Cholesterol Metabolism Related Protein Structure and Function by Electron Microsc

描述（由申请人提供）：胆固醇酯转移蛋白（CETP）介导中性脂质（包括胆固醇酯（CE）和甘油三酯（TG））在 高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）。人血浆中LDL-胆固醇（LDL-C）水平升高和/或HDL-胆固醇（HDL-C）水平降低是心血管疾病（CVD）的主要风险因素。由于增加的CETP可以降低HDL-C浓度，并且CETP缺乏与升高的HDL-C水平相关，因此已经在临床试验中研究了CETP抑制剂，包括torcetrapib、anacetrapib和dalcetrapib，用于治疗CVD。尽管临床上对CET抑制有着浓厚的兴趣，但关于CETP介导的脂蛋白间脂质转移的分子机制，甚至CETP如何与脂蛋白相互作用，我们知之甚少。使用结构方法研究CETP机制的困难在于与CETP的相互作用可以改变脂蛋白，特别是HDL的大小，形状和组成。我们建议使用我们验证的优化负染电子显微镜（NS-EM）协议，其中脂蛋白颗粒的闪光固定保留了接近天然状态的构象，用于直接可视化单个分子或大分子颗粒。我们还使用我们的“计算凝胶过滤”算法来选择同质的HDL粒子的亚群进行单粒子重建。结合CETP抗体，我们拟鉴定CETP与HDL、LDL和VLDL相互作用的区域，以进一步研究CETP与人血浆脂蛋白相互作用的机制。将通过单粒子技术获得CETP、HDL、CETP-HDL复合物和CETP-LDL复合物的三维（3D）重建。此外，我们建议研究CETP抑制剂如何影响CETP的结构和功能。最后，分子动力学（MD）模拟提出了评估的分子流动性CETP和预测可能的构象变化，与脂质转移。提出了三个具体目标：1）检查与脂蛋白结合的CETP的结构和形态，2）通过免疫电子显微镜和分子动力学模拟测试CETP隧道机制，3）CETP抑制剂对CETP构象和在各种脂蛋白之间CE转移中的功能的影响：

DESCRIPTION (provided by applicant): Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL). An elevated level of LDL-cholesterol (LDL-C) and/or a low level of HDL-cholesterol (HDL-C) in human plasma are major risk factors for cardiovascular disease (CVD). Since increased CETP can reduce HDL-C concentration and CETP deficiency is associated with elevated HDL-C levels, CETP inhibitors, including torcetrapib, anacetrapib and dalcetrapib have been investigated in clinical trials for treating CVD. Despite the intense clinical interest in CET inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. Difficulty while investigating CETP mechanisms using structural methods is interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We propose to use our validated optimized negative-staining electron microscopy (NS-EM) protocol in which flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. We also use our "computational gel-filtration" algorithms to select homogenous a subpopulation of HDL particles for single-particle reconstruction. Associating with CETP antibodies, we propose to identify the regions of CETP that interact with HDL, LDL, and VLDL, to further study the mechanisms by which CETP interacts with human plasma lipoproteins. Three-dimensional (3D) reconstructions of CETP, HDL, the CETP-HDL complex and the CETP-LDL complex will be obtained by single-particle techniques. In addition, we propose to investigate how CETP inhibitors affect CETP structure and function. Finally molecular dynamics (MD) simulation is proposed to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. Three specific aims are proposed: 1) Examine the structure and morphology of CETP bound to lipoprotein, 2) Test the CETP tunnel mechanism by immuno-electron microscopy and molecular dynamic simulation, 3) Effect of CETP Inhibitors on CETP conformation and function in CE transfer among various lipoproteins: