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 KK ，Davoll S ，Figueiredo-Pereira ME ，Correia MA ... -  《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae?

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.

Murray BP ，Correia MA 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

Cytochrome P450 3A degradation in isolated rat hepatocytes: 26S proteasome inhibitors as probes.

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.

Wang HF ，Figueiredo Pereira ME ，Correia MA 《-》

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.

Chun J ，Kent UM ，Moss RM ，Sayre LM ，Hollenberg PF ... -  《-》

Aryl acetylenes as mechanism-based inhibitors of cytochrome P450-dependent monooxygenase enzymes.

Foroozesh M ，Primrose G ，Guo Z ，Bell LC ，Alworth WL ，Guengerich FP ... -  《CHEMICAL RESEARCH IN TOXICOLOGY》