蛋白结构研究进行确证，同时锌在维系Tp0751其它生物学功能中的作用也尚有待进一步研究。为了验证这种设想，本项目在我们前期研究的基础上和结合国外最新研究进展，利用X 射线晶体学技术解析蛋白质的三维空间结构；将蛋白的锌依赖活性区HEVIH分别点突变为HQVIH、AEVIH和HEVIA，以分别考察199位天冬氨酸、198位和202位组氨酸在结合锌及及维持蛋白质结构与功能中的作用；设计抑制实验，研究加入金属抑制剂后对蛋白黏附、水解及促炎症活性的影响；总结以上信息，分析锌在维系Tp0751的结构及生物学功能如黏附、水解及促炎症活性中的作用及机制。本项目的完成不仅能为系统研究Tp0751的功能奠定基础，而且也为Tp致病机制研究及梅毒的治疗研究提新的方向和靶标。

中文关键词： 梅毒螺旋体；Tp0751；糖尿病；硫氧还蛋白互作蛋白；膜蛋白

英文摘要： T. pallidum membrane protein Tp0751 plays a role in degrading host components and stimulating inflammatory reactions, and is a crucial pathogenesis factor in the host tissue colonization and bacterial dissemination. Recent research indicates that the proteolytic activity of Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, whether Tp0751 is a zinc-dependent metalloprotease remains unknown and thus depends on its structure determination. Moreover, the role of zinc in maintaining the other function of Tp0751 such as attaching to host cells,stimulating inflammatory reactions is still need to be investigated. To test this prediction, based on our present research, we will use X-ray crystallography to analysis its three-dimensional structure, and further determine its function as a metalloprotease. And we will use site-directed mutagenesis by mutation of the HEXXH motif to HQXXH,AEVIH and HEVIA to analyze whether Glutamate at position 199 and Histidines at positions 198 and 202 bind to znic and maintains the stucture and funcion of Tp0751. Furthermore, we will design protease inhibition assays to probe the effect of various broad-spectrum protease inhibitors on the attachment, proteolytic and stimulating inflamatory activity of Tp0751. This project

英文关键词： Treponema pallidum；Tp0751；Diabetes mellitus；thioredoxin-interacting protein；membrane protein