Molecular mechanism of Rheb-dependent mTORC1 regulation

项目摘要/摘要 MTORC1是通过调节必要的细胞合成代谢来控制细胞生长/增殖的主控者 过程，包括蛋白质、脂肪和核苷酸的合成。对于mTORC1的激活，而氨基酸 通过RAG Small GTP酶(RAGS)的激活、生长将mTORC1招募到溶酶体膜 因子通过激活Rheb小GTP酶来激活溶酶体上的mTORC1，这是一种直接的 MTORC1的激活剂。然而，溶酶体mTORC1被招募的分子机制 活性RAG在溶酶体上发现并与Rheb相互作用尚不清楚。我们 最近报道，氨基酸刺激增加了多泛素化Rheb(Ub-Rheb)的水平，这是 与不含泛素的Rheb形成异多聚体并对mTORC1表现出强烈的结合偏好， 从而支持溶酶体上mTORC1的激活(姚等人，Mol Cell，2020)。我们确认了 Aaxin3是一种关键的溶酶体Ub-Rheb脱泛素酶，其溶酶体定位可通过 氨基酸诱导的活性RAGS。我们假设依赖泛素的更高组装顺序 Rheb可能增加其在溶酶体膜上的密度，有助于mTORC1及其受体的隔离。 用于激活的效应器。然而，Rheb的泛素连接酶会产生Rheb的多泛素化 并支持mTORC1激活，但尚未确定。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，我们鉴定了HUWE1和Cullin3泛素连接酶 复合体与Rheb相互作用，在不影响Akt的情况下正向调节细胞mTORC1活性 活动。我们的初步数据表明，HUWE1是Rheb与mTORC1相互作用所必需的 和CAD，它是从头合成嘧啶的关键酶，其活性由Rheb和Rheb刺激。 MTORC1-S6K1途径。在这项提案中，我们将研究通过哪些分子机制 HUWE1和CUL3泛素连接酶复合体配位泛素化Rheb及其作用 泛素连接酶对mTORC1活性、从头合成嘧啶和细胞生长的调节 控制力。我们提议的研究的具体目的是：调查这些泛素是否以及如何 连接酶配位泛素化Rheb形成催化活性Rheb多聚体刺激 MTORC1活动(Aim1)；确定这种Ub-Rheb-mTORC1依赖的作用和机制 特定下游通路的信号放大，如从头合成嘧啶(AIM2)； 研究HUWE1/CUL3-Ub Rhab-mTORC1-CAD通路在大鼠脑内的生理学意义 调节癌细胞增殖和组织动态平衡，如肝脏和肾小球上皮 细胞(Aim3)。拟议的研究将提供以前未被认识到的分子机制 MTORC1的激活和对理解Ub-Rheb中心无膜室的洞察 用于放大mTORC1依赖的信号，用于细胞生长/增殖控制。

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.