Modulation of ABCA1 Expression and Activity

描述(申请人提供)：人群研究表明，血浆高密度脂蛋白(高密度脂蛋白)水平与心血管疾病风险呈负相关，这意味着与高密度脂蛋白代谢相关的因素具有动脉粥样硬化保护作用。其中一个因素是细胞膜上的三磷酸腺苷结合盒(ABC)转运体ABCA1，它介导过量的胆固醇从细胞到高密度脂蛋白的运输。虽然ABCA1的表达在转录水平上受到高度调控，但ABCA1的蛋白表达和活性也受到鲜为人知的翻译后过程的调节。载脂蛋白与细胞的相互作用至少对ABCA1途径有三种不同的影响，包括ABCA1转运的胆固醇和磷脂的去除，ABCA1蛋白的稳定，以及ABCA1功能所需的酪氨酸激酶信号通路的激活。导致这些影响的载脂蛋白和ABCA1决定因素尚不清楚。这些研究和其他研究表明，多种细胞因素参与稳定ABCA1蛋白并调节其活性。我们建议使用功能分析和蛋白质发现策略来表征参与调节ABCA1蛋白质水平和功能的过程。我们将表征载脂蛋白A-I和载脂蛋白模拟肽对ABCA1表达和活性的影响。这些研究将使用各种合成肽和ABCA1突变体来建立负责这种调节的载脂蛋白和ABCA1的性质，表征载脂蛋白诱导的ABCA1构象的变化，并确定ABCA1中与载脂蛋白相互作用的位置。我们将在小鼠模型中测试ABCA1稳定肽预防动脉粥样硬化的能力。我们还将使用体外活性分析、蛋白质的质谱分析和插入突变基因发现来识别和表征调节ABCA1活性和蛋白质水平的细胞因子。这些研究将提示ABCA1通路可能的损伤部位，并发现治疗心血管疾病的潜在治疗靶点。

DESCRIPTION (provided by applicant): Population studies have shown an inverse relationship between plasma high-density lipoprotein (HDL) levels and risk for cardiovascular disease, implying that factors associated with HDL metabolism are atheroprotective. One of these factors is a cell membrane ATP-binding cassette (ABC) transporter called ABCA1, which mediates the transport of excess cholesterol from cells to HDL apolipoproteins. Although ABCA1 expression is highly regulated at the level of transcription, ABCA1 protein expression and activity are also modulated by poorly understood post-translational processes. The interaction of apolipoproteins with cells has at least three distinct effects on the ABCA1 pathway, including removal of ABCA1-transported cholesterol and phospholipids, stabilization of ABCA1 protein, and activation of a tyrosine kinase signaling pathway required for ABCA1 function. The apolipoprotein and ABCA1 determinants responsible for these effects are unknown. These and other studies have implicated multiple cellular factors as being involved in stabilizing ABCA1 protein and regulating its activity. We propose to use functional assays and protein discovery strategies to characterize processes involved in modulating ABCA1 protein levels and function. We will characterize the effects of apoA-I and apolipoprotein-mimetic peptides on ABCA1 expression and activity. These studies will use a variety of synthetic peptides and ABCA1 mutants to establish the apolipoprotein and ABCA1 properties responsible for this modulation, to characterize changes in ABCA1 conformation induced by apolipoproteins, and to identify sites in ABCA1 that interact with apolipoproteins. We will test for the ability of ABCA1-stabilizing peptides to protect against atherosclerosis in mouse models. We will also identify and characterize cellular factors that modulate ABCA1 activity and protein levels using in vitro activity assays, mass spectrometric analysis of proteins, and insertional mutagenic gene discovery. These studies will suggest possible sites of impairment of the ABCA1 pathway and uncover potential therapeutic targets for treating cardiovascular disease.