Molecular and Functional Analysis of Human ApoA-1

描述（由申请人提供）：ApoA-I是HDL的主要蛋白质组分，是HDL生物发生和功能所必需的。 无脂apoA-1和由ABCA 1依次脂化apoA-1形成的不同HDL物质似乎在胆固醇流出、脂质选择性摄取、LCAT活化以及HDL的其他可能功能中具有不同的功能。基于我们最近的发现，我们的假设是apoA-I结构的细微变化可能影响HDL生物发生和功能，包括ABCA 1和SR-BI介导的胆固醇流出、SR-BI介导的脂质选择性摄取和LCAT的活化。在本申请中，我们将使用体外和体内的方法来阐明apoA-I的结构和功能。体外研究将利用腺病毒感染后apoA-I-/-小鼠中产生的apoA-I和HDL的突变形式。体内研究将在apoA-I-/-小鼠和表达apoA-I突变体的转基因小鼠中利用腺病毒介导的基因转移。我们的具体目标是：1）通过物理化学方法确定apoA-I的特定结构域和残基的贡献，所述特定结构域和残基负责通过溶液中或与脂质结合时的分子内或分子间相互作用稳定apoA-I的构象和结构。apoA-I突变体的结构变化将与apoA-I的体外和体内功能相关，包括LCAT活化以及脂质和脂蛋白结合。2)研究脂质结合的apoA-1与SR-B1的功能相互作用以及apoA-1突变对SR-B1介导的胆固醇流出和选择性脂质摄取的影响。3)研究apoA-I突变对胆固醇流出的影响以及无脂质apoA-I与ABCA 1的功能相互作用，导致细胞磷脂和胆固醇流出。4)采用腺病毒介导的apoA-l-/-小鼠和转基因小鼠的基因转移研究apoA-l突变对不同HDL种类的生物发生和功能的影响。流行病学和遗传学数据，结合最近的转基因实验，表明apoA-I和HDL水平的增加可以预防动脉粥样硬化。相比之下，低apoA-I和HDL水平使人类容易患冠状动脉疾病（CAD），这是全球死亡的主要原因。了解apoA-I的分子结构和各种生物学功能可能会导致新的药理学方法来预防和/或治疗这些疾病。

DESCRIPTION (provided by applicant): ApoA-I is the major protein component of HDL and is required both for the biogenesis and the functions of HDL. Lipid-free apoA-1 and different HDL species formed by sequential lipidation of apoA-I by ABCA1 appear to have distinct functions in cholesterol efflux, selective uptake of lipids, activation of LCAT, and possibly other functions of HDL. It is our hypothesis based on our recent findings, that subtle changes in the apoA-l structure may affect HDL biogenesis and functions including ABCA1- and SR-BI-mediated cholesterol efflux, SR-Bl-mediated selective uptake of lipids, and activation of the LCAT. In this application we will use in vitro and in vivo approaches to elucidate the structure and functions of apoA-I. The in vitro studies will utilize mutant forms of apoA-I and HDL produced in apoA-l-/- mice following adenovirus infection. The in vivo studies will utilize adenovirus-mediated gene transfer in apoA-l-/- mice and transgenic mice expressing apoA-l mutants. Our specific aims are: 1) To determine by physicochemical methods the contribution of specific domains and residues of apoA-l that are responsible for stabilizing the conformation and structure of apoA-l through intra- or intermolecular interactions in solution, or when bound to lipids. Structural changes of the apoA-I mutants will be correlated with the in vitro and in vivo functions of apoA-l, including LCAT activation and lipid and lipoprotein binding. 2) To investigate the functional interactions of lipid-bound apoA-l with SR-Bl and the effect of apoA-l mutations in SR-Bl-mediated cholesterol efflux and selective lipid uptake. 3) To investigate the effect of apoA-I mutations in cholesterol efflux and the functional interactions of lipid-free apoA-I with ABCA1 that leads to efflux of cellular phospholipid and cholesterol. 4) To investigate the implications of apoA-l mutations on the biogenesis and the function of different HDL species using adenovirus-mediated gene transfer in apoA-l-/- mice as well as transgenic mice. 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 humans to coronary artery disease (CAD), a leading cause of mortality worldwide. Understanding the molecular structure and the various biological functions of apoA-I may lead to new pharmacological approaches to prevent and/or treat these conditions.