benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and 3-5-dicarbethoxy-2-6-dimethyl-4-ethyl-1-4-dihydropyridine

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.

Topics: Adenine; Adenosine Triphosphatases; Ammonium Chloride; Animals; Autophagy; Boronic Acids; Cell Cycle Proteins; Cross-Linking Reagents; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dexamethasone; Dicarbethoxydihydrocollidine; Formaldehyde; Hepatocytes; Leupeptins; Lysosomes; Male; Nuclear Proteins; Polymers; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Saccharomyces cerevisiae Proteins; Troleandomycin; Ubiquitin; Valosin Containing Protein

Mechanism-based inactivation of liver microsomal cytochromes P450 3A (CYP 3A, P450s 3A) in vivo and/or in 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.

Topics: Aclarubicin; Animals; Aryl Hydrocarbon Hydroxylases; Biological Transport; Brefeldin A; Cell Separation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Cytosol; Dicarbethoxydihydrocollidine; Drug Interactions; Endoplasmic Reticulum; Female; Leupeptins; Liver; Models, Biological; Oxidoreductases, N-Demethylating; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Ubiquitins