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.
Highlights
Cytochrome P450 enzymes (CYPs) are accountable for the metabolism of a variety of endogenous and exogenous substances
We demonstrated that heterologously expressed equine CYP3A94 is metabolically active and co-expression of P450 oxidoreductase (POR) in V79 cells enhances its activity
We could demonstrate that equine CYP3A94 is metabolically active and its activity is dependent on an adequate level of POR
Summary
Cytochrome P450 enzymes (CYPs) are accountable for the metabolism of a variety of endogenous and exogenous substances. These enzymes are involved in physiological processes such as the biosynthesis and degradation of steroids, cholesterol, and other endogenous compounds, but they are responsible for the biotransformation of drugs and toxins [1, 2]. 18 CYP families have been discovered in humans; the CYP families 1–3 are referred to as the most important enzymes for the biotransformation of drugs [3]. Since they play a key role in many drug-drug interactions, research on these CYPs is of prime importance. Complex medical care and multidrug treatment became more important in equines, knowledge about equine CYPs requires further in depth investigation
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