Project 2 - Structural Basis of HDL Maturation

摘要：项目2 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 对它们的结构的了解非常有限。此外，我们更不了解如何 主要的高密度脂蛋白，载脂蛋白A-I，与其他蛋白质相互作用，决定高密度脂蛋白的功能。我们会 验证以下假设：apoA-I与自身进行高度特异性的接触，形成一种分子支架， 稳定高密度脂蛋白，并通过特定的蛋白质：蛋白质相互作用，促进高密度脂蛋白伙伴的联系 蛋白质来定义粒子的功能。在我们之前的工作中，我们使用了交联化学和质量 利用光谱分析技术在重组颗粒中生成apoA-I的详细模型以及从人类中提取“真正的”高密度脂蛋白 血浆。尽管在大小和形状上有很大的差异，但这些结构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 高密度脂蛋白的三个主要成分：载脂蛋白A-II、对氧磷酶1(PON1)和胆固醇酯转移蛋白 (CETP)。具体目标是：1)测试三叶和其他模型的apoA-I在球形重组和 天然或真正的血浆高密度脂蛋白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协同的过程粒度分子动力学(MD)技术)以确定 载脂蛋白A-I和载脂蛋白A-II的分子相互作用及一种新的人类载脂蛋白A-II细菌 表达系统，以获得包含这两种蛋白质(也是协同作用)的第一个天然高密度脂蛋白颗粒模型 与Segrest)。3)确定apoA-I与两个重要的高密度脂蛋白对接之间的分子相互作用 蛋白质，PON1和CETP，使用化学交联和定点突变。一路走来，我们将 还使用我们的实验技术直接测试Segrest在 项目1并评估从卵磷脂：胆固醇中分离出的潜在增强功能高密度脂蛋白的结构 Heinecke在项目3中研究了酰基转移酶缺乏的主题。我们的方法独特地交织在新的 实验技术和最先进的MD方法产生的结构知识将是 直接应用于硬件描述语言功能。此外，我们的重点是真实的高密度脂蛋白颗粒的结构，这些颗粒 在正常人和有罕见遗传病的人中传播。载脂蛋白A-I的结构 毫无疑问，高密度脂蛋白可以调节高密度脂蛋白的代谢，并可能介导某些高密度脂蛋白的心脏保护作用。 亚种。因此，对其结构及其与其他蛋白质相互作用的分子理解， 特别是那些作为药物靶标被探索的药物，如LCAT和CETP，对于设计新的 利用胆固醇反向运输和高密度脂蛋白的抗炎作用的治疗。

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.