ABCA1 Transporter: In order to examine the necessary structural features for a protein to promote lipid efflux by the ABCA1 transporter, synthetic peptides were tested on ABCA1-transfected cells (ABCA1 cells) and on control cells. L-37pA, a 37 l amino acid peptide that contains two class-A amphipathic helices linked by proline, showed a 4-fold increase in cholesterol and phospholipid efflux from ABCA1 cells compared to control cells. The same peptide synthesized with a mixture of l and d amino acids was less effective than L-37pA in solubilizing dimyristoyl phosphatidyl choline vesicles and in effluxing cholesterol from ABCA1 transfected cells. In contrast, the 37pA peptide synthesized with all d amino acids (D-37pA) was as effective as L-37pA. Unlike apoA-I, L-37pA and D-37pA were also capable, although at a reduced rate, of facilitating cholesterol efflux independent of ABCA1 from control cells, Tangier disease cells, and paraformaldehyde fixed ABCA1 cells. The ability of peptides to bind to cells correlated with their lipid affinity. The combined results from these studies indicated that the amphipathic helix was the key structural motif for peptide-mediated lipid efflux from ABCA1, however there was no stereoselective requirement. In addition, unlike apoA-I, synthetic peptides can also efflux lipid by a passive, energy-independent pathway that does not involve ABCA1 but does depend upon their lipid affinity. ABCA1 Transporter: The ABCA1 transporter on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was ascertained by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. These findings are consistent with an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL. ABCG5-ABCG8: The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5?ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists however no classic LXRre was identified in the intergenic region. These combined results have identified several potential regulatory elements for modulation of the expression of the ABCG5 and ABCG8 genes, and the intergenic region was found to function act as a unique bidirectional promoter.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL002063-03
Application #
6817761
Study Section
(MDB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Wu, Justina E; Basso, Federica; Shamburek, Robert D et al. (2004) Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice. J Biol Chem 279:22913-25
Basso, Federica; Freeman, Lita; Knapper, Catherine L et al. (2003) Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations. J Lipid Res 44:296-302
Remaley, Alan T; Thomas, Fairwell; Stonik, John A et al. (2003) Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway. J Lipid Res 44:828-36
Brewer Jr, H Bryan; Santamarina-Fojo, Silvia (2003) Clinical significance of high-density lipoproteins and the development of atherosclerosis: focus on the role of the adenosine triphosphate-binding cassette protein A1 transporter. Am J Cardiol 92:10K-16K
Remaley, Alan T; Bark, Samantha; Walts, Avram D et al. (2002) Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster. Biochem Biophys Res Commun 295:276-82
Neufeld, Edward B; Demosky Jr, Steven J; Stonik, John A et al. (2002) The ABCA1 transporter functions on the basolateral surface of hepatocytes. Biochem Biophys Res Commun 297:974-9
Vaisman, B L; Lambert, G; Amar, M et al. (2001) ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice. J Clin Invest 108:303-9