Early Events in Lipoprotein Assembly

脂蛋白组装的早期事件

• 批准号: 7729753
• 负责人: Christopher James MCKNIGHT
• 金额: $ 37.5万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729753
• 关键词: Abetalipoproteinemia; Apolipoproteins; Apolipoproteins B; Atherosclerosis; Blood; Blood Circulation; Cardiovascular Diseases; Cause of Death; Cell secretion; Cell-Free System; Cells; Cholesterol; Cholesterol Esters; Chylomicrons; Complex; Coupled; Cytosol; Degradation Pathway; Diet; Dietary Fats; Effectiveness; Electrons; Elements; Emulsions; Endoplasmic Reticulum; Environment; Event; Fatty acid glycerol esters; Goals; Histidine; In Vitro; Label; Life; Lipids; Lipoproteins; Liver; Location; Low-Density Lipoproteins; Membrane; Methods; Microscopic; Molecular Chaperones; Myocardial Infarction; N-terminal; Particle Size; Pathway interactions; Pharmaceutical Preparations; Phospholipids; Play; Process; Prokaryotic Cells; Property; Proteins; Research; Role; Serum; Shapes; Stream; Stroke; System; Testing; Translations; Triglycerides; United States; Very low density lipoprotein; Vesicle; drug discovery; killings; microsomal triglyceride transfer protein; mortality; nanoGold; novel; particle; prevent; protein function; protein structure; single molecule; translational approach

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.

高水平的血清低密度脂蛋白(LDL)与动脉粥样硬化有关，动脉粥样硬化是美国的主要死亡原因。这项提议的长期目标是开发降低低密度脂蛋白水平和减轻心血管疾病的新疗法。低密度脂蛋白来源于一种叫做极低密度脂蛋白(VLDL)的前体颗粒，它在肝脏中组装并分泌。Soth低密度脂蛋白和极低密度脂蛋白由 脂肪、胆固醇、胆固醇酯、磷脂和单分子载脂蛋白S(APO)。肝脏分泌的极低密度脂蛋白的量不受APOS表达水平变化的调节，相对恒定。相反，在缺乏足够的脂质的情况下，APOS在细胞中被降解。这种降解途径代表了降低极低密度脂蛋白组装从而降低低密度脂蛋白水平的目标。极低密度脂蛋白的组装被认为是一个分两步进行的过程。在需要内质网驻留蛋白微粒体甘油三酯转移蛋白(MTP)的第一步中，APOS形成一个密集的“起始颗粒”，主要包含磷脂。在第二步中，起始颗粒与内质网中的脂滴融合，形成极低密度脂蛋白。第二步也需要MTP。引发粒子是如何形成的，以及MTP如何执行其功能尚不清楚。为了填补这一空白，这项提议将使用原核细胞自由翻译系统在体外产生起始粒子。该系统将用于测试脂类、MTP和APO结构在脂蛋白颗粒形成中的作用。 其具体目的是：1)在添加磷脂和磷脂-三酰甘油乳剂的无细胞体系中表达APOS的N-末端片段，以检验APOS与脂类的共翻译相互作用是自发的，并且对于起始复合体的形成是必要的。这些结果将揭示APO的连续结构域在脂质募集中的作用以及启动复合体的形成机制。2)利用无细胞系统确定MTP在APO N-末端结构域折叠和脂质募集中的作用。 这项提议的成功完成将提供有关极低密度脂蛋白组装途径的新的机械性细节，可用于靶向药物发现努力，通过阻止细胞分泌低密度脂蛋白来降低低密度脂蛋白水平。高水平的血清低密度脂蛋白(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。这项提议的长期目标是开发降低LOL水平和缓解心血管疾病的新疗法。这项拟议的研究将提供有关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努力，通过阻止细胞分泌VLOL来降低LOL水平。