Functions of apoE in cholesterol and triglyceride homeostasis

描述（由申请人提供）：载脂蛋白E是胆固醇转运系统的重要蛋白，在普通人群中有三种常见的亚型apoE2、apoE3和apoE4。ApoE促进受体介导的脂蛋白残留物清除循环，有助于脂质稳态，并防止动脉粥样硬化。apoE缺乏或特异性点突变干扰受体结合，阻碍动脉粥样硬化脂蛋白从循环中清除，导致III型高脂蛋白血症，这与人类和实验动物的过早动脉粥样硬化有关。载脂蛋白e的apoE4亚型也被认为是迟发性家族性阿尔茨海默病的一个危险因素。而apoE在生理范围内的表达清除脂蛋白残留物，apoE2、apoES或apoE4的高水平表达导致高甘油三酯血症。我们最近已经确定，高甘油三酯血症在很大程度上是由位于羧基端区氨基酸261至269之间的疏水残基介导的。这些残基也影响含有载脂蛋白e的高密度脂蛋白颗粒的形成。低密度脂蛋白受体的缺乏或apoE受体结合域的突变降低了小鼠诱导高甘油三酯血症所需的apoE阈值。体外和体内实验表明，apoE与ABCA1功能相互作用，促进含apoE的HDL的生物生成。此外，apoE与SR-BI相互作用并促进脂质外排。在这个应用中，我们建议利用我们在过去四年中获得的新知识来解决与载脂蛋白e的功能及其在血脂异常、动脉粥样硬化和高密度脂蛋白样载脂蛋白的生物发生中的作用有关的三个重要问题。我们的具体目标是：1：研究单个apoE残基L261， W264, F265, L268， V269在apoE体内功能中的作用，包括高甘油三酯血症的发展，防止动脉粥样硬化和HDL的形成。最终目标是产生具有动脉粥样硬化保护作用和改善生物学功能的重组apoE形式。2：研究与III型高脂蛋白血症相关的apoE突变体在受体识别、对高甘油三酯血症的敏感性和对动脉粥样硬化的易感性中的羧基末端结构域的相对贡献。这些研究可能确定apoE的功能不同于LDL受体结合，有助于其抗动脉粥样硬化特性。3：阐明apoE和ABCA1之间的功能相互作用如何促进含有高密度脂蛋白样apoE的脂蛋白颗粒的新生生物发生，这些颗粒通过随后与LCAT和SR-BI的相互作用而成熟，以及它们在总体胆固醇稳态中的作用。这些研究将采用单敲除或双敲除小鼠的apoa - 1、apoE、SR-BI基因转移和生化分析。

DESCRIPTION (provided by applicant): Apolipoprotein E is an important protein of the cholesterol transport system which has three common isoforms, apoE2, apoE3, and apoE4 in the general population. ApoE promotes receptor-mediated clearance of lipoprotein remnants from the circulation, contributes to lipid homeostasis, and protects from atherosclerosis. Deficiency or specific point mutations in apoE that interfere with receptor binding impede the clearance of atherogenic lipoproteins from the circulation, and result in type III hyperlipoproteinemia which is associated with premature atherosclerosis in humans and experimental animals. The apoE4 isoforms of apoE has been also implicated as a risk factor in late-onset familial Alzheimer's disease. Whereas expression of apoE within a physiological range clears lipoprotein remnants, high levels of expression of apoE2, apoES, or apoE4 causes hypertriglyceridemia. We have established recently that the hypertriglyceridemia is mediated to a large extent by hydrophobic residues located between amino acids 261 to 269 of the carboxy terminal region. These residues also influence the formation of apoE-containing HDL particles. Deficiency of the LDL receptor or mutations in the receptor binding domain of apoE decreases the threshold of apoE required for induction of hypertriglyceridemia in mice. In vitro and in vivo experiments have shown that apoE interacts functionally with ABCA1 and promotes the biogenesis of apoE-containing HDL. In addition, apoE interacts functionally with SR-BI and promotes lipid efflux. In this application, we propose to capitalize on the new knowledge we have acquired during the last four years to address three important questions pertinent to the functions of apoE and its role in dyslipidemia, in atherogenesis and the biogenesis of HDL-like apoE-containing lipoproteins. Our specific aims are: 1: To investigate the contribution of individual apoE residues L261, W264, F265, L268, V269 in the in vivo functions of apoE, including the development ofhypertriglyceridemia, protection from atherogenesis, and formation of HDL. The ultimate goal is to generate recombinant apoE forms that are atheroprotective and have improved biological functions. 2: To investigate the relative contribution of the carboxy terminal domain of apoE in receptor recognition, sensitivity to hypertriglyceridemia and susceptibility to atherosclerosis in apoE mutants that are associated with dominant forms of type III hyperlipoproteinemia. These studies may identify apoE functions different from the LDL receptor binding that contribute to its anti-atherogenic properties. 3: To elucidate how functional interactions between apoE and ABCA1 contribute to the de novo biogenesis of HDL-like apoE- containing lipoprotein particles, the maturation of these particles by subsequent interactions with LCAT and SR-BI, and their role in the overall cholesterol homeostasis. Gene transfer in single or double knockout mice for apoA-l, apoE, SR-BI and biochemical analyses will be employed in these studies.