ANALYSIS OF HUMAN APOLIPOPROTEIN A-I

载脂蛋白A-I是高密度脂蛋白的主要蛋白质组分，其生理功能 功能具有重大的生物学和医学重要性。在这 我们建议使用体外和体内方法来应用于 阐明载脂蛋白A-I的结构和功能。这是我们的假设 根据我们最近的发现，羧基中的疏水残基- 末端结构域(残基208-243)是与脂质结合所必需的 和高密度脂蛋白的形成。这也是我们的假设，带电残留物 在分离apoA-I螺旋的扭结区域中，可能有助于 与SR-Bl结合。对于体外研究，我们将利用现有的 表达不同形式载脂蛋白A-I的细胞系已建立 在前一个资助期内，以及我们为 细胞大规模生长、载脂蛋白A-I的纯化和分析基因 体内研究将使用转移和转基因方法。 我们的具体目标是：1)利用现有的永久细胞系 将上一次授权期产生的apoA-I表格表达为细胞 合成载脂蛋白A-I和含载脂蛋白A-I的工厂。 从这些细胞系产生的正常和突变的apoA-I形式将是 使用已建立的方法进行提纯和表征，并将 用于研究载脂蛋白A-的理化和功能性质 I.物理化学分析旨在绘制特定的结构域 和apoA-I残基参与分子内和分子间 溶液中的相互作用以及与负责的脂质结合时的相互作用 为了稳定载脂蛋白A-I的构象和结构。功能性 分析将包括LCAT激活和胆固醇外流特性 变种人的名字。2a)映射apoA-I的结构域和/或残基， 对于使用现有的清道夫SR-BI受体结合很重要 以及新的载脂蛋白A-I变种。这些研究的基本原理是基于 关于最近与Krieger博士的合作表明rHDL 颗粒竞争125I高密度脂蛋白与SRBI受体的结合。2B) 要将apoA-I的残基映射到羧基末端结构域中， 参与与脂类和脂蛋白的结合，这可能是必不可少的 对于HDL子类的组装，使用现有的以及新的apoA-I 变种。流行病学和遗传学数据结合最近的 转基因实验表明，载脂蛋白A-I和高密度脂蛋白水平增加 防止动脉粥样硬化。相比之下，低载脂蛋白A-I和高密度脂蛋白水平 易患冠心病(CAD)，这是 全球范围内的死亡率。了解载脂蛋白A-I的生物学功能 和与CAD发展相关的高密度脂蛋白可能会导致新的 预防和/或治疗这些疾病的药理学方法。

ApoA-I is the major protein component of HDL and its physiological functions are of major biological and medical importance. In this application we propose to use in vitro and in vivo approaches to elucidate the structure and functions of apoA-I. It is our hypothesis based on our recent finding, that hydrophobic residues in the carboxyl- terminal domain (residues 208-243) are essential for binding to lipids and formation of HDL. It is also our hypothesis that charged residues in the kink regions that separate the apoA-I helices may contribute to binding to SR-Bl. For in vitro studies we will capitalize on existing cell lines expressing variant forms of apoA-I that have been generated in the previous grant period and the methods we have developed for large-scale growth of cells, purification and analysis of apoA-I. Gene transfer and transgenic methodologies will be used for in vivo studies. Our specific aims are: 1) To utilize existing permanent cell lines expressing apoA-I forms generated in the previous grant period as cell factories for synthesis of apoA-I and apoA-I containing lipoproteins. Normal and mutant apoA-I forms produced from these cell lines will be purified and characterized using established methodologies and will be utilized to study the physicochemical and functional properties of apoA- I. The physicochemical analyses are designed to map specific domains and residues of apoA-I involved in intra-and inter-molecular interactions in solution and when bound to lipids that are responsible for stabilizing the conformation and structure of apoA-I. Functional analyses will include LCAT activation and cholesterol efflux properties of the mutants. 2a) To map the domains and/or residues of apoA-I that are important for binding to the scavenger SR-BI receptor using existing as well as new apoA-I variants. The rationale of these studies is based on recent collaborative work with Dr. Krieger showing that rHDL particles compete for the binding of 125I HDL to the SRBI receptor. 2b) To map the residues of apoA-I within the carboxyl-terminal domain that are involved in binding to lipids and lipoproteins that may be essential for the assembly of HDL subclasses, using existing as well as new apoA-I variants. Epidemiological and genetic data combined with recent transgenic experiments suggest that increased apoA-I and HDL levels protect from atherosclerosis. In contrast, low apoA-I and HDL levels predispose to coronary artery disease (CAD), a leading cause of mortality worldwide. Understanding the biological functions of apoA-I and HDL which are relevant to the development of CAD may lead to new pharmacological approaches to prevent and/or treat these conditions.