Structural Thermodynamics of Human Apolipoprotein C-1

描述(申请人提供)：本项目的长期目标是在分子细节上确定可交换的载脂蛋白和脂蛋白的能量-结构-功能关系，从而深入了解脂蛋白在动脉粥样硬化和其他脂蛋白相关疾病发病机制中的作用机制。可交换载脂蛋白是脂蛋白中的可溶性蛋白质组分，介导脂质和胆固醇的运输和代谢，在动脉粥样硬化、冠心病、中风等人类重大疾病的发病机制中起着至关重要的作用。脂蛋白的结构稳定性和组成的可变性对其功能至关重要，为了阐明正常和疾病状态下脂蛋白作用的分子机制，必须对其进行详细的了解。这项拟议的工作通过详细研究两个小的人类血浆载脂蛋白apoC-1和apoA-2的能量学、结构和脂结合功能来解决这一长期目标。载脂蛋白C-1通过抑制载脂蛋白E介导的低密度脂蛋白受体相关途径对可能导致动脉粥样硬化的甘油三酯颗粒的摄取，延缓了它们的清除。ApoC-1激活卵磷脂：胆固醇酰基转移酶(LCAT)的能力可能解释了LCAT主要激活剂apoA-1缺乏的受试者血浆胆固醇酯水平正常的原因。载脂蛋白A-2从高密度脂蛋白中取代载脂蛋白A-1的能力影响高密度脂蛋白的抗动脉粥样硬化功能。我们将结合远近紫外圆二色谱和荧光光谱、电子显微镜、差示扫描量热法和X射线衍射法，对脱脂和脂结合的人apoA-2、apoC-1和一系列针对关键结构区域的apoC-1突变体进行能量和结构分析。这种分析将在单个氨基酸水平上确定载脂蛋白C-1中关键结构元素对其折叠、稳定性和脂结合特性的作用；确定决定载脂蛋白稳定性的关键决定因素及其与脂蛋白代谢过程中载脂蛋白之间转移动力学的关系；结晶载脂蛋白C-1并分析其晶体结构，可能为功能性载脂蛋白构象提供模型。这些结果将为理解正常和疾病状态下脂蛋白作用的分子机制提供能量和结构基础。

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine in molecular detail the energetic-structure-function relationship in exchangeable apolipoproteins and lipoproteins, thereby providing an insight into molecular mechanisms of lipoprotein action in the pathogenesis of atherosclerosis and other lipoprotein-related diseases. Exchangeable apolipoproteins are soluble protein components of lipoproteins that mediate lipid and cholesterol transport and metabolism and play crucial roles in the pathogenesis of atherosclerosis, coronary heart disease, stroke and other major human disorders. Structural stability and compositional variability of lipoproteins are essential for their functions, and have to be understood in detail in order to elucidate molecular mechanisms of lipoprotein action in normal and in diseased states. The proposed work addresses this long-term goal through detailed studies of the energetics, structure and lipid binding function of two small human plasma apolipoproteins, apoC-1 and apoA-2. ApoC-1 delays the clearance of potentially atherogenic triglyceride-rich particles by inhibiting their uptake via the apoE-mediated low-density lipoprotein receptor-related pathway. The ability of apoC-1 to activate lecithin:cholesterol acyltransferase (LCAT) may account for normal plasma levels of cholesterol esters in subjects with deficiency of the major LCAT activator, apoA-1. ApoA-2 ability to displace apoA-1 from high-density lipoproteins (HDL) affects the antiatherogenic functions of HDL. Energetic and structural analyses of lipid-free and lipid-bound human apoA-2, apoC-1 and a series of apoC-1 mutants targeted towards key structural regions will be carried out by using a combination of far- and near-UV circular dichroism and fluorescence spectroscopy, electron microscopy, differential scanning calorimetry, and x-ray diffraction methods. Such analysis will determine, at the level of individual amino acids, the roles of key structural elements in apoC-1 for its folding, stability and lipid binding properties; identify critical determinants for the Lp stability and their relation to the kinetics of apolipoprotein transfer among lipoproteins in the course of lipoprotein metabolism; crystallize apoC-1 and analyze its crystal structure that may provide a model for a functional apolipoprotein conformation. The results will provide the energetic and structural basis for understanding molecular mechanisms of lipoprotein action in normal and diseased states.