A Molecular Study of Rag GTPase-Dependent Amino Acid Sensing

项目总结/摘要 雷帕霉素复合物1的机制靶点（mTORC 1）是细胞生长的关键调节因子， 增殖在有利的、营养丰富的环境中激活后，mTORC 1触发合成代谢反应 并抑制catalysts。营养信号，特别是氨基酸信号，通过一个 一系列蛋白质复合物，最终聚集在Rag GTP酶上，Rag GTP酶是一种异二聚体GTP酶， 直接将mTORC 1募集到溶酶体表面。Rag GTP酶及其调节因子的新发现 揭示了氨基酸充足和mTORC 1激活之间的关键中间体。但 这些蛋白质机器如何合作传递氨基酸信号的分子机制仍然是 难以捉摸。了解这一途径的机制细节将需要：（1）确定结构 关键蛋白质组分，以揭示对其生物学功能至关重要的结构域和残基，以及（2） 生物化学分析，以确定蛋白质功能的机制水平和量化的影响，具体 扰动在这项提案中，我们的目标是开发生物化学和生物物理工具来研究Rag依赖的 在分子水平上的氨基酸传感。具体来说，我们的目标是使用结构生物学工具， 可视化介导这一过程的蛋白质复合物（目的1），并进行酶动力学测定以定量 Rag GTP酶及其调节因子的功能（目的2）。此外，我们计划在体外重建一个 系统来重现Rag依赖的氨基酸传感（Aim 3），并直接可视化组织。 这些蛋白质机器在单分子水平（目标4）。这里开发的方法将提供一个 统一的模型，并产生新的见解，这一重要的生物过程。

Project Summary / Abstract The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a key regulator of cell growth and proliferation. Upon activation in a favorable, nutrient-rich environment, mTORC1 triggers anabolic reactions and inhibits catabolism. Nutrient signals, especially amino acid signals, are transmitted to mTORC1 through a series of protein complexes, which ultimately converge on the Rag GTPases, a heterodimeric GTPase that directly recruits mTORC1 to the lysosomal surface. Recent discovery of the Rag GTPases and their regulators has revealed a key intermediate between amino acid sufficiency and mTORC1 activation. However, the molecular mechanisms of how these protein machineries collaborate to transmit the amino acid signal are still elusive. Understanding the mechanistic details of this pathway will require: (1) determination of the structures of key protein components to reveal domains and residues that are critical for their biological functions, and (2) biochemical analyses to define protein functions at the mechanistic level and quantify the effect of specific perturbations. In this proposal, we aim to develop biochemical and biophysical tools to study Rag-dependent amino acid sensing at the molecular level. Specifically, we aim to use structural biology tools to directly visualize the protein complexes that mediate this process (Aim 1), and enzymatic kinetics assays to quantify the functions of the Rag GTPases and their regulators (Aim 2). Further, we plan to reconstitute an in vitro system to recapitulate Rag-dependent amino acid sensing (Aim 3), and directly visualize the organization of these protein machines at the single-molecule level (Aim 4). The approaches developed here will provide a unified model and yield novel insights into this important biological process.