DYNAMICS OF APOE BINDING TO LIPOPROTEINS AND RECEPTORS

APOE 与脂蛋白和受体结合的动力学

• 批准号: 3366035
• 负责人: Martha P. Mims
• 金额: $ 16.19万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3366035
• 关键词: apolipoproteins; blood lipoprotein; blood lipoprotein biosynthesis; blood lipoprotein transport; cell type; complementary DNA; fatty acylation; glycosylation; human subject; intracellular transport; laboratory rabbit; laboratory rat; liver cells; posttranslational modifications; protein structure function; radioimmunoassay; radiotracer; receptor binding; site directed mutagenesis; species difference; thrombin; transfection; very low density lipoprotein; western blottings

ApoE is found in a number of animal species and although it is synthesized by various cell types, most plasma apoE is produced by the liver. In the circulation, apoE participates in lipid transport by mediating lipoprotein binding to the B/E receptor. A key factor in plasma lipid transport is the capacity of apoE to exchange between lipoprotein surfaces; however, not all apoE is capable of transfer, and not all lipoprotein-associated apoE is receptor active. The factors which govern apoE transfer between lipoproteins and receptor binding capacity are unclear. Two lines of evidence demonstrate that apoE secreted by the liver may be modified by fatty acid acylation. Whether this acylation is related to the functional state of plasma apoE is not known. Recently it has been suggested that a portion of the apoE synthesized by hepatic cells is not secreted, but remains within the cell where it may participate in intracellular lipid transport. The signal which determines whether apoE is secreted or remains in the cell is undefined, however, in other systems, targeting of proteins to specific cellular sites is accomplished by fatty acid acylation. The possibility that fatty acid acylation of apoE might affect its functional capacities in the plasma and/or its trafficking within the cell is intriguing. The goal of this project is to understand the factors which affect apoE transferability and receptor binding capacity and control targeting of apoE to intra- and extracellular pools. This goal will be accomplished through four aims designed to examine the existence and origins of different physical states of apoE and to probe their influence on apoE functional capacity. First, the physical and physiological state of the apoE component of nascent VLDL (isolated from the liver) will be examined by determining the susceptibility of apoE to thrombin cleavage, the capacity of apoE to transfer off of VLDL, and the capacity of apoE to mediate binding of nascent VLDL to the B/E receptor. The second aim is to examine fatty acid acylation of apoE and determine the appearance of apoE acylation in differing cell types and animal species, the localization of acyl-apoE in a specific lipoprotein class, the fatty acid specificity and site(s) of acylation, and the physical and physiological consequences of apoE acylation. The third aim is to examine cellular aspects of apoE acylation and determine the role of acylation in targeting of apoE to intra- and extracellular pools, the temporal relationship of apoE acylation to protein synthesis and post-translational modifications, and factors which influence apoE acylation. Finally, site-directed mutagenesis will be used to modify the acylation site(s) of apoE, and the cDNA will be transfected into hepatocytes to examine the effect of the modifications on targeting of apoE to intra- and extracellular pools, association of apoE with specific lipoproteins, and apoE transferability and thrombin sensitivity.

ApoE存在于许多动物物种中，尽管它是 由多种细胞类型合成，大多数血浆apoE由 肝脏 在循环中，apoE通过以下途径参与脂质转运： 介导脂蛋白与B/E受体的结合。 的关键因素 血浆脂质转运是指apoE在血浆中进行脂质交换的能力。 脂蛋白表面;然而，并非所有的apoE都能够转移， 并非所有脂蛋白相关的apoE都是受体活性的。 的因素 其控制脂蛋白和受体结合之间的apoE转移 能力不清楚。 有两条证据表明apoE 由肝脏分泌的脂肪酸可以通过脂肪酸酰化来修饰。 是否 这种酰化与血浆apoE的功能状态无关， 知道的最近有人提出apoE的一部分 由肝细胞合成的蛋白质不被分泌，而是留在细胞内 在那里它可能参与细胞内脂质转运。 信号 它决定了apoE是分泌还是留在细胞内， 然而，在其他系统中，蛋白质靶向特定的 细胞位点通过脂肪酸酰化完成。 的可能性 apoE的脂肪酸酰化可能影响其功能 和/或其在细胞内的运输是有趣的。 的 本项目的目的是了解影响apoE的因素 转移性和受体结合能力以及控制靶向 apoE与细胞内和细胞外池。 这一目标将得以实现 通过四个目的，旨在研究的存在和起源， 探讨apoE的不同物理状态对apoE的影响 功能能力。 第一，身体和生理状态的 将检查新生VLDL（从肝脏分离）的apoE组分 通过测定apoE对凝血酶裂解的敏感性， apoE转移VLDL的能力，以及apoE转移VLDL的能力。 介导新生VLDL与B/E受体的结合。 第二个目标是 检查apoE的脂肪酸酰化，并确定 在不同的细胞类型和动物物种中apoE酰化， 酰基载脂蛋白E定位于特定脂蛋白类，脂肪酸 酰化的特异性和位点，以及 apoE酰化的后果。 第三个目的是检查细胞 载脂蛋白E酰化的方面，并确定酰化在 apoE靶向细胞内和细胞外池， 载脂蛋白E酰化与蛋白质合成的关系 翻译后修饰和影响apoE的因素 酰化反应。 最后，定点诱变将用于修饰 在apoE的一个或多个酰化位点处，将cDNA转染入 肝细胞，以检查修饰对靶向 apoE与细胞内和细胞外池的关系，apoE与特异性 脂蛋白和apoE转移性和凝血酶敏感性。