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Home / Equine Research Bank / PLoS One / Heterologous expression of equine CYP3A94 and investigation ...
PloS one2014; 9(11); e113540; doi: 10.1371/journal.pone.0113540

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

Abstract: The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.
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Publication Date: 2014-11-21 PubMed ID: 25415624PubMed Central: PMC4240602DOI: 10.1371/journal.pone.0113540Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research study aims to understand the activity of equine CYP3A94, a type of enzyme, through the use of a system that can control the levels of another protein in the body, the NADPH P450 oxidoreductase (POR).

Objective of the Study

  • The main goal of this study was to analyze the activity of a specific enzyme, the equine CYP3A94, by using a mechanism that permits the regulation of the POR protein levels inside mammalian cells.

Heterologous Expression and Investigation Approach

  • The researchers have used a heterologous expression of the CYP3A94 and the equine POR in V79 cells—a type of cell line used extensively in cell biology and genetics.
  • The system used to regulate the POR protein is based on a ‘destabilizing domain’ (DD) that transfers its instability to any fused protein. This fusion protein is then degraded by a process known as the ubiquitin-proteasome system (UPS).
  • In this setup, an addition of a stabilizing element, “Shield-1”, prevents the DD fusion protein from degradation, thereby maintaining an ideal level of the POR enzyme.

Experiment Observations and Results

  • The test involved monitoring the alterations in the levels of POR and the activity of CYP3A94 through various analytic processes such as cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry, at different concentrations of Shield-1.
  • The researchers established that equine CYP3A94 was metabolically active and its activity was significantly enhanced when POR was co-expressed.
  • Upon higher Shield-1 concentrations, there was a decline in POR level, but cytochrome c reduction did not significantly change and the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours.
  • Interestingly, when the researchers compared V79-CYP3A94 POR cells with or without Shield-1, no significant differences were found. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded in the absence of Shield-1.
  • The researchers deduced that Shield-1 decreases POR protein levels and CYP3A94 activity, hinting that Shield-1 might have unexplored effects on POR activity.

Conclusions and Further Research

  • While the mechanism of regulating POR with the pPTuner system did not yield expected results, the team concluded that the V79-CYP3A94/DD-POR cell line system can be useful in further experiments for investigating the equine CYP3A94.
  • These experiments could shed light on the character and responsive behavior of the equine CYP3A94, given that the enzyme’s activity was significantly boosted with the co-expression of POR.

Cite This Article

APA
Dettwiler R, Schmitz AL, Plattet P, Zielinski J, Mevissen M. (2014). Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels. PLoS One, 9(11), e113540. https://doi.org/10.1371/journal.pone.0113540

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 9
Issue: 11
Pages: e113540
PII: e113540

Researcher Affiliations

Dettwiler, Ramona
  • Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Schmitz, Andrea L
  • Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Plattet, Philippe
  • Division Neurological Sciences, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Zielinski, Jana
  • Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Mevissen, Meike
  • Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

MeSH Terms

  • Animals
  • Cell Line
  • Cloning, Molecular / methods
  • Cricetinae
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochromes c / metabolism
  • Female
  • Gene Expression Regulation
  • HEK293 Cells
  • Horses
  • Humans
  • Male
  • Morpholines / pharmacology
  • NADPH-Ferrihemoprotein Reductase / genetics
  • NADPH-Ferrihemoprotein Reductase / metabolism

Conflict of Interest Statement

The authors have declared that no competing interests exist.

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