Characterization of novel regulators of amino acid-sensitive mTORC1 signaling

描述（由申请人提供）：雷帕霉素的哺乳动物靶标（mTOR）是丝氨酸-苏氨酸激酶，是mTOR复合物1（mTORC 1）的催化亚基。mTORC 1是细胞生长和活力的主要调节因子，促进蛋白质合成、核糖体生物合成和线粒体生物合成，同时抑制自噬的分解代谢过程。毫不奇怪，该途径在癌症中失调，并且许多充分表征的肿瘤抑制剂是mTORC 1途径的负调节剂。这种广泛的合成代谢和分解代谢途径的调节需要mTORC 1作为细胞健康状态的监测器。它的活性在细胞中受到氨基酸水平、生长因子、能量水平以及DNA损伤和其他细胞应激的严格调节。在所有这些输入中，氨基酸是独特的;它们利用独立的机制来激活激酶，并且是活性所必需的，即使在存在其他激活刺激的情况下也是如此。尽管越来越多 尽管我们对控制mTORC 1活性的上游途径了解不多，但令人惊讶的是，我们对氨基酸如何被感知或它们的水平如何向mTORC 1发出信号知之甚少。阐明mTORC 1通路的这一关键方面将有助于为mTORC 1的药理学抑制提供新的靶点，同时可能最大限度地减少毒副作用。该项目的目标是识别和表征mTORC 1活性的新型调节剂，以响应氨基酸水平。我们已经使用蛋白质组学方法来鉴定与在氨基酸存在下激活mTORC 1的机制相互作用的蛋白质。在这个建议中，我们专注于一个特定的相互作用的mTORC 1通路中的蛋白质的特性。此外，该蛋白已被证明是B型肝炎病毒癌基因的细胞靶点，可能涉及mTORC 1在病毒感染和B型肝炎病毒相关肝细胞癌中的作用。为了表征这种新的蛋白质，我们提出了以下目标：1。使用敲低实验，通过其控制mTOR运输至溶酶体（激酶在溶酶体中被激活），确定该新途径成员在氨基酸激活mTORC 1中的作用。2.通过免疫沉淀和质谱鉴定与新途径成员相互作用的蛋白质，以充分阐明其在氨基酸传感途径中的功能。3.探讨氨基酸敏感性mTORC 1通路在B型肝炎病毒感染中的作用。

DESCRIPTION (provided by applicant): Mammalian Target of Rapamycin (mTOR) is serine-threonine kinase and the catalytic subunit of mTOR Complex 1 (mTORC1). mTORC1 is a master regulator of cellular growth and viability, promoting protein synthesis, ribosome biogenesis, and mitochondrial biogenesis while inhibiting the catabolic process of autophagy. Not surprisingly, this pathway is dysregulated in cancer, and a number of well characterized tumor suppressors are negative regulators of the mTORC1 pathway. Regulation of such broad anabolic and catabolic pathways requires that mTORC1 acts as a monitor of the health status of the cell. Its activity is tightly regulated in cells by the levels of amino acids, growth factors, nd energy levels, as well as DNA damage and other cellular stresses. Of all of these inputs, amino acids are unique; they utilize an independent mechanism to activate the kinase and are absolutely required for activity, even in the presence of other activating stimuli. Despite growing knowledge of the upstream pathways that control mTORC1 activity, surprisingly little is known about how amino acids are sensed or how their levels signal to mTORC1. Elucidation of this crucial aspect of the mTORC1 pathway will be instrumental in providing new targets for pharmacological inhibition of mTORC1 while potentially minimizing toxic side effects. The goal of this project is to identify and characterize novel regulators of mTORC1 activity in response to amino acid levels. We have used a proteomic approach to identify proteins that interact with the machinery that activates mTORC1 in the presence of amino acids. In this proposal, we focus on the characterization of one particular interacting protein in the mTORC1 pathway. Additionally, this protein has been shown to be a cellular target of a Hepatitis B virus oncogene, possibly implicating a role for mTORC1 in both viral infection and Hepatitis B virus-associated hepatocellular carcinoma. To characterize this new protein, we propose to following aims: 1. Define the role of this new pathway member in the activation of mTORC1 by amino acids through its control of mTOR trafficking to the lysosome, where the kinase is activated, using knockdown experiments. 2. Identify proteins that interact with the new pathway member using immunoprecipitation followed by mass spectrometry in order to fully elucidate its function within the amino-acid sensing pathway. 3. Interrogate the role of the amino acid-sensitive mTORC1 pathway in Hepatitis B virus infection.