高等真核生物氨基酸感应机制研究

Amino acids are not only the building blocks for proteins, but are also a type of specific cellular factors that regulate a variety of biological processes including biogenesis, autophagy, and metabolism, and consequently control cell growth and proliferation. Dysfunction of amino acid signaling is often related to pathogenesis of many diseases such as diabetes, obesity and cancers. Recently, a number of amino acid sensing proteins have been identified in higher eukaryote, including the arginine sensor CASTOR family, the leucine sensor Sestrin family and the SAM sensor SAMTOR, which can directly sense the cellular levels of different amino acids and regulate the downstream GATOR1, GATOR2 and Rag GTPases complexes, and thus play crucial roles in mTORC1 and AMPK regulation; however, the exact functions and their molecular mechanisms are still elusive. In this project, we will carry out detailed structural and functional studies of the aforementioned amino acid sensors using a combination of structural biology, biochemistry, molecular biology, and cell biology, elucidate the molecular basis of the substrate specificity of these sensing proteins with different amino acids, and uncover the molecular mechanisms of their functional roles in promoting amino acid-dependent activation of the mTORC1 signaling pathway. The results might also reveal insights into the functional roles of the mutations in the pathogenesis of related metabolic diseases, which will be useful in the design and development of diagnostics and therapeutics against these diseases. Thus, this study will have important biological significances and important biomedical implications.

氨基酸不仅是蛋白质合成的原材料，还是一种特殊的细胞因子调控细胞内与生长发育相关的多种生命过程，包括蛋白质合成与降解以及细胞自噬等，最终调节细胞生长、增殖以及生物体发育，其功能失调与多种疾病的发生发展相关，包括肥胖症、糖尿病和肿瘤等。近年来鉴定出一些氨基酸感应蛋白，包括精氨酸感应蛋白CASTOR家族、亮氨酸感应蛋白Sestrin家族和SAM感应蛋白SAMTOR等，通过直接感应细胞内氨基酸水平，调控下游GATOR1/2和Rag复合物的活性，进而影响下游mTORC1和AMPK等与生长发育相关的重要信号通路。但是这些感应蛋白的结构、功能以及发挥功能的分子机制还不清晰。本项目拟运用结构生物学、生物化学和细胞生物学等方法，系统研究这些感应蛋白的结构和功能，揭示它们特异性识别和结合不同氨基酸配体的分子基础，阐明它们发挥分子开关作用、调控下游信号通路的分子机制，探讨它们的功能失调与相关疾病发生发展的关系。

氨基酸是一种特殊的细胞因子调控细胞内与生长发育相关的多种生命过程，包括蛋白质合成与降解以及细胞自噬等，最终调节细胞生长、增殖以及生物体发育，其功能失调与多种疾病的发生发展相关，包括肥胖症、糖尿病和肿瘤等。近年来鉴定出一些氨基酸感应蛋白，通过直接感应细胞内氨基酸水平，调控下游GATOR1/2和Rag-Ragulator、EGOC复合物的活性，进而协同Rheb、Rab等小G蛋白影响下游mTORC1等与生长发育相关的重要信号通路活性。但是这些感应蛋白以及下游调控蛋白的结构、功能以及发挥功能的分子机制还不清晰。本项目以高等真核生物中氨基酸感应蛋白CASTOR、SAMTOR以及相关调控蛋白EGOC、小G蛋白Rheb和Rab9A等为研究对象，运用结构生物学、生物化学、分子生物学和细胞生物学等方法，系统研究这些蛋白在氨基酸感应和细胞生长调控过程中发挥生物学功能的结构基础和分子机制，测定了氨基酸信号通路调控中发挥重要功能的EGO-TC与酵母Rag小G蛋白的同源蛋白Gtr1-Gtr2共同形成的五元复合物（EGOC）的晶体结构，揭示了Rheb突变体Y35N和D60K调控mTORC1活性的分子机制，探讨它们的功能失调与相关疾病发生发展的关系，测定了参与氨基酸运输和内吞体代谢的Rab9A-Nde1复合物的结构，并通过测定SAM感应蛋白SAMTOR多个结构，阐明它们发挥分子开关作用、调控下游信号通路的分子机制，这些研究结果进一步阐释了真核生物中氨基酸等营养物质对mTORC1信号通路的调控机制和保守性，为后续靶向这些重要蛋白和下游mTORC1信号通路的新药研发建立了分子基础。