HEPATIC ASSEMBLY OF APOLIPOPROTEIN B CONTAINING LIPOPROTEINS

含有脂蛋白的载脂蛋白 B 的肝脏组装

• 批准号: 6338876
• 负责人: GREGORY S SHELNESS
• 金额: $ 19.92万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6338876
• 关键词: apolipoprotein B; apolipoproteins; atherosclerosis; blood lipoprotein metabolism; blood lipoprotein transport; chemical aggregate; coronary disorder; disease /disorder proneness /risk; endoplasmic reticulum; glycosylation; immunocytochemistry; immunoelectron microscopy; intracellular transport; liver cells; liver metabolism; posttranslational modifications; protein folding; protein purification; protein sequence; protein structure function

Hepatic apolipoprotein B (apoB)-containing lipoproteins are the predominant carriers of plasma cholesterol and, in elevated concentrations, represent a positive risk factor for the development of premature coronary artery atherosclerosis. Despite its importance of numerous aspects of lipoprotein metabolism, including the prevention and treatment of hyperlipidemias and their complications, the mechanism underlying the hepatic assembly of apoB-containing lipoprotein remains poorly defined. The overall goal of the proposed research is to characterized apoB's interactions with lipids, membranes and the microsomal triglyceride transfer protein (MTP) and to understand how these interactions results in the assembly and regulation of apoB-containing lipoproteins. The following specific aims will be addressed: I. Define how the amino-terminal domain of apoB functions to initiate lipoprotein formation in the hepatic endoplasmic reticulum (ER). To define how this domain functions, its properties will be analyzed using several in vitro assays designed to reflect potential functions during lipoprotein assembly in vivo. For each assay the behavior of native and chemically reduced apoB17 (amino-terminal 17% of apoB) was well as apoB17 harboring specific cysteine to serine mutations will be studied. The critical function of this domain will emerge by identifying which assay(s) displays a structure-function profile similar to that observed for apoB17's capacity to initiate lipoprotein assembly in vivo; II. Establish whether the initial co-translational phase of lipoprotein assembly requires recruitment by apoB of specific lipid classes. This question will be addressed by determining the relative lipid compositions of a series of C- terminally truncated, epitope-tagged forms of apoB that are though to represent static intermediates in the normal process of co-translational lipoprotein formation; III. Identify and characterize intermediates in the post-translational folding and assembly of apoB28. Since the post- translational assembly of apoB28 can be readily synchronized and manipulated, intermediates in this process can be identified as can the roles of cellular factors with known or proposed roles in the process of lipoprotein assembly; IV. Unequivocally establish the topology of apoB within the hepatic ER. The prevailing, through controversial view of the regulation of apoB secretion, involves the capacity of the hepatocyte to regulate the translocation of apoB across the R membrane in response to lipid availability. To establish unequivocally whether cytosolic, translocation arrested forms of apoB exist within the hepatic ER, the extent of glycosylation at specific sites within the C-terminal 50% of the protein will be monitored.

肝载脂蛋白B（APOB） - 含脂蛋白是 血浆胆固醇的主要载体和高架 浓度是发展的积极风险因素 过早的冠状动脉粥样硬化。尽管它的重要性 脂蛋白代谢的许多方面，包括预防和 治疗高脂血症及其并发症，机制 含有APOB的脂蛋白的肝组件的基础仍然存在 定义不佳。拟议研究的总体目标是 表征了Apob与脂质，膜和 微粒体甘油三酸酯转移蛋白（MTP），并了解这些 相互作用导致含APOB的组装和调节 脂蛋白。将解决以下特定目标：I。定义如何 APOB功能的氨基末端结构域启动脂蛋白 肝内质网（ER）中的形成。定义如何 域功能，将使用几种体外分析其特性 旨在反映脂蛋白组装过程中潜在功能的测定法 体内。对于每种测定，天然和化学降低的行为 APOB17（APOB的氨基末端17％）均为APOB17特定 将研究半胱氨酸到丝氨酸突变。关键功能的关键功能 该域将通过识别哪些测定显示 结构功能轮廓类似于APOB17的容量 在体内启动脂蛋白组件； ii。确定是否 脂蛋白组装的初始共同阶段需要 特定脂质类的APOB招募。这个问题将是 通过确定一系列C-的相对脂质组成来解决 终端截断，表位标记的APOB的形式 在共同翻译的正常过程中代表静态中间体 脂蛋白形成； iii。识别和表征中间体 APOB28的翻译后折叠和组装。 自从邮政 APOB28的翻译组装可以容易同步，并且 在此过程中操纵的中间体可以识别 细胞因子的作用，在此过程中具有已知或提出的作用 脂蛋白组件； iv。明确建立APOB的拓扑 在肝脏中。通过有争议的观点 APOB分泌的调节，涉及肝细胞的能力 调节APOB在R膜上的易位 脂质可用性。 明确地确定胞质，是否 易位逮捕的Apob形式存在于肝ER中， C端50％的特定位点的糖基化程度 蛋白质将被监测。