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衰老人群的代谢疾病和死亡率。尽管我们了解对脂质代谢的调节，但生物体如何感觉脂质仍然未知。可以想象的是，脂质的感应会为调节代谢，蛋白质抗议，压力反应和生长的细胞做出的下游决定提供信息，这些决定随着年龄的增长而失调。雷帕霉素（MTOR）的机械靶标是丝氨酸/苏氨酸激酶和氨基酸传感器，可驱动生长和增殖。最近，在没有氨基酸的情况下，已证明培养细胞中的MTOR被胆固醇和磷脂酸（PA）激活。整个生物体中的MTOR感觉是否尚不清楚。 MTOR作为两个主要复合物-MTORC1和MTORC2。 MTORC1的激活发生在氨基酸存在下的溶酶体表面，需要刺激其活性的关键调节蛋白。相比之下，MTORC2对调节细胞骨架组织的生长因素做出反应。 MTORC1的过度激活（以下称为MTOR）通过破坏自噬和促进生长的部分驱动衰老和与年龄有关的疾病。但是，如何随着年龄的增长而过度激活MTORC1仍然未知。已经表明，随着年龄的增长，膜脂质的定量和质量变化包括溶酶体膜脂质的变化，这是MTOR激活的主要部位。我们的初步数据表明，对小鼠进行口服玉米油会导致MTOR的激活及其转移到溶酶体膜上富含胆固醇的微分区（CRMS）/脂质筏。我们的初步数据还表明，来自油热小鼠肝脏的溶酶体膜的免疫沉淀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。由于溶酶体膜上的MTOR感觉营养素，我假设溶酶体膜上的MTOR感觉脂质，以及与年龄相关的溶酶体膜脂质组成的变化导致MTOR多激活。为了检验我们的假设，我们提出了以下特定目的：在AIM 1中，将使用潜水员方法来表征溶酶体膜上脂质驱动的MTOR激活。通过从溶酶体膜上对脂质组和蛋白质组学分析的Immunoprecipitiitiition，我将确定与MTOR及其相互作用伴侣结合的脂质物种。我将在体外使用siRNA屏幕来沉默每个相互作用的伴侣，这将确定脂质驱动的MTOR信号的新调节剂。在AIM 2中，我将表征溶酶体膜CRM的脂质组成的变化以及随着年龄的增长的溶酶体CRM的扩展。我将确定随着年龄的增长与MTOR活性增加相关的膜脂质组成的改变。然后，我将通过shrnas来确定是否会通过SHRNA对肝脏中相关的生物合成酶的特定膜脂质的合成是否会抑制年龄相关的MTOR过度活化。我还将确定靶向MTOR的关键相互作用伴侣是否会衰减与年龄相关的MTOR信号传导的过度激活和反向有害MTOR依赖性结果，即自体噬和蛋白抑制失败的阻塞。