The molecular basis for high density lipoprotein heterogeneity

描述（由申请人提供）：高密度脂蛋白（HDL）是由蛋白质和脂质组成的血源性大分子复合物，在预防心血管疾病（CVD）中发挥关键作用，心血管疾病是美国死亡的主要原因，目前正在深入研究中，作为现有降脂药物的潜在增强作用。这些复合物是多分散的，可以包含多达50种不同的蛋白质，每种蛋白质都可能调节宿主HDL颗粒的代谢和潜在的益处/病理。其中一些蛋白质在补体调节、蛋白酶抑制和先天免疫等过程中具有令人惊讶的作用。该应用的研究旨在了解HDL蛋白异质性的分子基础。我们将测试HDL由许多不同的颗粒亚群组成的假设，每个亚群都有独特的蛋白质组合，在预防心血管疾病方面发挥着不同的生理作用。在Aim 1中，HDL颗粒将通过四种正交色谱方法从正常人血浆中分离出来，每个分离组分的蛋白质组成将采用我们开发的基于质谱的新模式分析。在多种分离技术中持续存在的蛋白质相关性将成为不同HDL亚颗粒的候选物，并将使用免疫亲和下拉技术进行验证。在Aim 2中，从Aim 1中分离的HDL亚组分将受到同源双功能和新型亲和标记的交联剂的影响，以共价连接密切相互作用的蛋白质，并通过质谱法进行鉴定。该方法将获得相互作用蛋白的序列特定结构信息，并作为HDL亚粒子鉴定的补充模式。我们设想，这一信息将为未来的治疗研究奠定基础，旨在专门调节某些心脏保护HDL亚颗粒的水平或模拟它们的作用。该研究还可能确定HDL亚种，可作为预测心血管疾病保护或易感性的生物标志物。

DESCRIPTION (provided by applicant): High density lipoproteins (HDL) are blood-borne macromolecular complexes composed of protein and lipid that play critical roles in the prevention of cardiovascular disease (CVD), the major cause of mortality in the United States, and are under intense study as a potential enhancement to current lipid-lowering medications. These complexes are polydisperse and can contain up to 50 different proteins, each likely able to modulate the metabolism and potential benefit/pathology of the host HDL particle. Some of these proteins have surprising roles in processes such as complement regulation, protease inhibition and innate immunity. The studies in this application are aimed at understanding the molecular basis of HDL protein heterogeneity. We will test the hypothesis that HDL is composed of numerous distinct particle subpopulations, each with unique protein combinations, which play distinct physiological roles related to the protection against CVD. In Aim 1, HDL particles will be separated from normal human plasma by four orthogonal chromatographic approaches and the protein composition of fractions from each separation will be subjected to a new mass spectrometry-based pattern analysis we have developed. Protein correlations that persist across multiple separation techniques will become candidates for distinct HDL subparticles and will be verified using immunoaffinity pull-down techniques. In Aim 2, isolated HDL subfractions from Aim 1 will be subjected to homobifunctional and novel affinity-tagged cross-linking agents to covalently join closely interacting proteins to be identified by mass spectrometry. This approach will derive sequence specific structural information on the interacting proteins and serve as a complementary mode of HDL subparticle identification. We envision that this information will form a basis for future therapeutic investigations designed to specifically modulate the levels of certain cardioprotective HDL subparticles or to mimic their effects. The study may also identify HDL subspecies that can be used as biomarkers to predict protection or susceptibility to CVD. PUBLIC HEALTH RELEVANCE: High density lipoproteins (HDL) are the so-called "good cholesterol" because they protect against cardiovascular disease, but we don't understand why. The studies in this proposal will identify previously unknown HDL subspecies that may be particularly protective. This knowledge may be useful for developing predictive biomarkers and for developing preventative treatments for cardiovascular disease.