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: 2223250
• 负责人: Martha P. Mims
• 金额: $ 16.05万
• 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/2223250
• 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 由各种细胞类型合成，大多数血浆apoE是由 肝。 在循环中，apoE 通过以下方式参与脂质转运： 介导脂蛋白与 B/E 受体的结合。 一个关键因素是 血浆脂质转运是 apoE 之间交换的能力 脂蛋白表面；然而，并非所有 apoE 都能够转移，并且 并非所有脂蛋白相关的 apoE 都具有受体活性。 因素 控制脂蛋白和受体结合之间的 apoE 转移 容量不清楚。 两条证据表明 apoE 肝脏分泌的脂肪酸可通过脂肪酸酰化进行修饰。 无论 这种酰化与血浆 apoE 的功能状态无关 已知。最近有人建议 apoE 的一部分 肝细胞合成的不分泌，而是保留在细胞内 它可能参与细胞内脂质转运。 信号 决定apoE是否被分泌或保留在细胞中的是 未定义，然而，在其他系统中，将蛋白质靶向特定的 细胞位点是通过脂肪酸酰化完成的。 可能性 apoE 的脂肪酸酰化可能会影响其功能 在血浆中和/或其在细胞内的运输是有趣的。 这 该项目的目标是了解影响 apoE 的因素 可转移性和受体结合能力以及控制靶向 apoE 至细胞内和细胞外池。 这个目标将会实现 通过四个目的旨在检验的存在和起源 apoE的不同物理状态并探讨它们对apoE的影响 功能能力。 首先，身体和生理状态 将检查新生 VLDL（从肝脏分离）的 apoE 成分 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 不同细胞类型和动物物种中的 apoE 酰化 酰基-apoE 在特定脂蛋白类（脂肪酸）中的定位 酰化的特异性和位点，以及物理和生理学 apoE 酰化的后果。 第三个目标是检查细胞 apoE 酰化的各个方面并确定酰化在 将 apoE 靶向细胞内和细胞外池， apoE酰化与蛋白质合成的关系 翻译后修饰以及影响 apoE 的因素 酰化。 最后，将使用定点诱变来修改 apoE 的酰化位点，并且 cDNA 将被转染到 肝细胞以检查修饰对靶向的影响 apoE 与细胞内和细胞外池的关系，apoE 与特定的关联 脂蛋白、apoE 可转移性和凝血酶敏感性。