Cytochrome P450 3A Degradation in Isolated Rat Hepatocytes: 26S Proteasome Inhibitors as Probes

Abstract

Mechanism-based inactivation of liver microsomal cytochromes P450 3A (CYP 3A, P450s 3A)in vivoand/orin vitro,via heme modification of the protein, results in accelerated proteolytic degradation of the enzyme that is preceded by the ubiquitination of the protein, thereby implicating the ubiquitin-ATP-dependent 26S proteasomal system. In this study, this involvement is confirmed with the use of the proteasomal inhibitors aclarubicin and MG-132 as probes, in isolated rat hepatocytes treated with the P450 3A mechanism-based inactivator, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP). In addition, the findings reveal that during the course of this proteolysis, the endoplasmic reticulum (ER)-anchored DDEP-inactivated P450 3A is translocated from the ER to the cytosol in a brefeldin A-insensitive manner.