Neutral Lipid Transfer Research Articles

Structural insights on the deformations induced by various mutations on cholesteryl ester transfer protein

Cholesteryl Ester Transfer Protein (CETP) is a plasma glycoprotein that intervenes the reverse cholesterol transport (RCT) by equimolar exchange of Cholesteryl esters (CE) and Triglycerides (TGs) between anti-atherogenic High-Density Lipoproteins (HDLs) and pro-atherogenic Low-Density Lipoproteins (LDLs) resulting in the increased concentration of CEs in LDL. This is a potential cause for the formation of atherosclerotic plaques in blood vessels leading to fatality. Therefore, blocking the function of CETP has emerged as a novel strategy for suppressing atherosclerotic plaques. The crystal structure of CETP revealed two Cholesteryl esters (CEs) in the hydrophobic tunnel and two phospholipids (PLs) plugged on the concave surface. Previous lipid transfer assay experimental studies have shown a substantial reduction in the neutral lipid transfer in [R201S] and [I443W, V198W] mutants. However, the protein conformational arrangements due to the mutations present in the CETP system leading to a decrease in the transfer rate of neutral lipids is not explored. Thus, I explored the reason behind the decreased transfer rate in mutants using molecular dynamics (MD) simulations and free energy calculations. Resulting evidences show that R201S mutant induces unfavorable bending angle to CETP with a decreased binding efficiency between N-terminal phospholipid of CETP with S201. Also, an unfavorable conformation state of TGs is formed which makes them difficult to transfer across CETP. Likewise, [I443W, V198W] mutant induces unfavorable CE, TG, and bending angle conformation to CETP impeding neutral lipid transfer. Thus, my results provide sufficient insights on the causation for a decreased transfer rate as reported earlier. The detailed understanding obtained here could help in developing a new strategy in preventing the function of CETP by blocking the role of potential hot spot residues.

Bound Phospholipids Assist Cholesteryl Ester Transfer in the Cholesteryl Ester Transfer Protein.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that assists the transfer of cholesteryl esters (CEs) from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs), initiating cholesterol plaques in the arteries. Consequently, inhibiting the activity of CETP is therefore being pursued as a novel strategy to reduce the risk of cardiovascular diseases (CVDs). The crystal structure of CETP has revealed the presence of two CEs running in the hydrophobic tunnel and two plugged-in phospholipids (PLs) near the concave surface. Other than previous animal models that rule out the PL transfer by CETP and PLs in providing the structural stability, the functional importance of bound phospholipids in CETP is not fully explored. Here, we employ a series of molecular dynamics (MD) simulations, steered molecular dynamics (SMD) simulations, and free energy calculations to unravel the effect of PLs on the functionality of the protein. Our results suggest that PLs play an important role in the transfer of neutral lipids by transforming the unfavorable bent conformation of CEs into a favorable linear conformation to facilitate the smooth transfer. The results also suggest that the making and breaking interactions of the hydrophobic tunnel residues with CEs with a combined effort from PLs are responsible for the transfer of CEs. Further, the findings demonstrate that the N-PL has a more pronounced effort on CE transfer than C-PL but efforts from both PLs are essential in the transfer. Thus, we propose that the functionally important PLs can be considered with potential research interest in targeting cardiovascular diseases.

The architecture of Cidec-mediated interfaces between lipid droplets.

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100μm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs.

Abstract 14235: Hepatocyte Tissue Plasminogen Activator Limits Very-Low-Density Lipoprotein Production via Inhibiting ApoB-Microsomal Transfer Protein Interaction and ApoB Lipidation

Lower plasma tissue plasminogen activator (tPA), a key fibrinolytic enzyme, is associated with higher atherogenic cholesterol levels in humans, but the mechanism is unknown. Hepatocyte (HC) is the factory producing atherogenic apoB lipoproteins, also a major source of basal plasma tPA. Our hypothesis is that tPA limits apoB-VLDL production in HCs.Silencing tPA in HCs using AAV8-H1-sh-tPA in Ldlr -/- mice leads to increased plasma cholesterol, triglyceride, apoB, and exacerbated atherosclerosis compared with scramble-silenced controls. Fractionation of plasma lipoproteins by FPLC or ultracentrifugation shows increased cholesterol and apoB in VLDL and LDL fractions. Inhibiting VLDL hydrolysis by P407 leads to a faster plasma triglyceride-rising rate in HC-tPA-silenced wild type mice, suggesting higher hepatic VLDL production. In a pulse-chase assay, tPA-silenced human primary HC has higher apoB-associated radioactivity in cell medium and lysate, suggesting increased apoB production.Adequate apoB lipidation is required for its secretion. By electronic microscopy, the distribution of VLDL shifts to a larger diameter in plasma VLDL isolated from HC-tPA-silenced Ldlr -/- mice, suggesting higher lipid contents in VLDL. The endoplasmic reticulum (ER)-associated apoB is higher in tPA-silenced vs. control HCs, more prominent in the less denser density fractions. As density and lipidation are inversely related, these findings are consistent with the hypothesis that tPA limits apoB lipidation in ER. MTP is an ER chaperone incorporating neutral lipids onto apoB in HC. The tPA-silenced human primary HC has higher neutral lipid transfer activity than controls, despite similar MTP protein levels. Moreover, higher apoB is in the anti-MTP-precipitations from tPA-silenced vs. control HCs, suggesting that silencing tPA increases apoB-MTP interaction. Proximity ligation and immunoprecipitation assays show tPA interacts with apoB in HC. Solid-phase binding assay and native gel reveal purified human tPA binds to LDL, but not to MTP. tPA competes with MTP in binding to LDL. Moreover, tPA inhibits MTP-mediated neutral lipid transfer. These findings suggested tPA binds to apoB and inhibits MTP-dependent apoB lipidation and VLDL secretion.

Cargo Receptor-Mediated ER Export in Lipoprotein Secretion and Lipid Homeostasis.

APOB-containing lipoproteins are large, complex lipid carriers that ferry bulk lipids into the circulation via the secretory pathway, originating from the endoplasmic reticulum of specialized cells in the liver or the gut. Elevation of APOB-containing lipoproteins in the plasma represents a major risk factor for cardiovascular diseases. The production of these lipoproteins requires enzyme-catalyzed, cross-membrane transfer of neutral lipids and phospholipids to lipoproteins, in particular onto the structural component APOB. Transport of these lipid-bearing cargos relies on the COPII machinery and employs the transmembrane cargo receptor SURF4 and the small GTPase SAR1B, together constituting a selective transport program. Intriguingly, a number of factors implicated in lipoprotein production are also packaged into COPII vesicles and may be cotransported with APOB. These observations therefore point to a specialized produce-and-export itinerary during the secretion of these lipid-bearing cargos, warranting future investigations into this unique yet pivotal process at the crossroad of cell biology and physiology.

Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases.

Microsomal triglyceride transfer protein (MTP) was first identified as an endoplasmic reticulum (ER) resident protein that helps in the transfer of neutral lipids to nascent apolipoprotein B (apoB). Its critical role in the assembly and secretion of apoB-containing lipoproteins was identified in abetalipoproteinemia patients who have mutations in MTP and completely lack apoB-containing lipoproteins in the circulation. It has been established now that MTP not only is involved in the transfer of neutral lipids but also plays a role in cholesterol ester and cluster of differentiation 1d (CD1d) biosynthesis. Besides neutral lipids, MTP may also help in the transfer of sphingolipids such as ceramides and sphingomyelin to the apoB-containing lipoproteins. MTP is a multifunctional protein, and its deregulation during pathophysiological conditions gives rise to different metabolic conditions. This book chapter discusses the physiological role and regulation of MTP to maintain the homeostasis of lipids and lipoproteins. It also reviews the regulation of MTP during certain pathophysiological conditions and provides a brief overview of therapeutic interventions that can be possibly used to target its activity or expression to alleviate some of these metabolic diseases.

Steering the Lipid Transfer To Unravel the Mechanism of Cholesteryl Ester Transfer Protein Inhibition.

Human plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron microscopy studies have suggested that CETP penetrates its N- and C-terminal domains in HDL and LDL to form a ternary complex, which facilitates the lipid transfer between different lipoproteins. Inhibition of CETP lipid transfer activity has been shown to increase the plasma HDL-C levels and, therefore, became an effective strategy for combating cardiovascular diseases. Thus, understanding the molecular mechanism of inhibition of lipid transfer through CETP is of paramount importance. Recently reported inhibitors, torcetrapib and anacetrapib, exhibited low potency in addition to severe side effects, which essentially demanded a thorough knowledge of the inhibition mechanism. Here, we employ steered molecular dynamics simulations to understand how inhibitors interfere with the neutral lipid transfer mechanism of CETP. Our study revealed that inhibitors physically occlude the tunnel posing a high energy barrier for lipid transfer. In addition, inhibitors bring about the conformational changes in CETP that hamper CE passage and expose protein residues that disrupt the optimal hydrophobicity of the CE transfer path. The atomic level details presented here could accelerate the designing of safe and efficacious CETP inhibitors.

The crystal structure of human microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the "substrate-bound" and "free" states rather than differences arising from redox state.

Structural Plasticity of Cholesteryl Ester Transfer Protein Assists the Lipid Transfer Activity

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CEs) and triglycerides between different lipoproteins. Recent studies have shown that blocking the function of CETP can increase the level of HDL cholesterol in blood plasma and suppress the risk of cardiovascular disease. Hence, understanding the structure, dynamics, and mechanism by which CETP transfers the neutral lipids has received tremendous attention in last decade. Although the recent crystal structure has provided direct evidence of the existence of strongly bound CEs in the CETP core, very little is known about the mechanism of CE/triglyceride transfer by CETP. In this study, we explore the large scale dynamics of CETP by means of multimicrosecond molecular dynamics simulations and normal mode analysis, which provided a wealth of detailed information about the lipid transfer mechanism of CETP. Results show that the bound CEs intraconvert between bent and linear conformations in the CETP core tunnel as a consequence of the high degree of conformational flexibility of the protein. During the conformational switching, there occurred a significant reduction in hydrophobic contacts between the CEs and CETP, and a continuous tunnel traversing across the CETP long axis appeared spontaneously. Thus, our results support the recently proposed "tunnel mechanism" of CETP from cryo-EM studies for the transfer of neutral lipids between different lipoproteins. The detailed understanding obtained here could help in devising methods to prevent CETP function as a cardiovascular disease therapeutic.

Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP

Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies.

How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.

Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D470N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D470N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of amyloid fibrils in the highly flexible C-terminus domain of CETP.

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-(13)C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.

Crystal Structures of Cholesteryl Ester Transfer Protein in Complex with Inhibitors

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.

Abstract 46: Cholesteryl Ester Transfer Protein Interactions with Lipoproteins: Insights into Mechanisms by Electron Microscopy and Molecular Dynamics Simulation

Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between HDL, LDL and VLDL. Lipoprotein particles contain a neutral lipid core composed of CE and TG surrounded by a surface monolayer of phospholipids (PL), free cholesterol (FC), and apolipoproteins, most notably, apo B-100 in LDL and VLDL and apo A-I in HDL. 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 CETP inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. CETP is a hydrophobic glycoprotein of 476 amino acids (∼53 kDa, before posttranslational modification). Its crystal structure reveals a banana-shaped molecule with N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60 Å-long hydrophobic central cavity. Three CETP neutral lipid transfer hypotheses were proposed more than two decades ago: 1) a shuttle mechanism that involves CETP collecting CEs from one lipoprotein and delivering them through the aqueous phase to another lipoprotein; 2) a tunnel mechanism in which CETP bridges two lipoproteins forming a ternary complex, with lipids flowing from the donor to acceptor lipoprotein through the CETP molecule; and 3) a modified tunnel mechanism implicating a CETP dimer. One difficulty in investigating CETP mechanisms using structural methods is that interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We validated an 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 applied this protocol to study the mechanisms by which CETP interacts with spherical HDL, LDL and VLDL. Three-dimensional (3D) reconstructions of CETP, free and HDL-bound, were obtained by single-particle techniques. In addition, we used inhibitory CETP antibodies to identify the regions of CETP that interact with HDL and LDL. Finally molecular dynamics (MD) simulation was used to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. We discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into HDL and its C-terminal domain interacting with LDL or VLDL. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

CETP Inhibition: Does the Future Look Promising?

Based on epidemiologic studies conducted throughout the world, it is established that there is an inverse relationship between high-density lipoprotein cholesterol (HDL-C) and risk for coronary artery disease (CAD). The incidence of low HDL-C is high and increasing throughout the world. A variety of pharmacologic approaches are being developed to therapeutically modulate serum levels of HDL-C. One controversial approach to this is the use of molecules that inhibit the activity of cholesteryl ester transfer protein (CETP), an enzyme involved in neutral lipid transfer between lipoproteins. The inhibition of CETP can lead to substantial elevations in HDL-C. Based on a number of considerations, including the complex relationship between loss of function mutations in CETP and risk for CAD and the clinical experience with torcetrapib, it is difficult to predict if CETP inhibition will be associated with reductions in rates of atherosclerosis disease progression and risk for cardiovascular events.

Structural and biophysical insight into cholesteryl ester-transfer protein

CETP (cholesteryl ester-transfer protein) is essential for neutral lipid transfer between HDL (high-density lipoprotein) and LDL (low-density lipoprotein) and plays a critical role in the reverse cholesterol transfer pathway. In clinical trials, CETP inhibitors increase HDL levels and reduce LDL levels, and therefore may be used as a potential treatment for atherosclerosis. In this review, we cover the analysis of CETP structure and provide insights into CETP-mediated lipid transfer based on a collection of structural and biophysical data.

La elevación del colesterol unido a lipoproteínas de alta densidad: perspectiva futura. La CETP como diana terapéutica

Cholesteryl ester transfer protein (CETP) facilitates transfer of neutral lipids between lipoprotein classes. CETP plays a key role in cholesterol homeostasis, especially in the redistribution of cholesterol among particles and in reverse cholesterol transport. Consequently, modifying the activity of this protein could influence the development and progression of atheromatosis. The effects of inhibiting CETP include reductions in lowdensity lipoprotein (LDL)-cholesterol and cholesterol in triglyceride-rich particles and increases in high-density lipoprotein (HDL)-cholesterol, apolipoprotein A-1 and the size of LDL and HDL particles. The induction of expression of CETP activity in mice increases susceptibility to atherosclerosis, while inhibition of CETP activity in rabbits tends to reduce its development. However, the role of CETP inhibition in atheromatosis in humans remains to be established.

Cholesteryl Ester Transfer Protein Penetrates Lipoproteins For Cholesteryl Ester Transfer

Human cholesteryl ester transfer protein (CETP), a hydrophobic glycoprotein (476 amino acids, ∼74 kDa), mediates the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to apo-B containing lipoproteins (low-density lipoproteins - LDL; very low density lipoproteins - VLDL). The crystal structure of CETP reveals a banana-shaped protein consisting of N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60-A-long hydrophobic cavity. Since CETP deficiency is associated with elevated ‘good-cholesterol’ (HDL-associated) levels via delayed catabolism, as a result, CETP has become a high interest pharmacological drug target.Here, we investigated the mechanism of CETP-mediated neutral lipid transfer by electron microscopy (EM), image-processing, and molecular dynamic simulations (MDS). We discovered that: 1) CETP bridges HDL and LDL/VLDL to form a ternary complex; 2) the CETP C-terminal beta-barrel domain penetrates partially into the LDL/VLDL surface; 3) the N-terminal beta-barrel domain penetrates through the HDL surface and interacts with the CE core; and 4) MDS analysis suggested that the penetrating regions of CETP are highly mobile and form pores that connect to cavities running through the central axis of the CETP molecule to generate a tunnel. The tunnel was mainly hydrophobic and connected the distal ends through its central cavities. Thus, we proposed CETP resembles a Chinese finger trap, in which both beta-barrel domains penetrate the lipoprotein surface, resulting in pressure from both ends. The pressure may result in the anti-twisting of the two domains, leading to opening of the tunnel for CE transfer between HDL and LDL. The distal end of N-terminal beta-barrel domain could be particularly important for the design of pharmacological inhibitors, as blockage of the N-terminal beta-barrel domain would prevent CE transfer with a low probability of off target effects on normal HDL metabolism.