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Apolipoprotein A1 (apo A1), the major protein of high-density lipoprotein, is secreted as a proprotein and then cleaved by an uncharacterized metalloproteinase. Here this enzyme is identified as C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP-1). Studies with recombinant BMP-1, BMP-1 antibody, and BMP-1 siRNA establish this proteinase as the major or only apo A1-converting activity secreted by human liver-derived (HepG2) cells and CHO cells stably expressing human apo A1. BMP-1 stimulates the conversion of newly secreted proapo Alto its phospholipid- (PL-) binding form. In this way it promotes formation of functional HDL and reverse cholesterol transport, while inhibiting filtration and clearance of uncleaved proprotein. α2-Macroglobulin, a protease inhibitor secreted as part of the innate immune response, inhibits BMP-1 activity and blocks the maturation of proapo A1. The decrease in circulating apo A1 levels that is characteristic of the response to inflammation and infection may be mediated, at least in part, via BMP-1 by this novel mechanism. [ABSTRACT FROM AUTHOR]

In human vascular smooth muscle cells, inhibitors of protein kinase C activity reduced serine phosphorylation of caveolin-1 and increased sterol binding by this protein. This was measured after immunoprecipitation of caveolin-1 from cells labeled with tritiated cholesterol or the photoactivable cholesterol analogue FCBP [Fielding et al. (2002) Biochemistry 41, 4929-4937]. At the same time cellular sterol efflux was inhibited. Mutagenesis within a caveolin-1 central domain (residues 80-104) suggested a major role for serine-80 in mediating both of these effects. To perturb sterol binding, platelet-derived growth factor was added to the cells, leading to a transient loss of caveolin-1-associated sterol. Under these conditions, sterol efflux was stimulated, and caveolin-1 phosphorylation at tyrosine14, assayed with a selective antibody, was substantially increased above baseline levels. These changes were also blocked by inhibitors of protein kinase C activity. Selective inhibitors of the platelet-derived growth factor receptor and downstream kinases were used to show that loss of sterol from caveolin-1 preceded tyrosine phosphorylation, but relipidation was dependent on phosphotyrosine hydrolysis. [ABSTRACT FROM AUTHOR]

Describes a two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1. Concentration dependence of inhibition of phospholipid efflux by glyburide and vanadate; Transfer of phospholipids from human skin fibroblasts to extracellular medium in the presence or absence of apolipoprotein A-1.

Studies the kinetics of transport of low-density lipoprotein (LDL)-derived 3H-free cholesterol (FC) within the cell. Preparation of 3H-FC-labeled LDL; Cell culture; Uptake of FC from LDL; Transfer of FC between intracellular compartments; Transfer of intracellular FC to the cell surface.

Presents a study where a fibroblast caveolar membrane fraction was isolated by sucrose density gradient ultracentrifugation and its identity confirmed by protein markers. Skin fibroblast monolayers culture; Effects of cholesterol transfer from low density lipoprotein on caveolar cholesterol; Impact of metabolic inhibitors on FC influx and efflux.

Investigates the mechanism and specificity of free cholesterol influx from plasma and plasma lipoproteins into cultured cell monolayers. Effects of medium plasma concentration on rates of cholesterol influx and efflux; Effects of metabolic inhibitors on the uptake of H-cholesterol from low-density lipoprotein.

Determines the ability of mouse anti-apolipoprotein A-I (apo A-I) monoclonal antibodies to recognize pre-beta-high density lipoprotein (HDL) species in native plasma. Inhibition of the efflux of labeled cholesterol from cultured fibroblast monolayers; Broad specificity for HDL by several other antibodies.

Abstract: Syntheses are described of fatty acid analogs 5 and 6, and cholesterol (2) analogs 7 and 8 containing fluorenone groups, which are both photoactivable and fluorescent. The potential of the analogs of 2 as biochemical research tools has been demonstrated by the findings that 7 and 8 can replace 2 in apolipoprotein A-I-induced cellular efflux of 2 and that fluorescence is easily visible at the surface of smooth muscle cells equilibrated with 8. [Copyright &y& Elsevier]