Early Events in Lipoprotein Assembly

脂蛋白组装的早期事件

基本信息

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
项目类别：
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

项目摘要

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.

高血清低密度脂蛋白 (LDL) 水平与动脉粥样硬化有关，动脉粥样硬化是美国的主要原因。该提案的长期目标是开发降低低密度脂蛋白水平并减轻心血管疾病的新疗法。 LDL 源自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分泌。 Soth LDL 和 VLDL 的组成为脂肪、胆固醇、胆固醇酯、磷脂和单分子载脂蛋白 S (ApoS)。肝脏分泌的 VLDL 量不受 ApoS 表达水平变化的调节，ApoS 表达水平相对恒定。相反，在缺乏足够脂质的情况下，ApoS 在细胞中会被降解。这种降解途径代表了降低 VLDL 组装并从而降低 LDL 水平的目标。 VLDL 的组装被认为是一个两步过程。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 形成主要含有磷脂的致密“起始颗粒”。在第二步中，起始颗粒与内质网中的脂滴融合形成 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-三酰甘油乳液的无细胞系统中表达 ApoS 的 N 末端片段，以测试 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至关重要的假设。结果将揭示 ApoS 的连续结构域在脂质募集中的作用以及起始复合物的形成机制。 2) 使用无细胞系统确定 MTP 在 ApoS N 末端结构域的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，可用于靶向药物发现工作，通过阻止细胞分泌 LDL 来降低 LDL 水平。高血清低密度脂蛋白 (LOL) 水平与动脉粥样硬化有关，动脉粥样硬化是美国的主要原因之一。该提案的长期目标是开发降低 LOL 水平并缓解心血管疾病的新疗法。拟议的研究将提供有关 LOL（极低密度脂蛋白）前体组装的新机制细节，可用于靶向药物发现工作，通过阻止细胞分泌 VLOL 来降低 LOL 水平。