Characterization of novel regulators of amino acid-sensitive mTORC1 signaling

氨基酸敏感 mTORC1 信号传导新型调节剂的表征

• 批准号: 8311210
• 负责人: Lawrence Schweitzer
• 金额: $ 4.22万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8311210
• 关键词: Adverse effects; Amino Acids; Autophagocytosis; Binding; Biogenesis; Biological Assay; Catabolic Process; Catalytic Domain; Cells; Cellular Stress; Complex; DNA Damage; Data; Dimerization; Goals; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Health Status; Hepatitis B; Hepatitis B Virus; Immunoprecipitation; Infection; Knowledge; Light; Location; Lysosomes; Malignant Neoplasms; Mass Spectrum Analysis; Mitochondria; Molecular; Monitor; Nucleotides; Oncogenes; Pathway interactions; Phosphotransferases; Primary carcinoma of the liver cells; Process; Protein Biosynthesis; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Regulation; Ribosomes; Role; Signal Transduction; Stimulus; Testing; Therapeutic; Tumor Suppressor Proteins; Up-Regulation; Virus Diseases; Western Blotting; cell growth; cellular targeting; genetic regulatory protein; human FRAP1 protein; insight; mTOR inhibition; mTOR protein; member; novel; research study; response; trafficking

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.

描述(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和mTOR复合体1的催化亚单位(MTORC1)。MTORC1是细胞生长和活性的主要调节因子，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。不足为奇的是，这一途径在癌症中调节失调，一些特征良好的肿瘤抑制因子是mTORC1途径的负调节因子。对这种广泛的合成代谢和分解代谢途径的调节需要mTORC1作为细胞健康状态的监测器。它的活性在细胞内受到氨基酸水平、生长因子、能量水平以及DNA损伤和其他细胞应激的严格调控。在所有这些输入中，氨基酸是独一无二的；它们利用一个独立的机制来激活激酶，即使在存在其他激活刺激的情况下，也是激活所必需的。尽管不断增长 由于对控制mTORC1活性的上游通路的了解，令人惊讶的是，关于氨基酸是如何被感知的，或者它们的水平是如何向mTORC1发出信号的，人们知之甚少。阐明mTORC1途径的这一关键方面将有助于为mTORC1的药物抑制提供新的靶点，同时潜在地将毒副作用降至最低。该项目的目标是识别和表征mTORC1活性的新调节因子，以响应氨基酸水平。我们使用蛋白质组学的方法来识别在氨基酸存在的情况下与激活mTORC1的机制相互作用的蛋白质。在这个建议中，我们集中在mTORC1途径中一种特定的相互作用蛋白的特征上。此外，该蛋白已被证明是乙肝病毒癌基因的细胞靶点，可能暗示mTORC1在病毒感染和乙肝病毒相关性肝细胞癌中发挥作用。为了确定这一新蛋白的特性，我们提出了以下目标：1.通过敲除实验，通过控制mTOR转运到溶酶体，确定这一新的途径成员在氨基酸激活mTORC1中的作用。2.利用免疫沉淀和质谱仪鉴定与新途径成员相互作用的蛋白质，以便充分阐明其在氨基酸传感途径中的功能。3.探讨氨基酸敏感的mTORC1通路在乙肝病毒感染中的作用。