Cholesterol Metabolism Related Protein Structure and Function by Electron Microsc

电子显微镜研究胆固醇代谢相关蛋白质结构和功能

• 批准号: 8515519
• 负责人: Gang Ren
• 金额: $ 35.88万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-23 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515519
• 关键词: Affect; Algorithms; Antibodies; Arts; Binding; Biochemical; C-terminal; Cardiovascular Diseases; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Clinical; Clinical Trials; Complex; Computer Simulation; Cryoelectron Microscopy; Data; Drug Design; Electron Microscopy; Electrons; Gel Chromatography; Heterogeneity; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Human; Image Analysis; Imagery; Immunoelectron Microscopy; In Vitro; Individual; Investigation; LDL Cholesterol Lipoproteins; Length; Lipids; Lipoproteins; Low-Density Lipoproteins; Measurement; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Morphology; Nature; Negative Staining; Pharmaceutical Preparations; Phase III Clinical Trials; Plasma; Plasma Proteins; Process; Protein Binding; Protein Conformation; Protein Deficiency; Proteins; Protocols documentation; Risk Factors; Shapes; Structure; Techniques; Testing; Time; Triglycerides; Very low density lipoprotein; base; design; drug development; electron tomography; flexibility; human CETP protein; in vivo; inhibitor/antagonist; interest; molecular dynamics; particle; protein structure function; reconstruction; sample fixation; torcetrapib

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:

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））之间的转移 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增加可以降低HDL-C浓度，而CETP缺乏与HDL-C水平升高有关，因此CETP抑制剂，包括torcetrapib、anacetrapib和dalcetrapib，已在治疗CVD的临床试验中进行了研究。尽管临床上对 CET 抑制有浓厚的兴趣，但关于 CETP 介导的脂蛋白之间脂质转移的分子机制，甚至 CETP 如何与脂蛋白相互作用，人们知之甚少。使用结构方法研究 CETP 机制时的困难在于与 CETP 的相互作用可以改变脂蛋白（尤其是 HDL）的大小、形状和组成。我们建议使用经过验证的优化负染色电子显微镜 (NS-EM) 方案，其中脂蛋白颗粒的快速固定保留了接近天然状态的构象，以便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 颗粒子群进行单颗粒重建。结合CETP抗体，我们建议鉴定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。 CETP、HDL、CETP-HDL 复合物和 CETP-LDL 复合物的三维 (3D) 重建将通过单粒子技术获得。此外，我们建议研究CETP抑制剂如何影响CETP结构和功能。最后，提出分子动力学（MD）模拟来评估CETP的分子迁移率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抑制剂对各种脂蛋白之间CE转移中CETP构象和功能的影响：