Molecular mechanism of Rheb-dependent mTORC1 regulation

Rheb 依赖性 mTORC1 调节的分子机制

基本信息

批准号：
10416125
负责人：
Ken Inoki
金额：
$ 45.03万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10416125
关键词：
Ablation Affect Amino Acids Aspartate Binding Biogenesis Carbamyl Phosphate Cell Proliferation Cells Code Complex Data Databases Dihydroorotase Enzymes Epithelial Cells FRAP1 gene Functional disorder Generations Goals Growth Growth Factor Homeostasis Kidney Knockout Mice Ligase Lipids Liver Lysosomes Machado-Joseph Disease Mediating Membrane Molecular Monomeric GTP-Binding Proteins Mus Mutation NF-kappa B Nucleotides Organ Pathway interactions Physiological Play Polyubiquitination Post-Translational Protein Processing Process Protein Kinase Protein-Serine-Threonine Kinases Proteins Proteomics Pyrimidine Recruitment Activity Regulation Reporting Role Signal Transduction Starvation Structure Surface Tissues Ubiquitin cancer cell cell growth density in vivo insight knock-down lysosome membrane multicatalytic endopeptidase complex novel nucleotide metabolism podocyte preference recruit response transcarbamylase ubiquitin ligase

项目摘要

Project Summary/Abstract mTORC1 is a master controller of cell growth/proliferation by regulating essential cellular anabolic processes, including protein, lipid, and nucleotide synthesis. For mTORC1 activation, while amino acids recruit mTORC1 to the lysosomal membrane through Rag small GTPases (Rags) activation, growth factors execute mTORC1 activation on the lysosome by activating Rheb small GTPase, a direct activator of mTORC1. However, the molecular mechanisms by which lysosomal mTORC1 recruited by the active Rags finds and interacts with Rheb on the lysosome have not been well understood. We recently reported that amino acid stimulation increased polyubiquitinated Rheb (Ub-Rheb) levels, which forms heteromultimers with ubiquitin-free Rheb and displays a strong binding preference for mTORC1, thereby supporting mTORC1 activation on the lysosome (Yao et al., Mol Cell, 2020). We identified that Ataxin3 acts as a key lysosomal Ub-Rheb deubiquitinase whose lysosomal localization is mitigated by amino acid-induced active Rags. We hypothesized that ubiquitin-dependent higher-order of assembly of Rheb might increase its density on the lysosome membrane and help sequester mTORC1 and its effectors for activation. However, ubiquitin ligases for Rheb, which generate Rheb polyubiquitination and support mTORC1 activation, have not been identified. Using Ub-Rheb-interacting proteomics and lysosome proteomics databases, we identified that both HUWE1 and the Cullin3 ubiquitin ligase complex interact with Rheb and positively regulate cellular mTORC1 activity without affecting Akt activity. Our preliminary data indicate that HUWE1 is required for Rheb to interact with both mTORC1 and CAD, a key enzyme for de novo pyrimidine synthesis, of which activity is stimulated by Rheb and the mTORC1-S6K1 pathway. In this proposal, we will study the molecular mechanisms by which HUWE1 and the CUL3 ubiquitin ligase complex coordinately ubiquitinate Rheb and the role of these ubiquitin ligases in the regulation of mTORC1 activity, de novo pyrimidine synthesis, and cell growth control. The specific aims of our proposed studies are to: investigate whether and how these ubiquitin ligases coordinately ubiquitinate Rheb to form catalytically competent Rheb multimers to stimulate mTORC1 activity (Aim1); to determine the roles and mechanisms of this Ub-Rheb-mTORC1-dependent signal amplification for specific downstream pathways such as de novo pyrimidine synthesis (Aim2); study the physiological relevance of this HUWE1/CUL3-Ub Rhab-mTORC1-CAD pathway in the regulation of cancer cell proliferation and tissue homeostasis such as liver and glomerular epithelial cells (Aim3). The proposed studies will provide previously unappreciated molecular mechanisms for mTORC1 activation and insight into the understanding Ub-Rheb-centered membraneless compartment for amplifying mTORC1-dependent signals for cell growth/proliferation control.

项目概要/摘要 mTORC1 是细胞生长/增殖的主控制器，通过调节重要的细胞合成代谢过程，包括蛋白质、脂质和核苷酸的合成。对于 mTORC1 激活，而氨基酸通过 Rag 小 GTPases (Rags) 激活、生长，将 mTORC1 募集至溶酶体膜因子通过激活 Rheb 小 GTP 酶（一种直接的酶）在溶酶体上执行 mTORC1 激活 mTORC1 激活剂。然而，溶酶体 mTORC1 招募的分子机制活性 Rags 在溶酶体上发现并与 Rheb 相互作用尚未得到很好的了解。我们最近报道，氨基酸刺激增加了多泛素化 Rheb (Ub-Rheb) 水平，与不含泛素的 Rheb 形成异多聚体，并对 mTORC1 显示出强烈的结合偏好，从而支持溶酶体上的 mTORC1 激活（Yao 等人，Mol Cell，2020）。我们确定 Ataxin3 是一种关键的溶酶体 Ub-Rheb 去泛素酶，其溶酶体定位可通过以下因素减轻：氨基酸诱导的活性Rags。我们假设泛素依赖的高阶组装 Rheb 可能会增加其在溶酶体膜上的密度，并有助于隔离 mTORC1 及其用于激活的效应器。然而，Rheb 的泛素连接酶可产生 Rheb 多泛素化并支持mTORC1激活，尚未确定。使用 Ub-Rheb 相互作用蛋白质组学和在溶酶体蛋白质组数据库中，我们发现 HUWE1 和 Cullin3 泛素连接酶复合物与 Rheb 相互作用并正向调节细胞 mTORC1 活性而不影响 Akt 活动。我们的初步数据表明，Rheb 需要 HUWE1 才能与 mTORC1 相互作用 CAD，一种嘧啶从头合成的关键酶，其活性受到 Rheb 和 mTORC1-S6K1 通路。在本提案中，我们将研究其分子机制 HUWE1 和 CUL3 泛素连接酶复合物协同泛素化 Rheb 及其作用泛素连接酶在调节 mTORC1 活性、嘧啶从头合成和细胞生长中的作用控制。我们提出的研究的具体目的是：调查这些泛素是否以及如何连接酶协同泛素化 Rheb，形成具有催化能力的 Rheb 多聚体，从而刺激 mTORC1 活性（Aim1）；确定这种 Ub-Rheb-mTORC1 依赖性的作用和机制特定下游途径的信号放大，例如嘧啶从头合成 (Aim2)；研究 HUWE1/CUL3-Ub Rhab-mTORC1-CAD 通路在调节癌细胞增殖和组织稳态，例如肝脏和肾小球上皮细胞（目标3）。拟议的研究将提供以前未被重视的分子机制 mTORC1 激活和深入了解以 Ub-Rheb 为中心的无膜室用于放大 mTORC1 依赖性信号以控制细胞生长/增殖。