Asymmetric Distribution of Cholesterol in Membranes

虽然胆汁中高密度脂蛋白胆固醇的排泄是净排出胆固醇的主要途径 从体内，肝细胞如何如此快速地吸收和转移高密度脂蛋白衍生的胆固醇 胆道流出是未知的。了解这一过程可能会提供新的治疗方法 降低血浆胆固醇的目标，以改善不利的脂代谢。高密度脂蛋白 将胆固醇从组织输送到肝脏，与SRB1结合，将胆固醇输送到 肝细胞基底膜。由于自发的胆固醇解吸到胞浆中是 非常缓慢，必须存在一种未知的机制来快速吸收、解吸和大量 高密度脂蛋白-胆固醇的跨肝细胞转运。基因打靶研究表明，SCP-2和 L-FABP在不上调ORP、START或NPC的情况下促进胆汁胆固醇流出 家族胆固醇运输蛋白。作为候选蛋白，SCP-2和L-FABP是 假设通过以下方式调节这些快速步骤：结合基底外侧SRB1以增强 胆固醇的摄取/解吸和增加胞浆浓度或转运 胆固醇导致胆小管外流进入胆汁。这笔为期2年的ARRA R01拨款将考验最多 这一假设的重要方面，归结为两个目标，重点是结构性和功能性 SCP-2和L-FABP的肝细胞功能获得和功能丧失分析 转基因小鼠： 1)。胆固醇摄取：SCP-2或L-FABP是否影响高密度脂蛋白胆固醇摄取 通过与基底侧膜中的SRB1结合？初步数据显示，SCP-2 在体外结合SRB1，SCP-2过表达增加，SCP-2/SCP-x消融 降低肝细胞内SR-B1水平。高密度脂蛋白增加SRB1/SCP-2在 基底侧膜。 2)。胞内胆固醇转运：SCP-2或L-FABP能否促进 通过增加细胞质浓度或速率进行的跨肝细胞移动 高密度脂蛋白衍生胆固醇的转移？SCP-2和L-FABP增加了水的溶解度， 增强胞浆转运/扩散，并共转运另一种配体(脂肪酸) 肝细胞。 这些实验将解决肝脏胆固醇中一个关键的未解问题。 排泄-高密度脂蛋白-胆固醇是如何在基底外侧膜如此迅速地被吸收的 通过细胞质转移到肝细胞小管膜，用于胆道 排出？通过检验SCP-2和L-FABP介导快速摄取和转移的假设， 将获得关于调解这些快速增长的机制的根本性新见解 胆汁胆固醇排出的步骤--清除过量胆固醇的主要途径 身体。如果了解这一过程，增加治疗的新靶点 降低血浆胆固醇的胆固醇外流有可能被开发出来。

Although biliary excretion of HDL cholesterol is the major route for net cholesterol removal from the body, how hepatocytes so rapidly take up and transfer HDL-derived cholesterol for biliary efflux is unknown. Understanding this process may provide new therapeutic targets for lowering plasma cholesterol to improve adverse lipid metabolism. HDL transports cholesterol from tissues to liver, binding to SRB1 for cholesterol delivery to the hepatocyte basolateral membrane. Since spontaneous cholesterol desorption into cytosol is very slow, an unknown mechanism must exist for rapid uptake, desorption and massive transhepatocyte transport of HDL-cholesterol. Gene targeting studies show that SCP-2 and L-FABP enhance biliary cholesterol efflux without upregulating ORP, START, or NPC family cholesterol-trafficking proteins. As candidate proteins, SCP-2 and L-FABP are hypothesized to mediate these rapid steps by: binding basolateral SRB1 to enhance cholesterol uptake/desorption and increase cytosolic concentration or transport of cholesterol for canalicular efflux into bile. This 2 year ARRA R01 grant will test the most important aspects of this hypothesis, reduced to two aims, focusing on structural and functional analyses of hepatocytes from SCP-2 and L-FABP gain-of function and loss-of-function genetically-engineered mice: 1). Cholesterol uptake: Does SCP-2 or L-FABP impact HDL cholesterol uptake by binding with SRB1 in the basolateral membrane? Preliminary data show that SCP-2 binds SRB1 in vitro, SCP-2 overexpression increases, and SCP-2/SCP-x ablation decreases SR-B1 levels in hepatocytes. HDL increases SRB1/SCP-2 colocalization at the basolateral membrane. 2). Cytosolic cholesterol transport: Can SCP-2 or L-FABP facilitate transhepatocyte movement by increasing cytoplasmic concentration or rate of transfer of HDL-derived cholesterol? SCP-2 and L-FABP increase aqueous solubility, enhance cytosolic transport/diffusion, and cotransport another ligand (fatty acid) in hepatocytes. These experiments will address a key unanswered question in hepatic cholesterol excretion-how is HDL-cholesterol so rapidly taken up at the basolateral membrane and transferred through the cytoplasm to the hepatocyte canalicular membrane for biliary efflux? By testing the hypothesis that SCP-2 and L-FABP mediate rapid uptake and transfer, fundamental new insights will be gained regarding mechanisms mediating these rapid steps of biliary cholesterol efflux-the primary route for excess cholesterol removal from the body. If this process were understood, new therapeutic targets for increasing cholesterol efflux for lowering plasma cholesterol could potentially be developed.