High Density Lipoprotein Biogenesis and Speciation

心血管疾病(CVD)是一个公共卫生挑战，具有重大的经济和生活质量 对受害者及其家人的影响。在管理方面取得了令人印象深刻的进展 一些心血管疾病的血脂危险因素；他汀类降血脂药物通过以下方式减少心血管事件的数量 降低血浆低密度脂蛋白-胆固醇水平。通过改装减少心血管疾病 其他血脂风险因素一直是一个挑战，特别是提高高密度的血浆浓度 具有心脏保护作用的脂蛋白胆固醇(HDLC)；试图通过提高HDLC来减少心血管疾病 并不是一律成功的。许多科学家现在意识到，这种机械联系 高密度脂蛋白和动脉粥样硬化保护之间的关系比人们想象的要复杂得多，而且更多的关注 需要的是高密度脂蛋白的功能，而不仅仅是高密度脂蛋白的浓度。高密度脂蛋白在逆转胆固醇中的作用 运输(RCT)，胆固醇在外周组织中的运输，包括巨噬细胞在 动脉壁内皮下间隙，送至肝脏处理。RCT的主要步骤是巨噬细胞 胆固醇外流，血浆中高密度脂蛋白胆固醇的酯化，以及引导某些胆固醇的肝脏处理 排泄，从而减轻全身的胆固醇负担。最近的证据表明，并非所有的高密度脂蛋白都是 相同的，即，高密度脂蛋白是根据组成、结构和可能的功能来分类的。几项研究 已显示心血管疾病患者胆固醇流出到载脂蛋白B耗竭的血浆较低，与高密度脂蛋白胆固醇无关 浓度。这种差异一定是由于患者和那些患者在高密度脂蛋白的“质量”上的差异。 没有心血管疾病。一些高密度脂蛋白可能是更好的胆固醇受体；载脂蛋白AI可能在 外流。此外，基于优化的载脂蛋白AI结构的模拟可能是一种有前途的治疗方法 策略。 这项申请的目标集中在三个方面的高密度脂蛋白和载脂蛋白AI的结构和/或功能- 高密度脂蛋白与完整信号肽的物种生物发生；巨噬细胞外排的机制 将新生的高密度脂蛋白和微增溶作为新生的高密度脂蛋白形成和优化的替代 随机对照试验的载脂蛋白AI结构。这一背景和理论基础产生了三个目标： 目的1：验证含有完整信号肽(SPS)的高密度脂蛋白的生物发生 是独特的和物种的，并形成独特的高密度脂蛋白物种，具有不同的组成和功能。 目的2：验证载脂蛋白AI对磷脂微溶作用的一种新的假想模型， 这产生了重组高密度脂蛋白，这是由肝细胞产生的新生高密度脂蛋白和M-Ф胆固醇的体外类似物 外流。 目的3：验证载脂蛋白AI中保守的氨基酸残基是其结构所必需的假说 和功能。 这些目标的完成将揭示心脏保护性高密度脂蛋白疗法的新分子靶点。这些 AIMS将通过多学科的体外和体内方法来解决，其中包括化学 动力学、细胞生物学、脂肪和蛋白质分析、AAV介导的体内基因传递、高性能 薄层层析、免疫印迹和沉淀法、化学交联法、MALDI MS、定点突变、圆二色谱和动力学比浊法。

Cardiovascular disease (CVD) is a public health challenge with a major economic and quality-of-life impact on its victims and their families. Impressive inroads have been made in the management of some CVD lipid risk factors; the statin class of hypolipidemic drugs reduces the number of CVD events by reducing plasma levels of low density lipoprotein-cholesterol. Reduction in CVD via the modification of other lipid-risk factors has been a challenge, especially raising the plasma concentrations of high density lipoproteins-cholesterol (HDL-C), in a cardioprotective way; attempts to reduce CVD via raising HDL-C have not been uniformly successful. Many scientists now appreciate that the mechanistic connections between HDL and atheroprotection are more complex than once thought and that a greater focus on HDL function, rather that HDL-C concentrations alone, is needed. HDL plays a role in reverse cholesterol transport (RCT), the transport of cholesterol in peripheral tissues, including macrophages in the subendothelial space of the arterial wall, to the liver for disposal. The major RCT steps are macrophage cholesterol efflux, HDL-C esterification in plasma, and hepatic disposal, which directs some cholesterol to excretion thereby reducing the total body cholesterol burden. Recent evidence is that not all HDL are the same, i.e., HDL are speciated according to composition, structure, and likely function. Several studies have shown cholesterol efflux to apo B-depleted plasma is lower in CVD patients, independent of HDL-C concentrations. This difference must be due to differences in the “quality” of HDL in patients vs. those without CVD. Some HDL species may be better cholesterol acceptors; apo AI may play a direct role in efflux. Moreover, mimetics based on an optimized apo AI structure may be a promising therapeutic strategy. The aims of this application focus on three aspects of HDL and apo AI structure and/or function— speciated biogenesis of HDL with intact signal peptides (SP); the mechanism of efflux from macrophages giving nascent HDL and microsolubilization as a surrogate for nascent HDL formation, and optimization of apo AI structure for RCT. This background and rationale gave rise to three aims: Aim 1: To test the hypothesis that the biogenesis of HDL containing apos with intact signal peptides (SPs) is unique and speciated, and forms unique HDL species with distinct compositions and functions. Aim 2: To test a novel, hypothetical “pancake” model of phospholipid microsolubilization by apo AI, which yields rHDL, the in vitro analog of nascent HDL produced by hepatocytes and MФ cholesterol efflux. Aim 3: To test the hypothesis that conserved amino acid residues in apo AI are essential to its structure and function. Completion of these aims will reveal new molecular targets for cardioprotective HDL therapies. These aims will be addressed by multidisciplinary in vitro and in vivo approaches that includes chemical kinetics, cell biology, lipid and protein analyses, AAV-mediated gene delivery in vivo, high performance thin-layer chromatography, immune-blotting and precipitation methods, chemical cross-linking, MALDI MS, site-directed mutagenesis, circular dichroism, and kinetic turbidimetry.