Apolipoprotein A-I structure in high density lipoproteins

描述（由申请人提供）：虽然循环高密度脂蛋白（HDL）被认为是保护心血管疾病，我们有一个非常有限的了解他们的结构。此外，我们对主要的HDL蛋白，载脂蛋白（apo）A-I如何与其他蛋白相互作用以决定HDL功能的了解更少。该项目将测试apoA-I与它和其他HDL相关蛋白在人类血浆中常见的球形HDL颗粒中进行高度特异性接触的假设。在我们以前的工作中，我们使用交联化学和质谱法来产生重组颗粒中的apoA-I以及来自人血浆的“真实的”HDL的详细模型。尽管在大小和形状上有很大的差异，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，我们的目标是进一步评估这些和其他模型使用互补的结构技术，以及评估的基础上apoA-I的相互作用与三个主要的HDL组分：载脂蛋白A-II，卵磷脂：胆固醇酰基转移酶（LCAT），胆固醇酯转移蛋白（CETP）。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粗粒分子动力学（MD）技术来测试球形重构和天然血浆HDL中apoA-I的基于Trefoil的模型。2)利用交联和新的人apoA-II细菌表达系统确定apoA-I和apoA-II之间的分子相互作用，以获得含有两种蛋白质的天然HDL颗粒的第一个模型。3)通过检测LCAT使用定点诱变和交联进入apoA-I结构中的分子孔的概念，确定apoA-I和LCAT之间的分子相互作用。我们还将确定CETP是否特异性结合apoA-I，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方法结合在一起，这将放大我们对这些神秘粒子的结构理解。apoA-I的结构无疑调节HDL代谢。因此，对其结构及其与其他蛋白质的相互作用的分子理解，特别是那些正在探索作为药物靶点的蛋白质，如LCAT和CETP，对于设计利用胆固醇逆向转运和HDL抗炎作用的新疗法至关重要。

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.