Polycistronic expression of mitochondrial steroidogenic P450scc system in the HEK293T cell lineстатья

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Дата последнего поиска статьи во внешних источниках: 10 апреля 2019 г.

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[1] Polycistronic expression of mitochondrial steroidogenic p450scc system in the hek293t cell line / V. Efimova, L. Isaeva, A. Labudina et al. // Journal of Cellular Biochemistry. — 2019. — Vol. 120, no. 3. — P. 3124–3136. Abstract The cholesterol hydroxylase/lyase (CHL) system, which consists of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The FMDV 2A-based method allows for the expression of multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the co-expression 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. HEK293-T cells transfected with plasmid, containing cDNA for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, which was localized in mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in cytopsol. 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 the HEK293-T cells that didn’t express heterologous cytochrome P450scc were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. Following transfection of HEK293T cells with a vector directing the synthesis of only P450scc-2A, we discovered that these cells demonstrated cytochrome P450scc activity comparable to that of cells expressing all three components of the CHL system, and even comparable 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 electron transport proteins present in mitochondria of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, that indicates the absence of catalytically active 3β-HSD in these cells. Our findings suggest that HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics. [ DOI ]

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