A cytochrome c‐552 gene from a thermophilic hydrogen‐oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. A 780‐bp PstI –SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c‐552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. Cytochrome c‐552 is synthesized in vivo as a precursor having an N‐terminal signal sequence consisting of 18 amino acid residues. The cloned cytochrome c‐552 gene without its own signal sequence was introduced into the pKK223‐3 vector and expressed in E. coli upon induction with isopropyl β‐d‐thiogalactoside. An expressed cytochrome c‐552 protein had a methionine residue at the N‐terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c‐552. The heme c was attached to apo‐type cytochrome c‐552 in the cytoplasm of E. coli and the holoprotein had spectral properties, similar to the authentic cytochrome c‐552 from H. thermophilus.

Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ATP binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the efflux of phospholipid and cholesterol from cells to apolipoprotein A-I (apoA-I), reversing foam cell formation. This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells. After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time, cholesterol efflux, ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator, RT-PCR and Western blot, respectively. The mean ABCA1 fluorescence intensity on THP-1 macrophage-derived foam cells was detected by flow cytometry. Results showed that apoA-I markedly increased ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by an increase in the content of ABCA1. ApoA-I did not alter ABCA1 mRNA abundance. Significantly, thiol protease inhibitors increased the level of ABCA1 protein and slowed its decay in THP-1 macrophage-derived foam cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH4Cl showed such effects. The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors. Our results suggested that thiol protease inhibitors might provide an alternative way to upregulate ABCA1 protein. This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

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