人mTORC2复合物和相关蛋白的结构功能研究

mTORC2主要在PI3K通路下游接受胰岛素等细胞外生长因子信号，磷酸化AKT、PKC、SGK等AGC家族激酶，调控蛋白表达、细胞骨架重组、胞内钠离子浓度等信号通路促进细胞的增值和存活，与多种疾病相关。mTORC2复合物的核心由mTOR、mLST8、Rictor、mSin1构成，其中等分辨率4.9埃冷冻电镜结构最近被报道。我们前期的工作解析了3.0埃高分辨率mTORC2冷冻电镜结构，在此基础上本项目将综合运用结构生物学、生物化学等手段研究目前尚未观察到的mSin1 C端CRIM、RBD、PH结构域及其与AKT等底物、H-Ras等小G蛋白的相互作用，阐明mTORC2对底物的招募识别机理以及小G蛋白对mTORC2的分子调控机理。本项目同时还将通过冷冻电镜研究mTORC2和非核心组分Protor1/2的复合物结构，结合分子生物学，细胞生物学等方法阐明Protor1/2的功能和作用机制。

mTORC2, the rapamycin insensitive mTOR complex, acts downstream of PI3K pathway and primarily responds to growth factors like insulin, promoting protein expression, actin cytoskeleton polarization, sodium transport and subsequently anabolic output and cell survival. The major substrates of mTORC2 are AGC family kinases, including AKT, PKC, SGK; mTORC2 phosphorylation enhance their stability and activity. As a result disruption of the mTORC2 pathway leads to many human diseases involving AGC family kinases, especially cancer. The core components of mTORC2 are mTOR, mLST8, Rictor, mSin1. The medium resolution cryo-EM structures (4.9Å, and 6.2Å) of the mTORC2 core complex were recently reported. We also determined the cryo-EM structure of this complex but to a much higher resolution at 3.0Å. Based on our findings, we propose to further study the missing mSin1 CRIM, RBD, PH domains as well as their interactions with AGC family kinases, Ras family small G-proteins using a combination of X-ray crystallography, cryo-EM and biochemical assays. Our goal is to elucidate mechanistically how mTORC2 recognize its substrates and how its activity is regulated by certain small G-proteins. We also plan to use cryo-EM to determine mTORC2-Protor1/2 complex structures, as neither the structure nor the function of Protor1/2 is clear.