Elucidating the mechanism of leucine sensing by Sestrin2 upstream of mTORC1

项目摘要/摘要 好了！雷帕霉素复合体1的作用靶点是细胞的主要调控因子 在癌症、糖尿病和年龄1-5岁等人类疾病中经常被放松调控的生长。 MTORC1途径整合了一系列不同的输入信号，如生长因子信号、能量 水平和养分利用率，以协调细胞的合成代谢和分解代谢 环境条件和生物体生理1，2。 在mTORC1上游的输入中，亮氨酸一直被认为是 至关重要。在哺乳动物中，亮氨酸刺激多种重要的生理功能 包括肌肉生长、饱腹感和胰岛素分泌等现象，很大程度上是通过 MTORC117-19的激活。包括亮氨酸在内的氨基酸通过RAG向mTORC1发出信号 GTP酶，mTORC111的间接激活因子。此外，GATOR1复合体的功能还包括 GAP为RAGA/B，而GATOR2与GATOR115相互作用并抑制GATOR115。 最近的IP-MS分析表明，Sestrins(Sestrin1-3)与GATOR2相互作用 在没有氨基酸的情况下抑制mTORC1途径的蛋白质20，21。后续 生化分析表明Sestrin2-GATOR2相互作用对 亮氨酸在体外，Sestrin2与亮氨酸结合，解离常数(Kd)约为20 与细胞信号数据一起帮助证明Sestrin2在真正的 MTORC122的亮氨酸传感器。 为了了解Sestrin2感知亮氨酸的生化机制，我们有 解算了亮氨酸结合的Sestrin2的晶体结构，揭示了其结构基础 Sestrin223法检测亮氨酸。这一分析揭示了进化上独特的亮氨酸结合 口袋，以及亮氨酸结合所需的“盖锁”机构的存在。尽管 然而，这些见解引发了关于机制的几个更多问题 塞斯特林家族对亮氨酸的感知。在这项提案中，我们旨在通过以下方式解决这些问题 以下是具体目标： 1.研究了亮氨酸从Sestrin2中解离的动力学。 2.确定不同亮氨酸结合亲和力的结构基础 并列鹦鹉 3.表征GATOR2结合的Sestrin2的结构 好了！

Project Summary/Abstract ! The mechanistic target of rapamycin complex 1 (mTORC1) is a major regulator of cell growth that is frequently deregulated in human diseases such as cancer, diabetes, and aging1-5. The mTORC1 pathway integrates a diverse set of inputs such as growth factor signaling, energy levels, and nutrient availability in order to coordinate cell anabolism and catabolism with environmental conditions and organismal physiology1,2. Among the inputs upstream of mTORC1, the amino acid leucine has long been known to be of critical importance16. In mammals, leucine stimulates a variety of important physiological phenomena including muscle growth, satiety, and insulin secretion, in large part through activation of mTORC117-19. Amino acids, including leucine, signal to mTORC1 through the Rag GTPases, indirect activators of mTORC111. In addition, the GATOR1 complex functions as a GAP for RagA/B, while GATOR2 interacts with and inhibits GATOR115. Recent IP-MS analyses identified the Sestrins (Sestrin1-3) as GATOR2 interacting proteins that inhibit the mTORC1 pathway in the absence of amino acids20,21. Subsequent biochemical analyses revealed that the Sestrin2-GATOR2 interaction is directly sensitive to leucine in vitro, and Sestrin2 binds leucine with a dissociation constant (KD) of approximately 20 µM, which together with cell signaling data helped demonstrate that Sestrin2 in a bona fide leucine sensor for mTORC122. To understand the biochemical mechanism of leucine sensing by Sestrin2, we have solved the crystal structure of leucine-bound Sestrin2 at 2.7Å, revealing the structural basis for leucine detection by Sestrin223. This analysis revealed an evolutionarily unique leucine binding pocket, as well as the presence of a “lid-latch” mechanism required for leucine binding. Despite these insights however, the structure raised several more questions regarding the mechanism of leucine sensing by the Sestrins. In this proposal, we aim to address these questions through the following specific aims: 1. Characterize the kinetics of leucine dissociation from Sestrin2. 2. Determine the structural basis for the different leucine binding affinities of Sestrin paralogs 3. Characterize the structure of GATOR2-bound Sestrin2 !