DYNAMICS OF APOE BINDING TO LIPOPROTEINS AND RECEPTORS

载脂蛋白E在许多动物物种中被发现，尽管它是 由不同类型的细胞合成，大多数血浆载脂蛋白E是由 肝脏。在循环中，载脂蛋白E通过以下途径参与脂质转运 介导脂蛋白与B/E受体结合。其中的一个关键因素 血浆脂质转运是指载脂蛋白E之间交换的能力 脂蛋白表面；然而，并不是所有的apoE都能够转移，并且 并非所有与脂蛋白相关的载脂蛋白E都是受体活性的。影响因素 它们调控脂蛋白之间的载脂蛋白E转移和受体结合 产能尚不清楚。两条证据表明载脂蛋白E 肝脏分泌的脂肪酸可被脂肪酸酰化修饰。是否 这种酰化作用与血浆apoE的功能状态无关 为人所知。最近有人提出，载脂蛋白E的一部分 由肝细胞合成的物质不会分泌，而是留在细胞内。 在那里它可能参与细胞内的脂质运输。该信号 这决定了载脂蛋白E是分泌还是保留在细胞内 然而，在其他系统中，未确定的是将蛋白质靶向到特定的 细胞位置是通过脂肪酸酰化来完成的。这种可能性 载脂蛋白E的脂肪酸酰化可能影响其功能 在血浆中和/或它在细胞内的运输是耐人寻味的。这个 本项目的目标是了解影响载脂蛋白E的因素 转移性和受体结合能力与靶向性控制 APOE到细胞内池和胞外池。这个目标一定会实现的 通过四个目的来考察 载脂蛋白E的不同物理状态及其对载脂蛋白E的影响 功能能力。第一，老年人的身体和生理状态 将检查新生极低密度脂蛋白(从肝脏分离)的载脂蛋白E成分 通过测定载脂蛋白E对凝血酶裂解的敏感性， 载脂蛋白E对极低密度脂蛋白的转运能力和载脂蛋白E对 介导新生极低密度脂蛋白与B/E受体的结合。第二个目标是 检测载脂蛋白E的脂肪酸酰化反应并确定其形态 在不同细胞类型和动物物种中的APOE酰化， 酰基-载脂蛋白E在一种特殊的脂蛋白类--脂肪酸中的定位 酰化的特异性和位点(S)，以及物理和生理 载脂蛋白E酰化的后果。第三个目标是检查细胞 载脂蛋白E酰化的几个方面，并确定酰化在 载脂蛋白E靶向细胞内池和胞外池，时间 载脂蛋白E酰化与蛋白质合成和蛋白合成的关系 翻译后修饰及影响载脂蛋白E的因素 酰化反应。最后，定点突变将被用来修改 ApoE的酰化位点(S)，并将其转化入 肝细胞检测修饰对靶向性的影响 载脂蛋白E与胞内池和胞外池的关系 脂蛋白、载脂蛋白E转移性和凝血酶敏感性。

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.