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）介导中性脂质的转移，包括胆固醇酯（CEs）和甘油三酯（tg）

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: