How does mTOR sense lipid in vivo

mTOR如何感知体内脂质

• 批准号: 10415840
• 负责人: Henrietta J Bains
• 金额: $ 4.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415840
• 关键词: Age; Aging; Amino Acids; Autophagocytosis; BODIPY; Binding; Biochemical; Cells; Cholesterol; Complex; Corn Oil; Cultured Cells; Cytoskeletal Modeling; Data; Diabetes Mellitus; Diglycerides; Disease; Enzymes; FRAP1 gene; Failure; Fatty Acids; Geroscience; Growth; Growth Factor; Immunofluorescence Immunologic; In Vitro; Inflammatory; Label; Lead; Lipids; Liver; Longevity; Lysosomes; Malignant Neoplasms; Membrane; Membrane Lipids; Membrane Microdomains; Metabolic Diseases; Metabolism; Modeling; Mus; Nerve Degeneration; Nutrient; Oils; Oral; Organism; Outcome; Pathway interactions; Pharmacology; Phosphatidic Acid; Process; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Regulation; Signal Transduction; Sirolimus; Site; Small Interfering RNA; Surface; Testing; Tissue Preservation; Western Blotting; Whole Organism; adeno-associated viral vector; age related; aging population; biological adaptation to stress; cohesion; detection of nutrient; genetic regulatory protein; healthspan; in vivo; inhibitor; insight; lipid metabolism; lipidomics; lysosome membrane; mortality; new therapeutic target; novel; protein complex; proteostasis; sensor; small hairpin RNA

Alterations in lipid metabolism determine metabolic disease and mortality in the aging population. Despite our understanding of regulation of lipid metabolism, how organisms sense lipid remains unknown. It is conceivable that sensing of lipid will inform downstream decisions taken by the cell that modulate metabolism, proteostasis, stress response, and growth—each of which are dysregulated with age. The mechanistic target of rapamycin (mTOR), is a serine/threonine kinase and amino acid sensor, that drives growth and proliferation. More recently, mTOR in cultured cells has been shown to be activated by cholesterol and phosphatidic acid (PA) in absence of amino acids. Whether mTOR senses lipid in whole organisms is unclear. mTOR exists as two major complexes—mTORC1 and mTORC2. Activation of mTORC1 occurs at the lysosomal surface in presence of amino acids and requires key regulatory proteins that stimulate its activity. By contrast, mTORC2 responds to growth factors to regulate cytoskeletal organization. Hyperactivation of mTORC1 (hereafter, mTOR) drives aging and age-related diseases in part by disrupting autophagy and promoting growth. However, how mTORC1 is hyperactivated with age remains unknown. It has been shown that there are quantitative and qualitative changes in membrane lipids with age including changes in lysosomal membrane lipids—the major site of mTOR activation. Our preliminary data show that subjecting mice to an oral gavage of corn oil causes activation of mTOR and its translocation to distinct cholesterol-rich microdomains (CRMs)/lipid rafts in lysosome membranes. Our preliminary data also show that immunoprecipitating mTOR from lysosome membranes from livers of oil-gavaged mice reveal its binding to diacylglycerol. These data suggest that mTOR is a sensor of diacylglycerol, a membrane lipid. Since mTOR senses nutrients at lysosome membranes, I hypothesize that mTOR senses lipid at lysosomal membranes, and that age-related changes in lysosomal membrane lipid composition lead to mTOR hyperactivation. To test our hypothesis, we present the following specific aims: In Aim 1, diverse approaches will be used to characterize lipid-driven mTOR activation at lysosome membranes. By immunoprecipitating mTOR from lysosome membranes for lipidomic and proteomic analyses, I will identify lipid species that bind to mTOR and its interacting partners. I will use an siRNA screen in vitro to silence each of the interacting partners, which will identify novel regulators of lipid-driven mTOR signaling. In Aim 2, I will characterize the changes in lipid composition of lysosome membrane CRMs and expansion of lysosome CRMs with age. I will determine whether alterations in membrane lipid composition with age correlate with increased mTOR activity. I will then determine whether inactivating the synthesis of specific membrane lipids, e.g., PA and DG, by shRNAs against relevant biosynthetic enzymes in liver will dampen age-related mTOR hyperactivation. I will also determine whether targeting key interacting partners of mTOR in liver will dampen age-related hyperactivation of mTOR signaling and reverse deleterious mTOR-dependent outcomes, i.e., blockage of autophagy and proteostasis failure.

尽管我们对脂质代谢的调节有所了解，但脂质代谢的变化决定着细胞如何感知脂质，但可以想象，脂质的感知将为细胞做出调节代谢和蛋白质稳态的下游决策提供信息。 、应激反应和生长——每一个都随着年龄的增长而失调。雷帕霉素 (mTOR) 的机制靶标是一种丝氨酸/苏氨酸激酶和氨基酸传感器，可驱动生长和增殖。已证明在缺乏氨基酸的情况下，培养细胞中的 mTOR 会被胆固醇和磷脂酸 (PA) 激活。目前尚不清楚 mTOR 是否以两种主要复合物形式存在——mTORC1 和 mTORC2。溶酶体表面存在氨基酸，并且需要刺激其活性的关键调节蛋白，相比之下，mTORC2 响应生长因子来调节细胞骨架组织的过度激活。 mTORC1（以下简称 mTOR）部分通过破坏自噬和促进生长来驱动衰老和年龄相关疾病。然而，mTORC1 如何随着年龄的增长而过度激活仍不清楚，包括随着年龄的增长，膜脂质会发生量和质的变化。溶酶体膜脂质的变化——mTOR 激活的主要部位，我们的初步数据表明，给小鼠口服玉米油会导致 mTOR 的激活及其易位到不同的富含胆固醇的微域。 （CRM）/溶酶体膜中的脂筏。我们的初步数据还表明，从油饲小鼠肝脏的溶酶体膜中免疫沉淀 mTOR 揭示了其与二酰基甘油的结合，这些数据表明 mTOR 是二酰基甘油（一种膜脂质）的传感器。 mTOR 在溶酶体膜上感知营养物质，我追踪 mTOR 在溶酶体膜上感知脂质，以及与年龄相关的变化为了检验我们的假设，我们提出了以下具体目标：在目标 1 中，将使用多种方法通过免疫沉淀溶酶体膜上的 mTOR 来表征溶酶体膜上的 mTOR 激活。和蛋白质组学分析，我将鉴定与 mTOR 及其相互作用伙伴结合的脂质种类。我将使用体外 siRNA 筛选来沉默每个相互作用伙伴，这将确定新的调节因子。在目标 2 中，我将描述溶酶体膜 CRM 的脂质组成的变化以及溶酶体 CRM 随年龄的增长，我将确定膜脂质组成随年龄的变化是否与 mTOR 活性的增加相关。然后确定通过 shRNA 针对肝脏中相关生物合成酶来灭活特定膜脂（例如 PA 和 DG）的合成是否会抑制与年龄相关的 mTOR 过度激活。靶向肝脏中 mTOR 的关键相互作用伙伴是否会抑制与年龄相关的 mTOR 信号传导过度激活，并逆转有害的 mTOR 依赖性结果，即阻碍自噬和蛋白质稳态失败。