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Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line. | LitMetric

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line.

J Cell Biochem

Department of Molecular Basis on Ontogenesis, A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.

Published: March 2019

AI Article Synopsis

  • The cholesterol hydroxylase/lyase (CHL) system, involving cytochrome P450scc and associated proteins, converts cholesterol to pregnenolone, marking the first step in steroid hormone production in mammals.* -
  • A 2A-based multicistronic system was designed to coexpress bovine CHL proteins in HEK293T cells, allowing for effective conversion of steroid substrates into pregnenolone, despite spatial separation of the proteins.* -
  • The study indicates that HEK293T cells can be utilized to express steroidogenic enzymes and study their roles, as they demonstrated significant P450scc activity and produced pregnenolone without converting it to progesterone.*

Article Abstract

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 2А 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 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

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Source
http://dx.doi.org/10.1002/jcb.27577DOI Listing

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