Activation of rapamycin-sensitive TORC1 by endomembrane amino acid transporters

描述（由申请人提供）：rapamycin复合物1靶点（TORC1）在一个普遍保守的信号转导级联中起着重要作用，该信号转导级联响应营养物质和生长因子信号，以控制细胞生长和增殖。TORC1级联的失调导致多种类型的人类恶性肿瘤。TORC1的中心成员是Tor蛋白激酶，它们是在酵母中发现的，这是一个模型系统，在阐明TORC1信号级联过程中至关重要。基于其有效的抗增殖活性，雷帕霉素被用于多个临床领域，包括免疫抑制、癌症化疗和介入心脏病学。尽管进行了大量研究，但TORC1通路仍有部分特征。我们发现了一个意想不到的作用，膜囊运输系统在调节酵母TORC1信号；参与囊泡运输和蛋白质分选的蛋白质复合物的突变与非必需的Tor1激酶的突变相结合，使细胞无法存活或严重损害生长。我们证明了hop复合体是为有效的TORC1信号传导和TORC1调控基因的正常表达提供氨基酸稳态所必需的。这些研究还揭示了雷帕霉素作用机制的一个新方面：雷帕霉素绕过囊泡运输事件来激活TORC1控制的反激活因子。此外，我们发现自我复合体（EGOC）的突变会损害TORC1信号，而最佳细胞生长需要EGOC和TORC1活性。在酵母、昆虫和哺乳动物细胞中，EGOC具有进化保守的氨基酸敏感的GTPase亚基及其同源物（Gtr1、2和RagA-D蛋白）介导TORC1激活。然而，氨基酸传感器的身份和氨基酸激活EGOC GTPase的潜在机制尚不清楚。有趣的是，TORC1和EGOC与空泡氨基酸转运蛋白一起共定位于包括核内体和空泡在内的细胞膜上。本研究的目的是：1)阐明膜系统激活TORC1信号的作用和潜在机制；2)确定哺乳动物中保守的液泡氨基酸转运体是否被整合到传递氨基酸信号以激活TORC1的分子级联中。我们的工作模型是，内膜网络提供了一个平台，促进分子相互作用，激活和启用TORC1信号。与酵母TORC1类似，哺乳动物TORC1也定位于内膜，特别是溶酶体，这是酵母液泡的对应物。因此，我们认为我们的研究将继续揭示基本的、保守的TORC1信号传导方面，这些方面可以直接外推到哺乳动物模型中，并指导对人类癌症和其他疾病中选择TORC1通路缺陷的药物干预。

DESCRIPTION (provided by applicant): The target of rapamycin complex 1 (TORC1) plays a prominent role in a ubiquitously conserved signal transduction cascade that responds to nutrients and growth factor cues to control cell growth and proliferation. Dysregulation of the TORC1 cascade results in multiple types of human malignancies. The central TORC1 members are the Tor protein kinases, which were discovered in the yeast Saccharomyces cerevisiae, a model system that has been crucial in elucidating the TORC1 signaling cascade. Based on its potent antiproliferative activity, rapamycin is in use in several clinical areas including immunosuppression, cancer chemotherapy, and interventional cardiology. The TORC1 pathway remains partially characterized despite much study. We uncovered an unexpected role for the endomembrane vesicular trafficking system in regulating TORC1 signaling in yeast; mutations in protein complexes with roles in vesicular trafficking and protein sorting in combination with mutation of the nonessential Tor1 kinase, render cells inviable or severely growth impaired. We demonstrated that the HOPS complex is required to provide amino acid homeostasis for efficient TORC1 signaling and for normal expression of TORC1-governed genes. These studies also revealed a novel facet in the rapamycin mechanism of action: rapamycin bypasses vesicular trafficking events to activate TORC1- controlled transactivators. Moreover, we showed that mutations in the Ego complex (EGOC) compromise TORC1 signaling and both EGOC and TORC1 activity are required for optimal cell growth. The EGOC possesses evolutionary conserved amino acid-sensitive GTPase subunits and orthologs of these (Gtr1,2 and RagA-D proteins) mediate TORC1 activation in yeast, insect, and mammalian cells. However, the identity of the amino acid sensors and the underlying mechanisms by which amino acids activate EGOC GTPase are unknown. Intriguingly, TORC1 and the EGOC colocalize to endomembranes including those of endosomes and vacuoles, along with vacuolar amino acid transporters. The aims of this proposal are: 1) to elucidate the roles and underlying mechanism by which the endomembrane system enables TORC1 signaling and 2) to determine whether the vacuolar amino acid transporters, which are conserved in mammals, are integrated into the molecular cascade that conveys amino acid signals to evoke TORC1 activation. Our working model is that the endomembrane network provides a platform to facilitate molecular interactions that activate and enable TORC1 signaling. Similar to yeast TORC1, mammalian TORC1 is also localized to endomembranes, in particular lysosomes, which are the counterparts of yeast vacuoles. Thus, we submit that our research will continue to uncover fundamental, conserved TORC1 signaling aspects that could be directly extrapolated to mammalian models and guide pharmacological intervention in select TORC1 pathway defects in human cancer and other diseases. PUBLIC HEALTH RELEVANCE: The rapamycin sensitive-TORC1 is the central component of an evolutionary-conserved signaling cascade that in response to nutrients and growth factors regulates cell growth and proliferation. Defects in mTORC1 signaling lead to cancer and other human diseases. Rapamycin and its analogs are being developed as therapeutic agents for the treatment of a wide range of malignances and could also find indication in cognitive- and aging-related diseases. The model yeast S. cerevisiae has been crucial in the discovery of the TORC1 pathway and elucidation of rapamycin mechanism of action. Characterization of this pathway in yeast is continuing to reveal basic aspects of TORC1 signaling that could foster analogous studies in mammals and lead to improved anticancer therapies.