How does mTOR sense lipid in vivo

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，生物体如何感知脂质仍然未知。可以想象，对脂质的感知将为细胞调节代谢、蛋白质平衡、应激反应和生长的下游决策提供信息，而这些都随着年龄的增长而失调。雷帕霉素（mTOR）的机制靶点是一种丝氨酸/苏氨酸激酶和氨基酸传感器，它驱动生长和增殖。最近，培养细胞中的mTOR在缺乏氨基酸的情况下被胆固醇和磷脂酸（PA）激活。mTOR是否能感知整个生物体中的脂质尚不清楚。mTOR以两种主要复合物mtorc1和mTORC2存在。mTORC1的激活发生在氨基酸存在的溶酶体表面，需要关键的调节蛋白来刺激其活性。相比之下，mTORC2响应生长因子调节细胞骨架组织。mTORC1（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与年龄相关的疾病。然而，mTORC1如何随着年龄的增长而过度激活仍然未知。研究表明，随着年龄的增长，膜脂发生了定量和定性的变化，其中包括mTOR激活的主要部位溶酶体膜脂的变化。我们的初步数据表明，小鼠口服玉米油可激活mTOR，并将其转移到溶酶体膜中不同的富含胆固醇的微结构域(CRMs)/脂筏。我们的初步数据还表明，免疫沉淀的mTOR从油灌胃小鼠的肝脏溶酶体膜显示其结合二酰基甘油。这些数据表明mTOR是二酰基甘油（一种膜脂）的传感器。由于mTOR在溶酶体膜上感知营养，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过度激活。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，将使用多种方法来表征溶酶体膜上脂质驱动的mTOR激活。通过免疫沉淀溶酶体膜上的mTOR进行脂质组学和蛋白质组学分析，我将确定与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伙伴，这将确定脂质驱动的mTOR信号的新调节因子。在Aim 2中，我将描述溶酶体膜CRMs的脂质组成的变化以及溶酶体CRMs随年龄的增长。我将确定膜脂组成随年龄变化是否与mTOR活性增加相关。然后，我将确定shrna对肝脏中相关生物合成酶的抑制特定膜脂的合成，例如PA和DG，是否会抑制与年龄相关的mTOR过度激活。我还将确定靶向肝脏中mTOR的关键相互作用伙伴是否会抑制与年龄相关的mTOR信号过度激活，并逆转有害的mTOR依赖结果，即自噬阻断和蛋白酶抑制失败。

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.