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在许多动物物种中都发现，尽管它是 由各种细胞类型合成，大多数血浆APOE由 肝。 在循环中，Apoe通过 介导脂蛋白与B/E受体结合。 一个关键因素 等离子体脂质传输是APOE之间交换的能力 脂蛋白表面；但是，并非所有ApoE都能转移，并且 并非所有与脂蛋白相关的APOE都是活性的。 因素 控制脂蛋白与受体结合之间的APOE转移 容量尚不清楚。 两条证据表明APOE 由肝脏分泌可以通过脂肪酸酰化来改变。 无论 该酰基化与等离子体APOE的功能状态无关 已知。最近有人提出了APOE的一部分 由肝细胞合成的不是分泌，而是保留在细胞内 它可能参与细胞内脂质转运。 信号 哪个确定APOE是分泌还是保留在单元格中 但是，在其他系统中未定义，将蛋白质靶向特定 细胞位点是通过脂肪酸酰化来完成的。 可能性 APOE的脂肪酸酰化可能会影响其功能能力 在血浆和/或其在细胞内的运输中，很有趣。 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和控制靶向 APOE到细胞内和细胞外池。 这个目标将实现 通过四个目的旨在检查 APOE的不同物理状态并探究其对APOE的影响 功能能力。 首先，的身体和生理状态 将检查新生VLDL的APOE组件（从肝脏隔离） 通过确定APOE对凝血酶裂解的敏感性， APOE转移VLDL的能力，以及ApoE的能力 新生VLDL与B/E受体的介导结合。 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种类中的apoe酰基化， 在特定的脂蛋白类别（脂肪酸）中定位酰基-APOE 酰化的特异性和部位，以及物理和生理 APOE酰化的后果。 第三个目的是检查细胞 APOE酰化的各个方面并确定酰化在 靶向APOE到细胞内和细胞外池，时间为时间 Apoe酰化与蛋白质合成的关系 翻译后修改以及影响APOE的因素 酰化。 最后，将使用位置定向的诱变来修改 APOE的酰化位点，将cDNA转染到 肝细胞检查修饰对靶向的影响 APOE与细胞内和细胞外池，APOE与特定的关系 脂蛋白，APOE的转移性和凝血酶敏感性。