Apolipoprotein A-I structure in high density lipoproteins

描述(申请人提供)：高密度脂蛋白及其主要蛋白成分载脂蛋白A-L在预防人类心血管疾病中发挥着关键作用，每年在美国有近100万人死于心血管疾病。不幸的是，关于高密度脂蛋白心脏保护作用的分子基础仍有许多悬而未决的问题。阻碍详细了解这些影响的一个突出障碍是缺乏关于人体血浆中存在的高密度脂蛋白颗粒中载脂蛋白A-L结构的信息。我们建议检验这一假设，即在球形血浆高密度脂蛋白颗粒中apoA-L的结构与在简单高密度脂蛋白颗粒中发现的结构有关，这些简单高密度脂蛋白颗粒可以在体外创造和详细研究。在最初的资助期，我们使用交联化学和高分辨率质谱仪，在不同大小和形状的重组颗粒中生成了载脂蛋白A-L的详细模型。在接下来的应用中，我们建议将这些模型扩展到从人血浆中分离出来的真实高密度脂蛋白颗粒。使用我们已经计算出的结构，我们将监测载脂蛋白A-L的构象，因为粒子的复杂性是系统地增加的。作为第一步，将评估引入载脂蛋白A-11对载脂蛋白A-L结构的影响。载脂蛋白A-11是高密度脂蛋白中第二常见的蛋白质成分。然后，我们将监测从血浆高密度脂蛋白中分离出的脂组分生成的球形颗粒中载脂蛋白A-L和A-11的结构。最后，真实的人类血浆颗粒将被用来测试我们的模型。我们相信，这将是对真实人类高密度脂蛋白颗粒中载脂蛋白A-L和载脂蛋白A-11结构的最全面的研究。我们还将研究盘状和球形复合体中载脂蛋白A-L分子注册表转移的功能后果，这可能对高密度脂蛋白的组成如何调节其代谢具有令人兴奋的影响。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射线结晶学进行详细结构研究的可能性，至少在目前的技术下是这样。然而，我们的质谱学方法并不局限于对均匀样品的要求，因此是解决这一重要而复杂问题的最佳方法。我们相信，来自这些研究的信息将为未来设计的高针对性突变策略提供基础，以剖析载脂蛋白A-L的保护功能，并可能导致增强高密度脂蛋白保护作用的治疗。

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.