Early Events in Lipoprotein Assembly

高血清水平的低密度脂蛋白（LDL）与动脉粥样硬化有关，动脉粥样硬化是美国死亡的主要原因。这项提案的长期目标是开发新的治疗方法，降低LDL水平，减轻心血管疾病。LDL来源于一种称为极低密度脂蛋白（VLDL）的前体颗粒，该颗粒在肝脏中组装并从肝脏分泌。低密度脂蛋白和极低密度脂蛋白是由 脂肪、胆固醇、胆固醇酯、磷脂和单个载脂蛋白S（ApoS）分子。从肝脏分泌的VLDL的量不受相对恒定的ApoS表达水平的变化的调节。相反，在缺乏足够脂质的情况下，ApoS在细胞中被降解。这种降解途径代表了降低VLDL组装并由此降低LDL水平的靶点。VLDL的组装被认为是一个两步过程。在需要ER驻留蛋白微粒体甘油三酯转移蛋白（MTP）的第一步中，ApoS形成一个主要含有磷脂的致密“起始颗粒”。在第二步中，起始颗粒与ER中的脂滴融合以形成VLDL。第二步也需要MTP。引发粒子是如何形成的以及MTP如何发挥其功能尚不清楚。为了填补这一空白，该提案将使用原核细胞自由翻译系统在体外产生起始颗粒。该系统将用于测试脂质、MTP和ApoS结构在脂蛋白颗粒形成中的作用。 具体目标是：1）在补充有磷脂和磷脂-三酰甘油乳剂的无细胞系统中表达ApoS的N-末端片段，以检验ApoS与脂质的共翻译相互作用是自发的并且对于起始复合物的形成是必需的假设。这些结果将揭示ApoS连续结构域在脂质募集中的作用以及起始复合物形成的机制。2)利用无细胞系统研究MTP在ApoS N端结构域折叠和脂质募集中的作用。 该提案的成功完成将提供有关VLDL组装途径的新机制细节，该途径可用于靶向药物发现工作，以通过阻止其从细胞中分泌来降低LDL水平。高血清水平的低密度脂蛋白（LOL）与动脉粥样硬化有关，动脉粥样硬化是美国的主要死亡原因之一。该提案的长期目标是开发新的治疗方法，降低LOL水平并减轻心血管疾病。这项研究将提供关于LOL前体-极低密度脂蛋白（VLOL）组装的新机制细节，可用于靶向药物发现工作，通过阻止VLOL从细胞中分泌来降低LOL水平。

High serum levels of low density lipoprotein (LDL) are associated with atherosclerosis, the leading cause of death in the USA. The long term goal of this proposal is to develop new treatments that lower LDL levels and alleviate cardiovascular disease. LDL is derived from a precursor particle called very low density lipoprotein (VLDL) which is assembled in and secreted from the liver. Soth LDL and VLDL are composed of fats, cholesterol, cholesterol esters, phospholipids and a single molecule of apolipoprotein S (ApoS). The amount of VLDL secreted from the liver in not regulated by changes in the level of ApoS expression which is relatively constant. Instead, in the absence of sufficient lipid, ApoS is degraded in the cell. This degradation pathway represents a target for lowering VLDL assembly and thereby decreasing LDL levels. The assembly of VLDL is thought to be a two-step process. In the first step that requires the ER resident protein microsomal triglyceride transfer protein (MTP), ApoS forms a dense 'initiation particle' that contains primarily phospholipids. In the second step the initiation particle fuses with a lipid droplet in the ER to form VLDL. The second step also requires MTP. How the initiation particle is formed and how MTP performs its functions are not known. To fill this gap this proposal will use a prokaryotic cell free translation system to produce initiation particles in vitro. This system will be used to test the role of lipids, MTP and the structure of ApoS in the formation of lipoprotein particles. The specific aims are: 1) To express N-terminal fragments of ApoS in a cell free system supplemented with phospholipids and phospholipid-triacylglycerol emulsions to test the hypothesis that cotranslational interaction of ApoS with lipids is spontaneous and essential for the formation of the initiation complex. The results will reveal the role of successive domains of ApoS in lipid recruitment and the mechanism for the formation of the initiation complex. 2) To determine the role of MTP in the folding and lipid recruitment by the N-terminal domains of ApoS using the cell free system. The successful completion of this proposal will provide new mechanistic details about the VLDL assembly pathway that can be used to target drug discovery efforts to lower LDL levels by preventing its secretion from the cell. High serum levels of low density lipoprotein (LOL) are associated with atherosclerosis, one the leading causes of death in the USA. The long term goal of this proposal is to develop new treatments that lower LOL levels and alleviate cardiovascular disease. The research proposed will provide new mechanistic details about assembly of the precursor to LOL, very low density lipoprotein (VLOL), that can be used to target drug discovery efforts to lower LOL levels by preventing VLOL secretion from the cell.