Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.
ABSTRACT The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.
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ABSTRACT: One major mode of regulation of cytochrome P450 2E1 (CYP2E1) is at the posttranscriptional level, since many low-molecular-weight compounds stabilize the enzyme against proteolysis by the proteasome complex. In an in vitro system containing human liver microsomes, degradation of CYP2E1 in the microsomes required addition of the human liver cytosol fraction in a reaction sensitive to inhibitors of the proteasome complex. It is not clear how CYP2E1 in the microsomal membrane becomes accessible to the cytosolic proteasome. Since molecular chaperones play a role in protein folding and degradation, the possible role of heat shock proteins in CYP2E1 degradation by this reconstituted system was evaluated. Degradation of CYP2E1 required ATP; ATP-gammaS, a nonhydrolyzable analogue of ATP, did not catalyze CYP2E1 degradation by the cytosol fraction, indicating that ATP hydrolysis is required. Geldanamycin, a specific inhibitor of hsp90, inhibited the degradation of microsomal CYP2E1 by the cytosol fraction. Control experiments indicated that geldanamycin was not a substrate/ligand of CYP2E1 nor did it prevent microsomal lipid peroxidation, a process which increases CYP2E1 turnover. Inhibition by geldanamycin was prevented by molybdate. Both of these compounds have been shown to promote alterations in hsp90 structure and to modulate hsp90-protein interactions. The proteasome activity in the cytosol, as assayed by the cleavage of a fluorogenic peptide, was enhanced when ATP was added and inhibited by 30-40% by geldanamycin, effects that are similar, although less pronounced, to the degradation of CYP2E1 by the cytosol. Purified 20S proteasome could catalyze degradation of CYP2E1; however, in an assay using equal peptidase activity, the cytosol fraction was much more effective than the 20S proteasome in promoting CYP2E1 degradation. Immunodepletion of hsp90 from the cytosol resulted in prevention of the degradation of CYP2E1, a reaction that was reversed by the addition of pure hsp90 to this cytosol. These results suggest that in addition to the proteasome, the cytosol fraction contains other factors that modulate the efficiency of CYP2E1 degradation. The sensitivity to geldanamycin and molybdate and the immunodepletion experiments suggest that hsp90 is one of these factors that interact with CYP2E1 and/or with the proteasome to promote the degradation of this microsomal P450.Archives of Biochemistry and Biophysics 08/2000; 379(2):321-30. · 3.37 Impact Factor
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ABSTRACT: Hepatic cytochromes P450 (P450s) are monotopic endoplasmic reticulum (ER)-anchored hemoproteins that exhibit heterogenous physiological protein turnover. The molecular/cellular basis for such heterogeneity is not well understood. Although both autophagic-lysosomal and nonlysosomal pathways are available for their cellular degradation, native P450s such as CYP2B1 are preferentially degraded by the former route, whereas others such as CYPs 3A are degraded largely by the proteasomal pathway, and yet others such as CYP2E1 may be degraded by both. The molecular/structural determinants that dictate this differential proteolytic targeting of the native P450 proteins remain to be unraveled. In contrast, the bulk of the evidence indicates that inactivated and/or otherwise posttranslationally modified P450 proteins undergo adenosine triphosphate-dependent proteolytic degradation in the cytosol. Whether this process specifically involves the ubiquitin (Ub)-/26S proteasome-dependent, the Ub-independent 20S proteasome-dependent, or even a recently characterized Ub- and proteasome-independent pathway may depend on the particular P450 species targeted for degradation. Nevertheless, the collective evidence on P450 degradation attests to a remarkably versatile cellular sanitation brigade available for their disposal. Given that the P450s are integral ER proteins, this mechanistic diversity in their cellular disposal should further expand the repertoire of proteolytic processes available for ER proteins, thereby extending the currently held general notion of ER-associated degradation.Drug Metabolism Reviews 01/2003; 35(2-3):107-43. · 5.54 Impact Factor
- Archives of Biochemistry and Biophysics - ARCH BIOCHEM BIOPHYS. 01/1983; 225(1):216-236.