Molecular Mechanism of the Blockade of Plasma Cholesteryl Ester Transfer Protein by Its Physiological Inhibitor Apolipoprotein CI

Abstract

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal ␣-helix with significant electrostratic properties, whereas its N-terminal ␣-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal ␣-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.The cholesteryl ester transfer protein (CETP) 4 mediates the exchange of neutral lipids, i.e. cholesteryl esters and triglycerides between plasma lipoproteins (1, 2). Through its action, CETP can influence the atherogenicity of the lipoprotein profile (2-4), and recent studies (5-7) support a potential interest in inhibiting CETP activity in vivo by means of either anti-CETP immunotherapy, antisense oligonucleotides, or specific pharmacological inhibitors. In particular, the latest pharmacological interventions with small anti-CETP molecules in human populations demonstrated that CETP inhibition markedly increases HDL cholesterol levels and also decreases low density lipoprotein (LDL) cholesterol levels (8 -10). Most interestingly, previous studies (11-13) in human populations are consistent with the association of high CETP with an increase in coronary heart disease, in particular in subjects with elevated triglycerides.Besides interventional studies with exogenous compounds, a number of studies (14 -19) indicated that plasma CETP activity can be modulated by endogenous factors, among them the apolipoprotein components of circulating lipoproteins. Recently, apolipoprotein CI, i.e. a 6.6-kDa HDL apolipoprotein, was identified as a potent CETP inhibitor (20). In contrast to other putative apolipoprotein candidates that were identified only through in vitro experiments, the ability of apoCI to decrease specific CETP activity was documented in vivo through studies in CETPTg/apoCI-KO and CETPTg/HuapoCITg (transgenic mouse to both human CETP and human apolipoprotein CI) mice (21, 22). Although it was ...

Apolipoprotein CI is a physiological regulator of cholesteryl ester transfer protein activity in human plasma but not in rabbit plasma

“…Either total plasma or purifi ed CETP was used as a source of cholesteryl ester transfer activity, with the interference or not of endogenous lipoproteins, as previously described ( 19,23,24,29 ). In the lipoproteinindependent assay, a source of CETP was incubated with labeled liposome donors and exogenous lipoprotein acceptors in excess.…”

“…For apolipoprotein analysis, delipidated HDL proteins were applied on 4-12% SDS-polyacrylamide gels, and the apparent molecular weights of protein bands were determined by reference to protein standards ( 23 ). For the determination of electrophoretic mobility, native HDLs were applied on 0.5% agarose gels, and surface potentials (mV) were calculated from electrophoretic velocity as previously described ( 23,30 ).…”

“…This effect of apoCI on VLDL clearance has been shown to be of physiological relevance in humans ( 20,21 ). On the other hand, purifi ed human apoCI is a potent inhibitor of cholesteryl ester transfer protein (CETP) activity when associated with HDL, and as such it has the ability to reduce the net mass transfer of cholesteryl esters from HDL toward VLDL ( 22,23 ). The ability of apoCI to decrease specifi c CETP activity in vivo has been documented in CETPTg/apoCI-KO and CETPTg/HuapoCITg mice ( 19,24 ).…”

“…When rabbits are fed a cholesterol-enriched diet, the plasma concentration of apoB100-containing VLDL is dramatically increased, and this is associated with a marked redistribution of cholesteryl esters from HDL toward VLDL and LDL fractions ( 25 ). More interestingly, lyzed on an Ultrafl ex II MALDI-time-of-fl ight (TOF)/TOF mass spectrometer (Bruker Daltonique, Strasbourg, France) in the positive 25 kV linear mode ( 23 ). Fragments of purifi ed apoCI (5 µg) were obtained by partial hydrolysis with 300 ng of trypsin (Roche Diagnostics, Mannheim, Germany) for 10 to 150 min at 37°C, and they were desalted on ZipTip microcolumns (ZipTip µC18; Millipore, Molsheim, France) prior to MALDI-TOF mass spectrometer analysis.…”

Control of cholesteryl ester transfer protein activity by sequestration of lipid transfer inhibitor protein in an inactive complex

“…Apolipoprotein C-I, which resides primarily on HDL, has been reported to inhibit CETP in vitro, and studies with transgenic animals have demonstrated its ability to suppress CETP activity in vivo (6,7). Its mode of action is via modification of the surface charge of HDL, resulting in weakened CETP-HDL interactions and thus suppression of lipid transfer events with HDL (8). A second regulatory protein is lipid transfer inhibitor protein (LTIP), also known as apolipoprotein F (9).…”