Activation of rapamycin-sensitive TORC1 by endomembrane amino acid transporters

描述(申请人提供)：雷帕霉素复合体1(TORC1)的靶标在一个普遍保守的信号转导级联中起着重要作用，该信号转导系统响应营养物质和生长因子信号来控制细胞的生长和增殖。TORC1级联的失调导致多种类型的人类恶性肿瘤。TORC1的中心成员是在酵母中发现的Tor蛋白激酶，酿酒酵母是一个模型系统，在阐明TORC1信号级联中起着至关重要的作用。基于其强大的抗增殖活性，雷帕霉素被用于几个临床领域，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途径仍有部分特征。我们发现了膜内囊泡运输系统在调节酵母TORC1信号中的一个意想不到的作用；在囊泡运输和蛋白质分选中起作用的蛋白质复合体的突变，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，HOPS复合体是为有效的TORC1信号和TORC1控制的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中的一个新方面：雷帕霉素绕过囊泡转运事件激活TORC1控制的反式激活子。此外，我们发现自我复合体(EGOC)的突变损害了TORC1信号，并且EGOC和TORC1活性都是细胞最佳生长所必需的。EGOC具有进化保守的氨基酸敏感的GTPase亚基和这些亚基的同源蛋白(Gtr1，2和raga-D蛋白)，在酵母、昆虫和哺乳动物细胞中介导TORC1的激活。然而，氨基酸感受器的特性和氨基酸激活EGOC GTP酶的潜在机制尚不清楚。有趣的是，TORC1和EGOC共同定位于内膜，包括内小体和液泡的内膜，以及液泡氨基酸转运体。这一建议的目的是：1)阐明膜内系统激活TORC1信号的作用和潜在机制；2)确定在哺乳动物中保守的空泡氨基酸转运体是否整合到传递氨基酸信号的分子级联中，从而激活TORC1信号。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，它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信号方面，这些方面可以直接推断到哺乳动物模型，并指导对人类癌症和其他疾病中特定TORC1途径缺陷的药物干预。 公共卫生相关性：雷帕霉素敏感-TORC1是进化保守的信号级联反应的中心组成部分，该信号级联反应营养物质和生长因子，调节细胞的生长和增殖。MTORC1信号的缺陷会导致癌症和其他人类疾病。雷帕霉素及其类似物正在被开发为治疗多种恶性肿瘤的治疗剂，也可以在认知和衰老相关疾病中找到适应症。酿酒酵母模式菌在TORC1途径的发现和雷帕霉素作用机制的阐明中起着至关重要的作用。在酵母中对这一途径的表征正在继续揭示TORC1信号的基本方面，这可能会促进哺乳动物的类似研究，并导致改进的抗癌治疗。

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.