Apolipoprotein A-I structure in high density lipoproteins

描述(申请人提供)：尽管循环高密度脂蛋白(HDL)被认为对心血管疾病具有保护作用，但我们对其结构的了解非常有限。此外，我们对主要的高密度脂蛋白(Apo)A-I是如何与其他蛋白质相互作用来决定高密度脂蛋白功能的了解更少。这个项目将检验这样一个假设，即apoA-I与它和其他高密度脂蛋白相关的蛋白质在球形高密度脂蛋白颗粒中进行高度特异性的接触，这些蛋白质通常存在于人类血浆中。在我们之前的工作中，我们使用交联化学和质谱学来生成重组粒子中apoA-I的详细模型，以及从人类血浆中生成“真正的”高密度脂蛋白。尽管在大小和形状上有很大的差异，但这些结构都有一个反平行带状排列的主题。在这些发现的基础上，我们的目标是使用互补的结构技术进一步评估这些和其他模型，并评估apoA-I与三个主要高密度脂蛋白成分：载脂蛋白A-II、卵磷脂：胆固醇酰基转移酶(LCAT)和胆固醇酯转移蛋白(CETP)的相互作用。具体目标是：1)使用新的双同位素交联技术和最先进的全原子和过程颗粒分子动力学(MD)技术，在球形重组血浆和天然血浆高密度脂蛋白中测试基于三叶的载脂蛋白A-I模型。2)利用交联法和一种新的人类apoA-II细菌表达系统来确定apoA-I和apoA-II之间的分子相互作用，以获得包含这两种蛋白质的第一个天然高密度脂蛋白颗粒模型。3)通过检测LCAT通过定点突变和交联进入apoA-I结构中的分子孔这一概念来确定apoA-I和LCAT之间的分子相互作用。我们还将确定CETP是否与apoA-I特异性结合，并确定它们的接触点。这种方法独特地将新的实验技术与最先进的MD方法交织在一起，将放大我们对这些神秘粒子的结构理解。载脂蛋白A-I的结构无疑调节着高密度脂蛋白的代谢。因此，了解其结构及其与其他蛋白质的相互作用，特别是那些被探索为DRU靶点的蛋白质，如LCAT和CETP，对于利用胆固醇反向转运和高密度脂蛋白的抗炎作用的新疗法的设计至关重要。

DESCRIPTION (provided by applicant): Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. Furthermore, we understand even less about how the major HDL protein, apolipoprotein (apo)A-I, interacts with other proteins to dictate HDL function. This project will test the hypothesis that apoA-I makes highly specific contacts with it and other HDL associated proteins in spherical HDL particles commonly found in human plasma. In our previous work, we used cross- linking chemistry and mass spectrometry to generate detailed models of apoA-I in reconstituted particles as well as "real" HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt-like arrangement. Building on these discoveries, our goal is to further evaluate these and other models using complementary structural techniques as well as evaluate the basis of apoA-I's interactions with three major HDL components: apolipoprotein A-II, lecithin:cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein (CETP). The specific aims are: 1) To test the Trefoil-based models of apoA-I in spherical reconstituted and native plasma HDL using new dual isotope cross-linking techniques and state-of-the-art all-atom and course grained molecular dynamics (MD) techniques. 2) To determine the molecular interactions between apoA-I and apoA-II using cross-linking and a new human apoA-II bacterial expression system to derive the first models of native HDL particles containing both proteins. 3) To determine the molecular interaction between apoA-I and LCAT by testing the concept that LCAT accesses a molecular pore in the apoA-I structure using site-directed mutagenesis and cross-linking. We will also determine if CETP specifically binds apoA-I and identify their points o contact. This approach uniquely intertwines new experimental techniques with state-of-the-art MD approaches that will magnify our structural understanding of these enigmatic particles. The structure of apoA-I undoubtedly modulates HDL metabolism. Thus, a molecular understanding of its structure and its interactions with other proteins, particularly those being explored as dru targets such as LCAT and CETP, is critical for the design of new therapies exploiting reverse cholesterol transport and the anti-inflammatory roles of HDL.