Molecular mechanism of Rheb-dependent mTORC1 regulation

项目概要/摘要 mTORC 1是通过调节必需的细胞合成代谢来控制细胞生长/增殖的主控制器 过程，包括蛋白质，脂质和核苷酸合成。对于mTORC 1激活，而氨基酸 通过Rag小GTP酶（Rags）激活，将mTORC 1募集到溶酶体膜， 因子通过激活Rheb小GT3，直接激活溶酶体上的mTORC 1， mTORC 1的激活剂。然而，溶酶体mTORC 1通过其募集的分子机制， 活性Rags在溶酶体上发现并与Rheb相互作用尚未得到很好的理解。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形成异多聚体并显示出对mTORC 1的强结合偏好， 从而支持溶酶体上的mTORC 1活化（Yao等，Mol Cell，2020）。我们发现 共济失调蛋白3作为一种关键的溶酶体Ub-Rheb去泛素化酶，其溶酶体定位通过以下方式减轻： 氨基酸诱导的活性Rags。我们假设，泛素依赖的高阶组装 Rheb可能增加其在溶酶体膜上的密度，并有助于隔离mTORC 1及其受体。 用于激活的效应器。然而，产生Rheb多聚泛素化的Rheb的泛素连接酶， 并支持mTORC 1激活，尚未确定。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HUWE 1和Cullin 3泛素连接酶 复合物与Rheb相互作用并正向调节细胞mTORC 1活性而不影响Akt 活动我们的初步数据表明HUWE 1是Rheb与mTORC 1相互作用所必需的。 CAD是嘧啶从头合成的关键酶，其活性受Rheb刺激， mTORC 1-S6 K1通路。在这个建议中，我们将研究分子机制， HUWE 1和CUL 3泛素连接酶复合物协同泛素化Rheb，以及它们的作用 泛素连接酶在mTORC 1活性、从头嘧啶合成和细胞生长调节中的作用 控制我们提出的研究的具体目标是：调查是否以及如何这些泛素 连接酶协同泛素化Rheb以形成催化活性的Rheb多聚体，以刺激 mTORC 1活性（Aim 1）;以确定这种Ub-Rheb-mTORC 1依赖性 特异性下游途径如从头嘧啶合成（Aim 2）的信号放大; 研究HUWE 1/CUL 3-Ub Rhab-mTORC 1-CAD通路在肿瘤中的生理相关性。 调节癌细胞增殖和组织稳态，如肝脏和肾小球上皮 细胞（Aim 3）。拟议的研究将提供以前未被重视的分子机制， mTORC 1激活和对Ub-Rheb中心无膜区室的理解 用于放大mTORC 1依赖性信号以控制细胞生长/增殖。

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.