Project Summary/Abstract Cholesteryl ester transfer protein (CETP) mediates the transfer of lipids, including cholesteryl esters (CEs), triglycerides (TGs) and phospholipids (PLs) 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) and familial hyperlipidemia disease. Since increased CETP can reduce HDL-C concentration and since CETP deficiency is associated with elevated HDL-C levels, five CETP inhibitors, i.e. Torcetrapib, Anacetrapib, Dalcetrapib, Evacetrapib and Obicetrapib have been investigated in clinical trials for treating CVD. However, the fact that three inhibitors failed in large clinical trials reflects our knowledge is little concerning the molecular mechanisms of CETP- mediated lipid transfer among lipoproteins. In the last funding period, we discovered the CETP penetrated to HDL and LDL/VLDL, forming a tunnel for CE transfer between lipoproteins. In this period, we propose to test highlighted residues that play key roles in CE transfer. It can be difficult to investigate CETP mechanisms using structural methods as interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. Thus, we propose i) to study the mutated CETP in CE transfer by all-atom molecular dynamic (MD) simulation and our validated optimized negative-staining electron microscopy (OpNS-EM) protocol. In OpNS, the flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. ii) In the last period, we identified a CETP interface that binds to lipoprotein and patented antibodies as CETP inhibitors for treatment of CVD. In this period, we propose to validate the antibodies’ function in regulating plasma lipid levels in a rabbit dyslipidemia model. iii) To further understanding the CETP function in plasma phospholipid transfer, we propose to study the PLTP interaction with lipoprotein as PLTP has a similar sequence to CETP but a stronger function in phospholipid transfer among lipoproteins.