High density lipoprotein (HDL) is the major atheroprotective lipoprotein of human plasma. Successful formation of high density lipoprotein (HDL) from its major protein, apolipoprotein A-1 (apo A-1) depends on an initial, energy-dependent transfer of phospholipid (PL) from the cell surface which is catalyzed by the ATP-binding cassette-1 (ABC 1) transporter protein. PL transfer precedes FC transfer to apo A-1. In the absence of ABC-1 activity (as in human Tangier Disease) normal HDL are completely absent, despite the ability of PL to bind spontaneously to apo A-1 in vitro. Here the hypothesis is tested that ABC 1 modifies the conformation of apo A-1 binding to the cell surface to promote contact between PL and one or more specific PL-binding targets within its amino acid sequence. These contacts prime the nascent complex for the transfer of additional PL, and facilitate the efflux of cell surface FC. This is subsequently stabilized by direct FC-apo A-1 binding. In this Project, the binding sites of PL and FC to apo A- I in lipid-poor HDL formed at the surface of vascular cells will be characterized from the lipid-protein crosslinks generated by photoactivation from FC and PL analogs containing benzophenone groups at different points in their structure. Lipid-protein crosslinks will localized by protease digestion and HPLC/mass spectrometry. Lipid/lipid crosslinks will be identified by HPLC and TLC. The data obtained will be extended and confirmed using apo A-1 proteins from which individual PL and FC binding sites have been deleted by site- directed mutagenesis. Finally this project will identify the point at which nascent HDL become direct substrates for lecithin: cholesterol acyltransferase and cholesterol ester transfer protein, when cell-derived PL and FC are metabolized and appear in mature acceptor lipoproteins. These analyses, carried out at intervals during the initial lipidation of apo A- 1, will provide the most detailed picture available of the genesis of HDL at the surface of peripheral cells. Novel information on the spatial organization of FC and PL in lipoprotein complexes will be obtained, highly relevant to the molecular analysis of human HDL deficiency.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Program Project Review Committee (HLBP)
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University of California San Francisco
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Chau, Phuonglan; Fielding, Phoebe E; Fielding, Christopher J (2007) Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding. Biochemistry 46:8445-50
Spencer, Thomas A; Wang, Pingzhen; Popovici-Muller, Janeta V et al. (2006) Preparation and biochemical evaluation of fluorenone-containing lipid analogs. Bioorg Med Chem Lett 16:3000-4
Chau, Phuonglan; Nakamura, Yasushi; Fielding, Christopher J et al. (2006) Mechanism of prebeta-HDL formation and activation. Biochemistry 45:3981-7
Gan, Yonghong; Spencer, Thomas A (2006) Cholesterol surrogates incorporating a benzophenone as part of the sterol tetracycle. J Org Chem 71:5870-5
Gan, Yonghong; Wang, Pingzhen; Spencer, Thomas A (2006) Synthesis of benzophenone-containing fatty acids. J Org Chem 71:9487-90
Huuskonen, Jarkko; Vishnu, Meeta; Fielding, Phoebe E et al. (2005) Activation of ATP-binding cassette transporter A1 transcription by chromatin remodeling complex. Arterioscler Thromb Vasc Biol 25:1180-5
Huuskonen, Jarkko; Vishnu, Meeta; Pullinger, Clive R et al. (2004) Regulation of ATP-binding cassette transporter A1 transcription by thyroid hormone receptor. Biochemistry 43:1626-32
Nakamura, Yasushi; Kotite, Leila; Gan, Yonghong et al. (2004) Molecular mechanism of reverse cholesterol transport: reaction of pre-beta-migrating high-density lipoprotein with plasma lecithin/cholesterol acyltransferase. Biochemistry 43:14811-20
Huuskonen, Jarkko; Fielding, Phoebe E; Fielding, Christopher J (2004) Role of p160 coactivator complex in the activation of liver X receptor. Arterioscler Thromb Vasc Biol 24:703-8
Fielding, Phoebe E; Chau, Phuonglan; Liu, Dong et al. (2004) Mechanism of platelet-derived growth factor-dependent caveolin-1 phosphorylation: relationship to sterol binding and the role of serine-80. Biochemistry 43:2578-86

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