Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?
The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.
Korsmeyer
K K
KK
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Davoll
S
S
Figueiredo-Pereira
M E
ME
Correia
M A
MA
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001555:Benzene Derivatives / Q000494:pharmacology; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D006418:Heme / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D051381:Rats; D014452:Ubiquitins / Q000378:metabolism*
D001555:Benzene Derivatives; D065607:Cytochrome P-450 Enzyme Inhibitors; D008565:Membrane Proteins; D014452:Ubiquitins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C007164:cumene hydroperoxide