Cholesterol Metabolism Related Protein Structure and Function by Electron Microsc

描述(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(CES)和甘油三酯(TGS)在 高密度脂蛋白(高密度脂蛋白)、低密度脂蛋白(低密度脂蛋白)和极低密度脂蛋白(VLDL)。血浆低密度脂蛋白-胆固醇(LDL-C)水平升高和/或高密度脂蛋白-胆固醇(HDL-C)水平低是心血管疾病(CVD)的主要危险因素。由于CETP升高可降低高密度脂蛋白胆固醇浓度，而CETP缺乏则与高密度脂蛋白胆固醇水平升高相关，因此CETP抑制剂，包括torcetRapib、anacetRapib和dalcetRapib已被用于治疗CVD的临床试验中。尽管CET抑制在临床上引起了浓厚的兴趣，但关于CETP介导的脂蛋白之间的脂转移的分子机制，甚至CETP如何与脂蛋白相互作用，人们还知之甚少。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的大小、形状和组成，特别是高密度脂蛋白。我们建议使用我们经过验证的优化负染电子显微镜(NS-EM)方案，在该方案中，脂蛋白颗粒的闪光固定保留了近自然状态的构象，以便直接显示单个分子或大分子颗粒。我们还使用我们的“计算凝胶过滤”算法来选择同质的一个亚群的高密度脂蛋白颗粒进行单粒子重建。结合CETP抗体，我们建议确定CETP与高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白相互作用的区域，以进一步研究CETP与人血浆脂蛋白相互作用的机制。CETP、高密度脂蛋白、CETP-高密度脂蛋白复合体和CETP-低密度脂蛋白复合体的三维(3D)重建将通过单粒子技术获得。此外，我们还建议研究CETP抑制剂如何影响CETP的结构和功能。最后，分子动力学(MD)模拟被用来评估CETP的分子流动性，并预测与脂质转移相关的可能的构象变化。提出了三个具体的目标：1)检测CETP与脂蛋白结合的结构和形态；2)用免疫电子显微镜和分子动力学模拟测试CETP的隧道机制；3)CETP抑制剂对CETP构象和CETP在不同脂蛋白之间转移功能的影响：

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: