与死亡结构域蛋白TRADD\FADD\RIP1互作的牛分枝杆菌蛋白的鉴定和功能研究

项目名称： 与死亡结构域蛋白TRADD\FADD\RIP1互作的牛分枝杆菌蛋白的鉴定和功能研究

项目编号： No.31502034

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 杨杨

作者单位： 浙江农林大学

项目金额： 20万元

中文摘要： 牛分枝杆菌是重要的人畜共患病原菌并能抑制肿瘤坏死因子（TNF）介导的细胞凋亡。 TNF可以使其受体TNFR三聚化并招募死亡结构域相关蛋白TRADD、FADD和RIP1形成凋亡诱导复合体，从而导致细胞凋亡的发生。那么，牛分枝杆菌中哪些蛋白参与了凋亡诱导复合体形成的调控呢？因此，本项目拟从TNF信号通路中三个重要的效应蛋白TRADD、FADD和RIP1入手，通过酵母双杂交技术，筛选可与其互作的牛分枝杆菌蛋白，进而通过原核表达和免疫印迹等分子生物学和免疫学手段，研究牛分枝杆菌蛋白对细胞凋亡的抑制作用，然后通过突变分析、蛋白互作、免疫共沉淀等方法系统研究所筛选的牛分枝杆菌蛋白对TRADD和FADD寡聚化以及RIP1磷酸化、泛素化的影响，进一步阐明其在TNF介导的细胞凋亡信号传导中的作用和机制。本研究为揭示死亡结构域相关蛋白在牛分枝杆菌感染引起的凋亡中的作用提供理论基础和实验证据。

中文关键词： 牛分枝杆菌；细胞凋亡；肿瘤坏死因子；死亡结构域

英文摘要： Mycobacterium bovis (M. bovis), the causative agent of tuberculosis in a range of animal species and human, has the ability to inhibit tumor necrosis factor (TNF) mediated apoptosis. Trimerization of TNFR1 by TNFα brings together the TNFR1 to recruit TNFR1-associated death domain protein (TRADD), FAS-associated death domain protein (FADD) and receptor-interacting protein 1 (RIP1), which in turn activates or blocks apoptosis and necrosis through protein interactions and modifications. Dependent of this signaling pathway, are there any M. bovis proteins involved in the formation of appoptosis-inducing complex, triggering subsequent cell death? Thus, potential M. bovis proteins that interact with TRADD, FADD or RIP1 will be screened by yeast-two-hybrid system from a random genomic library. Candidate proteins will be further characterized by expression, pull-down and blotting using molecular and immunological bio-techniques to reveal whether these proteins are essential for TNF mediated apoptosis. Apoptosis inhibition by M. bovis proteins toward THP-1 cells will be demonstrated in the nonpathogenic Mycobacterium smegmatis. Dynamic modifications that include phosphorylation, acetylation, and multiple forms of ubiquitination of target proteins will be analyzed to evaluate their interplays in downstream signaling pathways. This will provide insight into the interactions between DDs and M. bovis proteins as a trigger of apoptosis or necrosis signaling.

英文关键词： Mycobacterium bovis;cell apoptosis;tumor necrosis factor;death domain