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一种重要蛋白质，在一般人群中有三种常见的同种型：apoE 2、apoE 3和apoE 4。ApoE促进受体介导的脂蛋白残余物从循环中清除，有助于脂质稳态，并防止动脉粥样硬化。干扰受体结合的apoE缺陷或特定点突变阻碍了致动脉粥样硬化脂蛋白从循环中的清除，并导致与人类和实验动物中的过早动脉粥样硬化相关的III型高脂蛋白血症。apoE的apoE 4亚型也被认为是迟发性家族性阿尔茨海默病的危险因素。尽管apoE在生理范围内的表达清除了脂蛋白残余物，但apoE 2、apoES或apoE 4的高水平表达引起高脂血症。我们最近已经确定，高胆固醇血症在很大程度上是由位于羧基末端区域的氨基酸261至269之间的疏水残基介导的。这些残基也影响含apoE的HDL颗粒的形成。LDL受体缺陷或apoE受体结合结构域突变可降低诱导小鼠高脂血症所需的apoE阈值。体外和体内实验表明，apoE在功能上与ABCA 1相互作用，并促进含apoE的HDL的生物合成。此外，apoE在功能上与SR-BI相互作用并促进脂质流出。在本申请中，我们建议利用我们在过去四年中获得的新知识，以解决三个重要的问题，apoE的功能及其在血脂异常中的作用，在动脉粥样硬化和HDL样apoE含脂蛋白的生物发生。我们的具体目标是：一曰：研究apoE残基L261、W264、F265、L268、V269在apoE体内功能中的作用，包括高脂血症的发生、动脉粥样硬化的保护作用和HDL的形成。最终目标是产生具有动脉粥样硬化保护作用并具有改善的生物学功能的重组apoE形式。第二章：研究与III型高脂蛋白血症显性形式相关的apoE突变体中apoE羧基末端结构域在受体识别、高脂血症敏感性和动脉粥样硬化易感性中的相对作用。这些研究可能会确定apoE功能不同的LDL受体结合，有助于其抗动脉粥样硬化的性质。第三章：阐明apoE和ABCA 1之间的功能性相互作用如何促进含HDL样apoE脂蛋白颗粒的从头生物合成，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。在这些研究中，将在单或双敲除小鼠中进行基因转移，用于apoA-I、apoE、SR-BI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.