Project 2 - Structural Basis of HDL Maturation

摘要：项目2 虽然循环高密度脂蛋白（HDL）被认为是保护心血管疾病，我们 对它们的结构了解非常有限此外，我们更不了解 主要的HDL蛋白，载脂蛋白（apo）A-I，与其它蛋白相互作用以决定HDL功能。我们将 测试apoA-I与自身高度特异性接触以形成分子支架的假设， 稳定HDL，并通过特定的蛋白质：蛋白质相互作用促进HDL伴侣的结合 蛋白质来定义粒子功能。在我们之前的工作中，我们使用了交联化学和质量 光谱法以产生重构颗粒中的apoA-I以及来自人的“真实的”HDL的详细模型 等离子体尽管在大小和形状上有很大的差异，这些建筑都有一个共同的主题， 反平行带状排列。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 使用互补结构技术的模型，以及评估apoA-I与 三种主要的HDL组分：载脂蛋白A-II、对氧磷酶1（PON 1）和胆固醇酯转运蛋白 （CETP）。具体目的是：1）检测三叶草和其他apoA-I模型在球形重构和 天然或“真实的”血浆HDL，使用新的双同位素交联技术和最先进的全原子和 与Segrest和Core B协同的课程粒度分子动力学（MD）技术。2)确定 使用交联的apoA-I和apoA-II之间的分子相互作用和一种新的人apoA-II细菌 表达系统以获得含有两种蛋白质（也协同作用）的天然HDL颗粒的第一模型 Segrest）。3)确定apoA-I与两个重要HDL对接的分子相互作用 蛋白质，PON 1和CETP，使用化学交联和定点诱变。沿着，我们将 我还使用我们的实验技术，直接测试LCAT的结构模型正在产生的Segrest在 项目1和评估的结构可能增强功能高密度脂蛋白分离卵磷脂：胆固醇 Heinecke在项目3中研究的酰基转移酶缺陷受试者。我们的方法独特地将新的 实验技术与国家的最先进的MD方法产生的结构知识，将 直接应用于HDL函数。此外，我们的重点是真正的HDL颗粒的结构， 在正常人和罕见遗传病患者中传播。apoA-I的结构 毫无疑问，调节HDL代谢，并可能介导某些HDL的心脏保护作用。 亚种。因此，对它的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的药物靶点，如LCAT和CETP，对于设计新的 利用胆固醇逆向转运和HDL的抗炎作用的疗法。

ABSTRACT: PROJECT 2 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. We will test the hypothesis that apoA-I makes highly specific contacts with itself to form a molecular scaffold that stabilizes HDL and facilitates, through specific protein:protein interactions, the association of HDL partner proteins to define particle function. 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, paraoxonase 1 (PON1) and cholesteryl ester transfer protein (CETP). The specific aims are: 1) To test the Trefoil and other models of apoA-I in spherical reconstituted and native or “real” plasma HDL using new dual isotope cross-linking techniques and state-of-the-art all-atom and course grained molecular dynamics (MD) techniques in synergy with Segrest and Core B. 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 (also in synergy with Segrest). 3) To determine the molecular interactions between apoA-I and two important HDL docking proteins, PON1 and CETP, using chemical cross-linking and site-directed mutagenesis. Along the way, we will also use our experimental techniques to directly test structural models of LCAT being generated by Segrest in Project 1 and evaluate the structure of potentially enhanced functional HDL isolated from lecithin:cholesterol acyl transferase deficient subjects studied by Heinecke in Project 3. Our approach uniquely intertwines new experimental techniques with state-of-the-art MD approaches resulting in structural knowledge that will be directly applied to HDL function. Furthermore, our focus is on the structure of authentic HDL particles that are circulating in normal individuals as well as those with rare genetic disorders. The structure of apoA-I undoubtedly modulates HDL metabolism, and possibly mediates cardioprotective effects of some HDL subspecies. Thus, a molecular understanding of its structure and its interactions with other proteins, particularly those being explored as drug 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.