Apolipoprotein A-I structure in high density lipoproteins

描述（由申请人提供）：高密度脂蛋白（HDL）及其主要蛋白质成分载脂蛋白（apo） a - 1在预防人类心血管疾病中起着至关重要的作用，在美国每年夺去近100万人的生命。不幸的是，关于HDL的心脏保护作用的分子基础仍有许多未解之谜。详细了解这些影响的一个突出障碍是缺乏关于人血浆中存在的高密度脂蛋白颗粒中apoA-l结构的信息。我们提出验证球形血浆HDL颗粒中的apoA-l结构与可以在体外创建和详细研究的简单HDL颗粒中的apoA-l结构相关的假设。在最初的资助阶段，我们使用交联化学和高分辨率质谱法在不同大小和形状的重组颗粒中生成apoa - 1的详细模型。在这个延续应用中，我们建议将这些模型扩展到从人血浆中分离的真实HDL颗粒。利用我们已经计算出的结构，我们将监测apoa - 1的构象，因为粒子的复杂性是系统地增加。作为第一步，将评估引入载脂蛋白a - all（高密度脂蛋白中第二常见的蛋白质成分）对载脂蛋白a - 1结构的影响。然后，我们将监测从血浆HDL分离的脂质部分生成的球形颗粒中的apoa - 1和a - all的结构。最后，真正的人体等离子体粒子将被用来测试我们的模型。我们相信这将是迄今为止对真实人类HDL颗粒中apoa - 1和apoa - all结构进行的最全面的研究。我们还将研究盘状和球形复合体中apoA-l分子登记的变化的功能后果，这可能对HDL组成如何调节其代谢具有令人兴奋的意义。很明显，血浆高密度脂蛋白的性质排除了使用核磁共振和x射线晶体学进行详细的结构研究，至少以目前的技术。然而，我们的质谱分析方法不受均匀样品要求的限制，因此是解决这一重要而复杂问题的最佳方法。我们相信这些研究的信息将为未来高针对性的突变策略提供基础，旨在剖析apoa - 1的保护功能，可能导致增强HDL保护作用的治疗方法。

DESCRIPTION (provided by applicant): High density lipoprotein (HDL) and its major protein constituent, apolipoprotein (apo)A-l, play critical roles in the prevention of human cardiovascular disease, which claims nearly a million lives in the United States every year. Unfortunately, there are still many unanswered questions 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-l in HDL particles that exist in human plasma. We propose to test the hypothesis that the structure of apoA-l in spherical plasma HDL particles is related to that found in simple HDL particles that can be created and studied in detail in vitro. In the initial funding period, we used cross-linking chemistry and high-resolution mass spectrometry to generate detailed models of apoA-l in reconstituted particles of various size and shape. In this continuation application, we propose to extend these models to real HDL particles isolated from human plasma. Using the structures we have already worked out, we will monitor the conformation of apoA-l as the complexity of the particles is systematically increased. As a first step, the effects of introducing apolipoprotein A-ll, the second most common protein constituent of HDL, on the structure of apoA-l will be evaluated. We will then monitor the structure of apoA-l and A-ll in spherical particles generated with lipid fractions isolated from plasma HDL. Finally, true human plasma particles will be used to test our models. We believe that this will be the most comprehensive study of apoA-l and apoA-ll structure in authentic human HDL particles ever attempted. We will also study the functional consequences of a shift in apoA-l molecular registry in discoidal and spherical complexes that may have exciting implications for how HDL composition modulates its metabolism. It is clear that the nature of plasma HDL precludes the use of NMR and X-ray crystallography for detailed structural studies, at least with current technology. However, our mass spectrometry approach is not limited by the requirement of a homogeneous sample and is thus the best available for addressing this important and complex problem. We believe that the information from these studies will provide the basis for future highly targeted mutagenesis strategies designed to dissect out the protective functions of apoA-l, perhaps leading to therapies for enhancing the protective effects of HDL.