How does mTOR sense lipid in vivo

脂代谢的改变决定了老年人口中的代谢性疾病和死亡率。尽管我们对脂类代谢的调节有所了解，但生物体如何感知脂类仍然是未知的。可以想象，对脂质的感知将为细胞做出的下游决策提供信息，这些决策调节新陈代谢、蛋白质平衡、应激反应和生长--每一种决策都随着年龄的增长而失调。雷帕霉素(MTOR)的作用靶点是一种丝氨酸/苏氨酸激酶和氨基酸感受器，可以驱动生长和增殖。最近，培养细胞中的mTOR被证明在没有氨基酸的情况下被胆固醇和磷脂酸(PA)激活。目前还不清楚mTOR是否在整个生物体中感觉到脂肪。MTOR以两种主要的复合体形式存在--mTORC1和mTORC2。在氨基酸存在的情况下，mTORC1的激活发生在溶酶体表面，需要关键的调节蛋白来刺激其活性。相比之下，mTORC2对生长因子做出反应，以调节细胞骨架组织。MTORC1(以下简称mTOR)的过度激活在一定程度上通过破坏自噬和促进生长来推动衰老和与年龄相关的疾病。然而，mTORC1是如何随着年龄的增长而过度激活的仍不清楚。研究表明，随着年龄的增长，膜脂有定量和定性的变化，包括mTOR激活的主要部位溶酶体膜脂的变化。我们的初步数据显示，小鼠口服玉米油会激活mTOR，并将其移位到溶酶体膜上不同的富含胆固醇微域(CRM)/脂筏。我们的初步数据还显示，免疫沉淀来自石油灌胃小鼠肝脏的溶酶体膜的mTOR揭示了它与二酰甘油的结合。这些数据表明，mTOR是一种膜脂二酰甘油的传感器。由于mTOR感觉溶酶体膜上的营养物质，我假设mTOR感觉溶酶体膜上的脂质，并且与年龄相关的溶酶体膜脂组成的变化导致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，将使用不同的方法来表征溶酶体膜上脂质驱动的mTOR激活。通过免疫沉淀法从溶酶体膜上分离mTOR进行脂质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类。我将在体外使用siRNA筛选使每个相互作用的伙伴沉默，这将识别脂质驱动的mTOR信号的新调节因子。在目标2中，我将表征溶酶体膜CRM的脂质组成的变化以及溶酶体膜CRM的膨胀随年龄的变化。我将确定膜脂成分随年龄的变化是否与mTOR活性增加相关。然后，我将确定针对肝脏相关生物合成酶的shRNAs是否会抑制与年龄相关的mTOR过度激活，从而抑制特定膜脂的合成，例如PA和DG。我还将确定在肝脏中定位mTOR的关键相互作用伙伴是否会抑制与年龄相关的mTOR信号的过度激活，并逆转有害的mTOR依赖的结果，即阻止自噬和蛋白抑制失败。

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.