Elucidating the mechanism of mTORC1 activation by amino acids

阐明氨基酸激活 mTORC1 的机制

• 批准号: 8905995
• 负责人: Laura Alice Stransky
• 金额: $ 3.89万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905995
• 关键词: ATPase Domain; Amino Acids; Amino Acids Activation; Area; Biological; Breast; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer Treatment; Breast Cancer cell line; Bypass; CDK6-associated protein p18; Cancer Biology; Cancer Etiology; Cell Fractionation; Cell Line; Cell physiology; Cell-Free System; Cells; Cessation of life; Characteristics; Co-Immunoprecipitations; Complex; Data; Development; Disease; Dissociation; Drug Targeting; EGF gene; Enzymes; Equilibrium; Event; Fluorescence Microscopy; Genes; Glucose; Goals; Growth; Growth Factor; Holoenzymes; Homeostasis; Knowledge; Libraries; Light; Luciferases; Lysosomes; Malignant Neoplasms; Mammary Neoplasms; Mediator of activation protein; Membrane; Molecular; Nutrient; Output; Pathway interactions; Peripheral; Play; Process; Proteins; Proton Pump; Protons; Recruitment Activity; Regulation; Role; Signal Pathway; Signal Transduction; Sirolimus; Starvation; Stimulus; Surface; System; Testing; Tumor Tissue; United States; Woman; Work; cancer type; cell growth; combat; drug development; human FRAP1 protein; improved; malignant breast neoplasm; neoplastic cell; novel; novel therapeutics; public health relevance; research study; response; screening; small hairpin RNA; therapeutic target; transmission process; tumor initiation; tumor progression; tumorigenic; vacuolar H+-ATPase

 DESCRIPTION (provided by applicant): Novel treatments for breast cancer are urgently needed to limit the impact of this deadly disease. In depth biological knowledge of signaling pathways is critical to the selection of appropriate targets for drug development. To that end, the goal of this work is to elucidate the biological contributions of the vacuolar H+- ATPase (V-ATPase) rotary proton pump to mTORC1 signaling. mTORC1 is a hub of cellular growth control that is often deregulated in cancer. These experiments will elucidate new ways to alter mTORC1 signaling and combat breast cancer. Specifically, the proposed study will evaluate the mechanism by which the V-ATPase participates in amino acid-induced mTORC1 activation. We hypothesize that reversible disassembly of the V-ATPase is a key event in amino acid signal transduction, and that this mechanism is altered in breast cancer. We will assess V-ATPase assembly in response to amino acid availability in both immortalized, but untransformed, cell lines and in a panel of breast cancer cell lines using both a cell fractionation approach and fluorescence microscopy. We expect that amino acid depletion will result in a reversible increase in assembly of the V-ATPase, which then leads to a decrease in mTORC1 signaling. The full network of factors necessary for amino acid sensing and subsequent mTORC1 activation is unknown. We hypothesize that the amino acid sensing machinery is contained within the lysosome, and will seek to identify the proteins that contribute to this process using a shRNA screening approach. We will screen a library shRNAs targeting lysosomal genes for the ability to modulate amino acid sensing. To facilitate screening in high throughput, we will employ a split luciferase system, consisting of subunits of the V-ATPase and the Ragulator (a key mediator of mTORC1 activation by amino acids) known to associate in an amino acid dependent manner, each tagged with a portion of the luciferase enzyme. Knockdown of a factor necessary for amino acid sensing would mimic the amino acid starved state, leading to constitutive association of the V- ATPase with the Ragulator and sustained luciferase activity. We will validate candidates by co- immunoprecipitation of the V-ATPase and Ragulator and assessment of downstream markers of mTORC1 activity after shRNA knockdown. These studies will identify the other lysosomal factors that are necessary for amino acid sensing. Together, accomplishment of this study will deepen our knowledge of events critical to breast tumor initiation and progression, and reveal new ways to target the mTOR pathway in cancer.

 描述（由申请人提供）：迫切需要新的乳腺癌治疗方法来限制这种致命疾病的影响。深入了解信号通路的生物学知识对于选择药物开发的适当靶点至关重要。为此， 本工作的目的是阐明液泡H+-ATP酶（V-ATP酶）旋转质子泵对mTORC 1信号转导的生物学贡献。mTORC 1是细胞生长控制的中心，通常在癌症中失调。这些实验将阐明改变mTORC 1信号传导和对抗乳腺癌的新方法。 具体而言，拟定的研究将评价V-ATP酶参与氨基酸诱导的mTORC 1激活的机制。我们假设V-ATP酶的可逆性分解是氨基酸信号转导的关键事件，并且这种机制在乳腺癌中发生了改变。我们将评估V-ATP酶组装在两个永生化的，但未转化的，细胞系和乳腺癌细胞系的面板中使用细胞分级分离的方法和荧光显微镜的氨基酸可用性。我们预计氨基酸消耗将导致V-ATP酶组装的可逆增加，然后导致mTORC 1信号转导的减少。 氨基酸传感和随后的mTORC 1激活所需的因子的完整网络是未知的。我们假设氨基酸传感机制包含在溶酶体中，并将寻求使用shRNA筛选方法来鉴定有助于此过程的蛋白质。我们将筛选靶向溶酶体基因的文库shRNA以调节氨基酸传感的能力。为了促进高通量筛选，我们将采用裂解荧光素酶系统，该系统由已知以氨基酸依赖性方式缔合的V-ATP酶和Ragulator（氨基酸激活mTORC 1的关键介质）亚基组成，每个亚基均标记有荧光素酶的一部分。氨基酸传感所必需的因子的敲低将模拟氨基酸饥饿状态，导致V-ATP酶与Ragulator的组成型缔合和持续的荧光素酶活性。我们将通过V-ATP酶和Ragulator的免疫共沉淀以及评估shRNA敲低后mTORC 1活性的下游标志物来验证候选物。这些研究将确定其他溶酶体的因素是必要的氨基酸传感。总之，这项研究的完成将加深我们对乳腺肿瘤发生和进展的关键事件的了解，并揭示靶向癌症中mTOR通路的新方法。