Molecular mechanism of Rheb-dependent mTORC1 regulation

Rheb 依赖性 mTORC1 调节的分子机制

• 批准号: 10641878
• 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
• 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/10641878
• 关键词: Ablation; Affect; Amino Acids; Aspartate; Binding; Biogenesis; Carbamyl Phosphate; Cell Proliferation; Cells; Code; Complex; Cullin Proteins; 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; Proliferating; 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 isopeptidase; ubiquitin ligase

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.

项目摘要/摘要 MTORC1是通过调节必需细胞合成代谢的细胞生长/增殖的主控制器 过程，包括蛋白质，脂质和核苷酸合成。用于MTORC1激活，而氨基酸 通过抹布小GTPases（抹布）激活，生长，将mTORC1募集到溶酶体膜到溶酶体膜 因素通过激活Rheb小GTPase（直接）在溶酶体上执行MTORC1激活 MTORC1的激活剂。但是，溶酶体MTORC1募集的分子机制 活跃的破布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的RHEB（UB-RHEB）水平，该水平是 与无泛素的Rheb形成异植物，并显示出对MTORC1的强烈结合偏好， 从而支持溶酶体上的MTORC1激活（Yao等，Mol Cell，2020）。我们确定了这一点 ataxin3充当关键的溶酶体UB-RHEB去素化酶，其溶酶体定位被减轻 氨基酸诱导的活性破布。我们假设泛素依赖于组装的高阶 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 激活的效应器。但是，Rheb的泛素连接酶，产生Rheb多泛素化 尚未确定支持MTORC1激活。使用UB-Rheb相互作用蛋白质组学和 溶酶体蛋白质组学数据库，我们确定了HUWE1和Cullin3泛素连接酶 复合物与Rheb相互作用并积极调节细胞MTORC1活性而不影响AKT 活动。我们的初步数据表明，Huwe1是Rheb与两个MTORC1相互作用所必需的 和CAD，是从头嘧啶合成的关键酶，其中Rheb和Rheb刺激其活性 MTORC1-S6K1途径。在此提案中，我们将研究分子机制 HUWE1和CUL3泛素连接酶复合物协调泛素脉络及其作用 MTORC1活性，从头嘧啶合成和细胞生长的调节中的泛素连接酶 控制。我们拟议的研究的具体目的是：研究这些泛素是否以及如何 连接酶协同泛素的Rheb形成催化胜任的Rheb多聚体以刺激 MTORC1活动（AIM1）;确定该UB-RHEB-MTORC1依赖性的作用和机制 特定下游途径的信号扩增，例如从头嘧啶合成（AIM2）； 研究此HUWE1/CUL3-UB RHAB-MTORC1-CAD途径的生理相关性 调节癌细胞增殖和组织稳态，例如肝脏和肾小球上皮 细胞（AIM3）。拟议的研究将提供先前未批准的分子机制 MTORC1激活和对理解UB-RHEB中心的无膜隔室的洞察力 用于扩增MTORC1依赖性信号以进行细胞生长/增殖控制。