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, cytochro mes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia ct al,, Arch. Biochem. Biophys. 297, 228, 1992; and Tierney ct al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that i n intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inacti vation. Our findings with intact rat hepatocytes and ER preparations contai ning native P450s and P450s inactivated via heme modification of the protei n have revealed that the proteolytic degradation of heme-modified P450s req uires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia ct al., Arch. Biochem. Biophys. 297, 228, 1992). Usi ng purified cumene hydroperoxide-inactivated P450s (rat Liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document th at these heme-modified enzymes are indeed ubiquitinated and then proteolyze d by the 26S proteasome, but not by its 20S proteolytic core, In addition, our studies indicate that the ubiquitination of these heme-modified P450s i s preceded by their phosphorylation, It remains to be determined whether, i n common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings sugges t that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation . (C) 1999 Academic Press.