INTRACELLULAR ASSEMBLY, TRANSPORT, AND DEGRADATION OF APOLIPOPROTEIN B

载脂蛋白 B 的细胞内组装、运输和降解

• 批准号: 6242225
• 负责人: GREGORY S SHELNESS
• 金额: $ 21.03万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242225
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; apolipoproteins; atherosclerosis; blood lipoprotein metabolism; blood lipoprotein transport; chemical aggregate; coronary disorder; crosslink; endoplasmic reticulum; glycosylation; immunocytochemistry; immunoelectron microscopy; intracellular transport; membrane biogenesis; membrane proteins; protein degradation; protein folding; protein transport; secretory protein; transcription factor; transfection; very low density lipoprotein

Apolipoprotein B-100 (apo B) is essential for the hepatic secretion of triglyceride-rich very low density lipoproteins (VLDL). The positive correlation between plasma concentrations of apo B and the risk of premature coronary heart disease suggests that an understanding of the intracellular assembly and transport of VLDL may have important implications for the prevention, pathogenesis, and management of atherosclerosis. The overall goals of the proposed research are to characterize early events in the biogenesis of VLDL and to determine how these events may contribute to the regulation of VLDL secretion. The following specific questions will be addressed: 1. Is intracellular turnover of apo B the predominant mechanism for the regulation of apo B secretion? It is known that a large percentage of newly synthesized apo B is degraded intracellularly, most likely in the endoplasmic reticulum (ER). In some cases, alterations in intracellular apo B turnover rates are responsible for the regulation of apo B secretion. To establish the generality of this mechanism, pre- and post- trans Golgi pools of apo B in HepG2 cells will be quantitated using a lectin affinity binding assay. Changes in these apo B intracellular pool sizes in response to mediators of apo B secretion will reveal if the amount of apo B shunted to a degradative compartment can quantitatively account for regulation of secretion and, if so, the general intracellular site(s) at which degradation occurs. 2. What features of the apo B molecule are responsible for its transient or stable association with the ER membrane? The ability of apo B to transiently or permanently associate with the ER membrane may be necessary for both VLDL assembly and regulation of VLDL secretion. To identify domains of apo B which are responsible for its association with the ER membrane, defined segments of apo B will be fused to a soluble secretory protein and expressed in cells of both hepatic (apo B- producing) and non-hepatic (non-apo B-producing) origin. The ability of apo B domains to direct transient or permanent membrane association and/or ER retention and degradation will be assessed using both biochemical and immunocytochemical criteria. Once identified, the consequences of altering or deleting these sequences in an intact apo B protein will be assessed. 3. Do trans-acting factors interact with apo B during its assembly into presecretory VLDL particles? The folding, assembly, and transport of many proteins within the secretory pathway requires transient assembly factors. To identify both general and specific factors which may be involved in the biogenesis and regulation of apo B-48 CDNA will be subjected to chemical cross-linking. Proteins which form cross-links with apo B as a function of metabolic labeling times will be examined in cells of both hepatic and non-hepatic origin.

载脂蛋白B-100（APO B）对于肝分泌至关重要 甘油三酸酯富密度脂蛋白（VLDL）。 积极 APO B的血浆浓度与存在的风险 过早的冠心病表明对 VLDL的细胞内组件和运输可能具有重要 对预防，发病机理和管理的影响 动脉粥样硬化。 拟议研究的总体目标是 表征VLDL生物发生中的早期事件，并确定如何 这些事件可能有助于对VLDL分泌的调节。 这 以下具体问题将被解决： 1。是Apo B的细胞内周转率 APO B分泌的调节？ 众所周知，很大一部分 新合成的apo b被细胞内降解，最有可能在 内质网（ER）。 在某些情况下，细胞内改变 APO B周转率负责APO B的调节 分泌。 建立这种机制的普遍性，前后 HepG2细胞中Apo B的反式高尔基池将使用A进行定量 凝集素亲和力结合测定法。 这些apo B细胞内池的变化 响应APO B分泌介质的大小将揭示是否是否 分流到降解室的Apo B量可以定量 解释分泌的调节，如果是的，则是一般细胞内的 发生降解的部位。 2。apo b分子的什么特征是其瞬态的原因 还是与ER膜的稳定关联？ Apo B的能力 瞬时或永久与ER膜相关 VLDL组装和VLDL分泌的调节所必需的。 到 确定负责与其关联的Apo B的域 ER膜，定义的Apo B段将融合到可溶性 分泌蛋白，并在两个肝细胞中表达（apo b- 生产）和非肝脏（非APO B产生）起源。 能力 直接瞬态或永久膜关联的APO B域 和/或ER保留和降解将使用两者进行评估 生化和免疫细胞化学标准。 一旦确定， 在完整的apo B中更改或删除这些序列的后果 将评估蛋白质。 3。在组装过程中与Apo B相互作用的反式作用因子与 预偏vldl颗粒？ 折叠，组装和运输 分泌途径中的许多蛋白质都需要短暂组装 因素。 确定可能是一般和特定因素 参与APO B-48 cDNA的生物发生和调节将是 进行化学交联。 形成交联的蛋白质 将使用APO B作为代谢标记时间的函数 肝和非肝脏起源的细胞。