Asymmetric Distribution of Cholesterol in Membranes

虽然高密度脂蛋白胆固醇的胆汁排泄是胆固醇从体内净排出的主要途径，但肝细胞如何如此快速地摄取和转移高密度脂蛋白衍生的胆固醇以供胆道外流尚不清楚。了解这一过程可能为降低血浆胆固醇以改善不良脂代谢提供新的治疗靶点。高密度脂蛋白将胆固醇从组织运送到肝脏，并与SRB1结合，将胆固醇运送到肝细胞基底膜。由于胆固醇自发解吸到胞浆中的速度非常慢，因此高密度脂蛋白-胆固醇的快速吸收、解吸和大量跨肝细胞转运一定存在一个未知的机制。基因打靶研究表明，SCP-2和L-FABP促进胆汁胆固醇外流，而不上调ORP、START或鼻咽癌家族胆固醇转运蛋白。作为候选蛋白，SCP-2和L-FABP被认为通过与基底外侧SRB1结合来促进胆固醇的摄取/解吸，并增加细胞内胆固醇的浓度或胆固醇的转运，从而介导这些快速的步骤。这项为期两年的ARRAR01拨款将检验这一假设的最重要方面，主要有两个目标，重点是对SCP-2和L-FABP功能获得和功能丧失基因工程小鼠的肝细胞的结构和功能分析： 1)。胆固醇摄取：SCP-2或L-FABP是否通过与基底膜上的SRB1结合而影响高密度脂蛋白的摄取？初步数据显示，SCP-2在体外与SRB1结合，SCP-2过表达增加，SCP-2/SCP-x消融降低肝细胞SR-B1水平。高密度脂蛋白增加SRB1/SCP-2在基底膜的共定位。 2)。胞浆胆固醇转运：SCP-2或L-FABP能否通过增加高密度脂蛋白衍生胆固醇的胞浆浓度或转移率来促进跨肝细胞运动？SCP-2和L-FABP增加了肝细胞的水溶性，促进了胞浆的转运/扩散，并共转运了另一种配体(脂肪酸)。 这些实验将解决肝脏胆固醇排泄中一个关键的悬而未决的问题--高密度脂蛋白-胆固醇是如何如此迅速地被基底膜吸收并通过细胞质转移到肝细胞管膜进行胆道外流的？通过检验SCP-2和L-FABP介导快速摄取和转移的假设，将对调节胆汁胆固醇快速流出的机制--过量胆固醇从体内清除的主要途径--获得根本的新见解。如果了解了这一过程，就有可能开发新的治疗靶点来增加胆固醇外流以降低血浆胆固醇。

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.