Apolipoprotein A-I Structure in High Density Lipoprotein

描述(由申请人提供)： 高密度脂蛋白(HDL)及其主要蛋白成分载脂蛋白(Apo)A-I可能起作用 在防止可导致人类心血管疾病的血管中的胆固醇积聚方面发挥关键作用，在美国每年夺去近一百万人的生命。不幸的是，人们对高密度脂蛋白心脏保护作用的分子基础知之甚少。详细了解这些影响的一个突出障碍是缺乏关于高密度脂蛋白中载脂蛋白A-I结构的信息。我们建议检验这一假设，即在球形人类血浆高密度脂蛋白颗粒中apoA-I的结构与在体外可以创建的最简单的盘状颗粒中的结构相关。该方法将利用重组的盘状MDL颗粒边缘固有的几何约束来生成这些颗粒上apoA-I组织的非常详细的模型。以这种结构为基准，然后将监测apoA-I区域之间的空间关系的变化，因为颗粒的复杂性从盘状系统地增加到定义良好的重组球形颗粒，最后到孤立的人类高密度脂蛋白颗粒。二 将使用补充方法来监测颗粒上apoA-I分子内部和之间的距离参数。它们是：A)使用apoA-I的色氨酸和半胱氨酸突变体的综合电池来研究荧光能量转移，以及B)利用可逆的硫醇交联剂来确定脂蛋白中apoA-I的不同区域的接近程度的新型质谱学/肽图技术的应用。这些方法将被用来生成不同颗粒形态下高密度脂蛋白结合的载脂蛋白A-I的详细“接近图”。这一信息将为产生apoA-I突变体的高度靶向性突变策略提供基础，这些突变体可以在体内用于剖析apoA-I的心脏保护功能。

DESCRIPTION (provided by applicant): High density lipoprotein (HDL) and its major protein constituent, apolipoprotein (apo)A-I, may play critical roles in the prevention of cholesterol accumulation in blood vessels that can lead to human cardiovascular disease, which claims nearly a million lives per year in the United States. Unfortunately, relatively little is known about the molecular basis for the cardio-protective effects of HDL. A prominent obstacle in the way of a detailed understanding of these effects is the lack of information on the structure of apoA-I in HDL. We propose to test the hypothesis that the structure of apoA-I in spherical human plasma HDL particles is related to that in the simplest discoidal particles that can be created in vitro. The approach will be to take advantage of the geometric constraints inherent to the edge of reconstituted discoidal MDL particles to generate a highly detailed model of apoA-I organization on these particles. Using this structure as a benchmark, changes in spatial relationships between regions of apoA-I will then be monitored as the complexity of the particles is systematically increased from discs, to well-defined reconstituted spherical particles, and finally to isolated human HDL particles. Two complementary approaches will be used to monitor the distance parameters within and between apoA-I molecules on the particles. These are: A) the use of a comprehensive battery of tryptophan and cysteine mutants of apoA-I to study fluorescence energy transfer, and B) the application of a novel mass spectrometry/peptide mapping technique that takes advantage of reversible thiol cross-linkers to determine the proximity of various regions of apoA-I in lipoproteins. These methods will be used to generate a detailed "proximity map" of HDL-bound apoA-I in different particle morphologies. This information will provide a basis for highly targeted mutagenesis strategies resulting in apoA-I variants that can be used in vivo to dissect out the cardio-protective functions of apoA-I.