Functions of apoE in cholesterol and triglyceride homeostasis

描述(申请人提供)：载脂蛋白E是胆固醇运输系统的重要蛋白质，在普通人群中有三种常见的亚型，载脂蛋白2，载脂蛋白E3和载脂蛋白E4。APOE促进受体介导的脂蛋白残留物从循环中清除，有助于脂质稳态，并保护动脉粥样硬化。载脂蛋白E基因的缺陷或特定的点突变干扰了受体结合，阻碍了致动脉粥样硬化脂蛋白从循环中的清除，并导致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。ApoE的apoE4亚型也被认为是晚发性家族性阿尔茨海默病的危险因素。在生理范围内表达载脂蛋白E可以清除脂蛋白残留物，而高水平表达载脂蛋白2、载脂蛋白ES或载脂蛋白E4会导致高甘油三酯血症。我们最近证实，高甘油三酯血症在很大程度上是由位于羧基末端第261到269个氨基酸之间的疏水残基介导的。这些残基也影响含有apoE的高密度脂蛋白颗粒的形成。低密度脂蛋白受体缺陷或载脂蛋白E受体结合域的突变降低了诱导小鼠高甘油三酯血症所需的载脂蛋白E阈值。体外和体内实验表明，apoE与ABCA1在功能上相互作用，并促进含有apoE的高密度脂蛋白的生物发生。此外，apoE与SR-BI在功能上相互作用，促进脂质外流。在这一应用中，我们建议利用我们在过去四年中获得的新知识来解决三个重要问题，这些问题与apoE的功能及其在血脂异常中的作用、在动脉粥样硬化形成中的作用以及含HDL样apoE的脂蛋白的生物发生有关。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生、对动脉粥样硬化的保护以及高密度脂蛋白的形成。最终目标是产生具有抗动脉粥样硬化和改善生物学功能的重组载脂蛋白E形式。2：研究载脂蛋白E的羧基末端结构域在受体识别、对高甘油三酯血症的敏感性和动脉粥样硬化易感性中的相对作用。这些研究可能确定载脂蛋白E不同于低密度脂蛋白受体结合的功能，这些功能有助于其抗动脉粥样硬化特性。3：阐明载脂蛋白E和ABCA1之间的功能相互作用如何促进高密度脂蛋白样脂蛋白颗粒的从头生物发生，这些颗粒通过随后与LCAT和SR-BI的相互作用而成熟，以及它们在整体胆固醇稳态中的作用。在这些研究中，将采用单基因或双基因敲除小鼠对载脂蛋白A-L、载脂蛋白E、SR-BI的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.