mTOR信号通路关键调节蛋白Rheb临近蛋白的筛选及其在细胞衰老中的功能研究

RheB是mTOR信号通路的重要调节蛋白，它定位于多种细胞器并被报道可以不依赖mTOR信号通路调节衰老，但其调节的具体机制未明。鉴于信号通路网络的本质是蛋白质相互作用，本项目采用临近标记技术利用团队前期开发、具有快速酶学动力学的生物素连接酶H-BPL，选取RheB分子作为蛋白诱饵，鉴定其在胰岛素模拟的生长因子刺激下短时间发生响应的临近蛋白网络作为本研究的出发点，进一步深入探讨mTORC1信号通路的调节方式及细胞内同期发生的其它响应过程。通过前期探索现已获得胰岛素刺激15分钟时RheB临近蛋白的质谱数据，已从中筛选得到若干可能具有GEF酶活的候选RheB调节分子，并初步判定了RheB在接收刺激后的细胞定位走向；本研究将继续鉴定RheB蛋白和mTORC1信号通路的重要调节蛋白和细胞中多细胞器协同性响应胞外信号的作用机制，及进一步讨论其在衰老过程中的变化，以说明衰老发生的普遍机制。

RheB, which locates in several organelles, is a key modulator in mTORC1 signaling. It is also reported RheB protein has a dual role in fasting-induced longevity of mTORC1 dependent and independent manner, and the latter is largely unknown. Considering the nature of signaling pathway stays at protein-protein interactions, here we propose to take RheB (central kinase in mTORC1 activation) as the screening bait using proximal labeling to identify its proximal protein network upon short-time insulin stimulation. Specifically we take advantage of our newly identified biotin ligase H-BPL with faster kinetics and stronger labeling activity, to explore the way mTORC1 signaling reacting to insulin and other spontaneous cellular response. Our pilot results based on the proximity mass spectrum data suggested several RheB modulating candidates with GEF activity, as well as showing the cellular distribution of RheB upon insulin stimulus. Further effort would be given to reveal modulators of classical mTORC1 signaling and cellular response synergy mechanism, as well as to investigate what are the alterations during aging in order to figure out the universal principle of cellular senescence.