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

Dettwiler, Ramona (2013). Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels. (Dissertation, Department of Clinical Research and Veterinary Public Health, Veterinär-Pharmakologie und -Toxikologie, Vetsuisse Fakultät Bern)

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It is well known that the activity of cytochrome P450 enzymes (CYPs) depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 heterologously expressed in V79 cells. A system allowing POR protein regulation based on a destabilizing domain (DD) that transfers its instability to a fused protein was used. Addition of ‘Shield-1’ protects the DD fusion protein from degradation.
Equine CYP3A94 was demonstrated to be metabolically active using a fluorescent substrate (BFC) and co-expression of POR elevated its activity. POR activity was also significantly increased in V79-CYP3A94/DD-POR cells. Surprisingly, although POR activity did not change significantly, different Shield-1 concentrations resulted in a decrease in detectable POR protein and CYP3A94 activity. The basal POR and CYP3A94 activity levels were unexpectedly high indicating that the DD/POR protein was not degraded. Decreasing POR protein levels and CYP3A94 activity after Shield-1 incubation suggest that Shield-1 impairs POR activity by an unknown mechanism.
Although regulation of POR could not be achieved, the cell line V79-CYP3A94/DD-POR can be used to further characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

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