Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line
The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR) initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot and mouth disease virus 2A based method allows to express multiple proteins from a single transcript. We developed a 2A based multicistronic system for the coexpression of three bovine CHL system proteins as the self processing polyprotein pCoxIV P450scc 2A Adx 2A AdR GFP (pCoxIV CHL GFP) with a cleavable N terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV CHL GFP, efficiently performed the expression of P450scc 2A, targeted to mitochondria, and Adx 2A, AdR GFP and the fusion protein Adx 2A AdR GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2A peptide residue on the C terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc 2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3b hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.
Journal of cellular biochemistry