How does mTOR sense lipid in vivo

mTOR如何感知体内脂质

• 批准号: 10625465
• 负责人: Henrietta J Bains
• 金额: $ 2.05万
• 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/10625465
• 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; Immunoprecipitation; 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; Phosphatidic Acid; Process; Proliferating; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Regulation; Serine; 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; detection of nutrient; genetic regulatory protein; healthspan; in vivo; inhibitor; insight; lipid metabolism; lipidomics; lysosome membrane; mortality; new therapeutic target; novel; pharmacologic; proteostasis; prototype; 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.

脂质代谢的改变决定了老年人群的代谢性疾病和死亡率。尽管我们了解脂质代谢的调节，但生物体如何感知脂质仍然是未知的。可以想象的是，脂质的感知将告知细胞调节代谢、蛋白质稳态、应激反应和生长的下游决定，这些决定中的每一个都随着年龄的增长而失调。雷帕霉素（mTOR）的机制靶标是丝氨酸/苏氨酸激酶和氨基酸传感器，其驱动生长和增殖。最近，培养细胞中的mTOR已显示在不存在氨基酸的情况下被胆固醇和磷脂酸（PA）激活。mTOR是否在整个生物体中感知脂质尚不清楚。mTOR以两种主要复合物mTORC 1和mTORC 2存在。mTORC 1的激活在存在氨基酸的情况下发生在溶酶体表面，并且需要刺激其活性的关键调节蛋白。相比之下，mTORC 2响应于生长因子来调节细胞骨架组织。mTORC 1（以下简称mTOR）的过度激活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，mTORC 1是如何随着年龄的增长而过度激活的仍然未知。已经表明，随着年龄的增长，膜脂质存在定量和定性的变化，包括溶酶体膜脂质的变化-mTOR活化的主要位点。我们的初步数据表明，小鼠经口灌胃玉米油导致mTOR活化及其易位到溶酶体膜中不同的富含胆固醇的微区（CRM）/脂筏。我们的初步数据还表明，免疫沉淀mTOR从溶酶体膜从灌油小鼠的肝脏揭示其结合二酰基甘油。这些数据表明，mTOR是一个传感器的二酰基甘油，膜脂质。由于mTOR在溶酶体膜上感知营养物质，我假设mTOR在溶酶体膜上感知脂质，并且溶酶体膜脂质组成的年龄相关变化导致mTOR过度活化。为了验证我们的假设，我们提出了以下具体目标：在目标1中，将使用不同的方法来表征溶酶体膜上脂质驱动的mTOR激活。通过从溶酶体膜上免疫沉淀mTOR进行脂质组学和蛋白质组学分析，我将鉴定与mTOR及其相互作用伙伴结合的脂质种类。我将在体外使用siRNA筛选来沉默每个相互作用的伴侣，这将识别脂质驱动的mTOR信号转导的新调节因子。在目标2中，我将描述溶酶体膜CRMs的脂质组成的变化和溶酶体CRMs随年龄的扩张。我将确定膜脂质组成随年龄的变化是否与mTOR活性增加相关。然后，我将确定是否灭活特定膜脂质的合成，PA和DG通过针对肝脏中相关生物合成酶的shRNA将抑制与年龄相关的mTOR超活化。我还将确定靶向肝脏中mTOR的关键相互作用伴侣是否会抑制年龄相关的mTOR信号转导过度激活并逆转有害的mTOR依赖性结果，即，自噬的阻断和蛋白质稳定的失败。