Elucidating the mechanism of leucine sensing by Sestrin2 upstream of mTORC1

阐明 mTORC1 上游 Sestrin2 的亮氨酸传感机制

• 批准号: 9325187
• 负责人: Robert Andrew Saxton
• 金额: $ 4.4万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325187
• 关键词: Acids; Address; Affinity; Amino Acids; Anabolism; Binding; Biochemical; Biological Assay; Catabolism; Cells; Complex; Crystallization; Data; Detection; Development; Diabetes Mellitus; Dissociation; FRAP1 gene; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Kinetics; Leucine; Lysosomes; Malignant Neoplasms; Mammals; Medical; Modeling; Molecular Conformation; Molecular Profiling; Mus; Muscle; Nucleotides; Nutrient; Pathway interactions; Physiological; Proteins; Recruitment Activity; Satiation; Signal Transduction; Site; Structure; Surface; Tissues; Work; cell growth; detection of nutrient; human disease; in vivo; insight; insulin secretion; interest; mutant; new therapeutic target; paralogous gene; sensor

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 !

项目概要/摘要 ！雷帕霉素复合物 1 (mTORC1) 的机制靶标是细胞的主要调节因子 在癌症、糖尿病和衰老等人类疾病中，生长经常失控1-5。 mTORC1 通路整合了多种输入，例如生长因子信号传导、能量 水平和营养可用性，以协调细胞合成代谢和分解代谢 环境条件和机体生理学1,2。 在 mTORC1 上游的输入中，长期以来人们都知道氨基酸亮氨酸可以 至关重要 16.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在内的现象，很大程度上是通过 mTORC117-19 的激活。氨基酸（包括亮氨酸）通过 Rag 向 mTORC1 发出信号 GTPases，mTORC111 的间接激活剂。此外，GATOR1 复合物的功能是 RagA/B 的 GAP，而 GATOR2 与 GATOR115 相互作用并抑制 GATOR115。 最近的 IP-MS 分析确定 Sestrins (Sestrin1-3) 与 GATOR2 相互作用 在缺乏氨基酸的情况下抑制 mTORC1 通路的蛋白质20,21。随后的 生化分析表明 Sestrin2-GATOR2 相互作用直接敏感 在体外，Sestrin2 与亮氨酸结合，解离常数 (KD) 约为 20 µM，与细胞信号传导数据一起帮助证明 Sestrin2 真正有效 mTORC122 的亮氨酸传感器。 为了了解 Sestrin2 亮氨酸传感的生化机制，我们有 解析了 2.7Å 亮氨酸结合的 Sestrin2 的晶体结构，揭示了 通过 Sestrin223 检测亮氨酸。该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”机制的存在。尽管 然而，这些见解，该结构提出了有关机制的更多问题 Sestrins 的亮氨酸感应。在本提案中，我们旨在通过以下方式解决这些问题 具体目标如下： 1. 表征 Sestrin2 亮氨酸解离的动力学。 2. 确定 Sestrin 不同亮氨酸结合亲和力的结构基础 旁系同源物 3. 表征 GATOR2 结合的 Sestrin2 的结构 ！