Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability-increasing protein share a similar tertiary structure.
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Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central beta-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxy-terminal beta-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies. (+info)
Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice.
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Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport. (+info)
Low density lipoproteins develop resistance to oxidative modification due to inhibition of cholesteryl ester transfer protein by a monoclonal antibody.
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Although numerous studies have investigated the relationship between cholesteryl ester transfer protein (CETP) and high density lipoprotein (HDL) remodeling, the relationship between CETP and low density lipoproteins (LDL) is still not fully understood. In the present study, we examined the effect of the inhibition of CETP on both LDL oxidation and the uptake of the oxidized LDL, which were made from LDL under condition of CETP inhibition, by macrophages using a monoclonal antibody (mAb) to CETP in incubated plasma. The 6-h incubation of plasma derived from healthy, fasting human subjects led to the transfer of cholesteryl ester (CE) from HDL to VLDL and LDL, and of triglycerides (TG) from VLDL to HDL and LDL. These net mass transfers of neutral lipids among the lipoproteins were eliminated by the mAb. The incubation of plasma either with or without the mAb did not affect the phospholipid compositions in any lipoproteins. As a result, the LDL fractionated from the plasma incubated with the mAb contained significantly less CE and TG in comparison to the LDL fractionated from the plasma incubated without the mAb. The percentage of fatty acid composition of LDL did not differ among the unincubated plasma, the plasma incubated with the mAb, and that incubated without the mAb. When LDL were oxidized with CuSO4, the LDL fractionated from the plasma incubated with the mAb were significantly resistant to the oxidative modification determined by measuring the amount of TBARS and by continuously monitoring the formation of the conjugated dienes, in comparison to the LDL fractionated from the plasma incubated without the mAb. The accumulation of cholesteryl ester of oxidized LDL, which had been oxidized for 2 h with CuSO4, in J774.1 cells also decreased significantly in the LDL fractionated from the plasma incubated with mAb in comparison to the LDL fractionated from the plasma incubated without the mAb. These results indicate that CETP inhibition reduces the composition of CE and TG in LDL and makes the LDL resistant to oxidation. In addition, the uptake of the oxidized LDL, which was made from the LDL under condition of CETP inhibition, by macrophages also decreased. (+info)
Action of atorvastatin in combined hyperlipidemia : preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles.
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Combined hyperlipidemia (CHL) is characterized by a concomitant elevation of plasma levels of triglyceride-rich, very low density lipoproteins (VLDLs) and cholesterol-rich, low density lipoproteins (LDLs). The predominance of small, dense LDLs contributes significantly to the premature development of coronary artery disease in patients with this atherogenic dyslipoproteinemia. In the present study, we evaluated the impact of atorvastatin, a newly developed inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, on the cholesteryl ester transfer protein (CETP)-mediated remodeling of apolipoprotein (apo) B-containing lipoprotein subspecies, and more specifically, the particle subpopulations of VLDL and LDL in CHL. In parallel, we evaluated the atorvastatin-induced modulation of the quantitative and qualitative features of atherogenic apo B-containing and cardioprotective apo AI-containing lipoprotein subspecies. Atorvastatin therapy (10 mg/d for a 6-week period) in patients with a lipid phenotype typical of CHL (n=18) induced reductions of 31% (P<0.0001) and 36% (P<0.0001) in plasma total cholesterol and LDL cholesterol, respectively. In addition, atorvastatin significantly reduced VLDL cholesterol, triglycerides, and apo B levels by 43% (P<0.0001), 27% (P=0.0006), and 31% (P<0.0001), respectively. The plasma concentrations of triglyceride-rich lipoproteins (VLDL1, Sf 60 to 400; VLDL2, Sf 20 to 60; and intermediate density lipoproteins, Sf 12 to 20) and of LDL, as determined by chemical analysis, were markedly diminished after drug therapy (-30% and -28%, respectively; P<0.0007). Atorvastatin significantly reduced circulating levels of all major LDL subspecies, ie, light (-28%, P<0.0008), intermediate (-27%, P<0.0008), and dense (-32%, P<0.0008) LDL; moreover, in terms of absolute lipoprotein mass, the reduction in dense LDL levels (mean -62 mg/dL) was preponderant. In addition, the reduction in plasma dense LDL concentration after therapy was significantly correlated with a reduction in plasma VLDL1 levels (r=0.429, P=0.0218). Atorvastatin induced a significant reduction (-7%, P=0.0039) in total CETP-dependent CET activity, which accurately reflects a reduction in plasma CETP mass concentration. Total CETP-mediated CET from high density lipoproteins to apo B-containing lipoproteins was significantly reduced (-26%, P<0.0001) with drug therapy. Furthermore, CETP activity was significantly correlated with the atorvastatin-induced reduction in plasma VLDL1 levels (r=0.456, P=0. 0138). Indeed, atorvastatin significantly and preferentially decreased CET from HDL to the VLDL1 subfraction (-37%, P=0.0064), thereby reducing both the levels (-37%, P=0.0001) and the CE content (-20%, P<0.005) of VLDL1. We interpret our data to indicate that 2 independent but complementary mechanisms may be operative in the atorvastatin-induced reduction of atherogenic LDL levels in CHL: first, a significant degree of normalization of both the circulating levels and the quality of their key precursors, ie, VLDL1, and second, enhanced catabolism of the major LDL particle subclasses (ie, light, intermediate, and dense LDL) due to upregulation of hepatic LDL receptors. (+info)
New functional promoter polymorphism, CETP/-629, in cholesteryl ester transfer protein (CETP) gene related to CETP mass and high density lipoprotein cholesterol levels: role of Sp1/Sp3 in transcriptional regulation.
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A new polymorphism located at position -629 (CETP/-629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The -629A allele was associated with lower CETP mass (P<0. 0001) and higher high density lipoprotein cholesterol (P<0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position -629 showed significantly lower luciferase activity with the promoter fragment of the A allele (-25%, P<0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the -629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%, P<0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression. (+info)
Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element.
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The cholesterol ester transfer protein (CETP) facilitates the transfer of HDL cholesterol esters from plasma to the liver. Transgenic mice expressing human CETP, controlled by its natural flanking region, increase expression of this gene in response to hypercholesterolemia. We established a CETP promoter-luciferase reporter assay in differentiated 3T3-L1 adipocytes to map the sterol upregulatory element. Promoter mutagenesis suggested that a direct repeat of a nuclear receptor binding sequence separated by 4 nucleotides (DR4 element, -384 to -399) was responsible for this activity. Using mice carrying normal or mutated promoter sequences, we confirmed the importance of this element for gene induction by dietary sterol. A gel retardation complex containing LXR/RXR was identified using the CETP DR4 element and adipocyte nuclear extracts. Both LXRalpha/RXRalpha and LXRbeta/RXRalpha transactivated the CETP promoter via its DR4 element in a sterol-responsive fashion. Thus, the positive sterol response of the CETP gene is mediated by a nuclear receptor binding site that is activated by LXRs. That Cyp7a, the rate-limiting enzyme for conversion of cholesterol into bile acids in the liver, is also regulated by LXRalpha suggests that this class of nuclear receptor coordinates the regulation of HDL cholesterol ester catabolism and bile acid synthesis in the liver. (+info)
Ferroverdins, inhibitors of cholesteryl ester transfer protein produced by Streptomyces sp. WK-5344. I. Production, isolation and biological properties.
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Streptomyces sp. WK-5344, a soil isolate, was found to produce structurally related inhibitors of cholesteryl ester transfer protein (CETP). New active compounds, designated ferroverdins B and C, were isolated along with known ferroverdin A from the fermentation broth by solvent extraction, ODS column chromatography and silica gel column chromatography. All ferroverdins showed a dose-dependent inhibitory activity against human CETP. The IC50 values were 21, 0.62 and 2.2 microM for ferroverdins A, B and C, respectively, indicating that ferroverdin B is one of the most potent CETP inhibitors of microbial origin. (+info)
Ferroverdins, inhibitors of cholesteryl ester transfer protein produced by Streptomyces sp. WK-5344. II. Structure elucidation.
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The structures of ferroverdins B and C, novel inhibitors of cholesteryl ester transfer protein, were elucidated by spectroscopic studies including various NMR measurements. They are the complex between one Fe2+ and three ligands, that is, two common p-vinylphenyl-3-nitroso-4-hydroxybenzoates and one hydroxy p-vinylphenyl-3-nitroso-4-hydroxybenzoate for ferroverdin B and one carboxylic acid p-vinylphenyl-3-nitroso-4-hydroxybenzoate for ferroverdin C. (+info)