Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland 97239-3098, USA. Toxicology and Applied Pharmacology
(Impact Factor: 3.71).
10/2004; 199(3):332-43. DOI: 10.1016/j.taap.2003.12.019
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.
Available from: dmd.aspetjournals.org
- "Membrane proteins, in particular, are degraded by the lysosomes through fusion of ER with lysosomal membranes, a process that is slower than that of the proteosome pathway. Indeed, it has been shown in cell lines that CYP2E1 degradation can occur via a ubiquitin-independent proteosomal pathway (Goasduff and Cederbaum, 1999; Huan et al., 2004). In the context of the established biphasic nature of CYP2E1 turnover found in vivo, it is tempting to speculate that, in the presence of substrate, CYP2E1 may not be subject to the more rapid phase of turnover via proteosomes due either to preferential sequestration of the enzyme in certain regions of the ER or to altered conformation of the protein as a result of substrate binding, or a combination of both. "
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ABSTRACT: Bernard B. Brodie's laboratory was the first to examine the mechanisms of drug-induced toxicity at the molecular level. They found that acetaminophen hepatotoxicity was due to the metabolic activation of the drug to a highly reactive toxic metabolite that depleted cellular glutathione and covalently bound to protein. Subsequent studies revealed that activation of acetaminophen to an active metabolite is primarily carried out by CYP2E1, an ethanol-inducible cytochrome P450 that was first suggested by characterization of the microsomal ethanol oxidation system. CYP2E1 is developmentally regulated, under liver-specific control, and undergoes substrate-induced protein stabilization. It is also regulated by starvation and diabetes through insulin-dependent mRNA stabilization. In addition to acetaminophen, CYP2E1 metabolically activates a large number of low M(r) toxicants and carcinogens and thus is of great toxicological importance. The mechanism of regulation CYP2E1 and its role in acetaminophen toxicity will be discussed.
Available from: jpet.aspetjournals.org
- "The addition of various proteasome inhibitors potentiated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells) by AA/Fe-NTA treatment, and this was associated with accumulation of oxidized and nitrated proteins (Perez and Cederbaum, 2003). CYP2E1 turnover is mediated by the proteasome, because several lowmolecular weight inducers of CYP2E1 such as ethanol, dimethyl sulfoxide, and pyrazole elevate CYP2E1 by stabilizing the enzyme against proteasome-catalyzed degradation (Roberts, 1997; Yang and Cederbaum, 1997; Huan et al., 2004). Moreover, the compromised proteasome function is believed to be involved in the formation of Mallory bodies, a hallmark of alcohol-induced liver disease (Bardag-Gorce et al., 2004). "
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ABSTRACT: A reduction in proteasome activity and accumulation of oxidized proteins may play a role in alcoholic liver disease. The current study assessed proteasome peptidase activities and oxidative modifications of proteasomes during oxidative stress generated by CYP2E1. The model of toxicity by arachidonic acid (AA) and iron [ferric-nitrilotriacetate (Fe-NTA)] in HepG2 cells overexpressing CYP2E1 (E47 cells) and control C34 cells was used. AA/Fe-NTA treatment decreased trypsin-like (T-L) activity of the proteasome in E47 cells but not in C34 cells. This inhibition was abolished by antioxidants. Chymotrypsin-like activity of the proteasome was increased in E47 cells, and activity was not altered by AA/Fe-NTA treatment. There were no changes in content of subunits of 20S proteasomes or 19S regulator ATPase subunits S4 and p42 by AA/Fe-NTA treatment. An increased content of the PA28alpha subunit of the 11S regulator of proteasomes was detected in E47 cells. In proteasome pellets, the decline of T-L activity was accompanied by increased content of carbonyl adducts, suggesting oxidative modification of proteasomes. Higher levels of ubiquitinated, 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins and lower levels of free ubiquitin were detected in untreated E47 cells in comparison with C34 cells. Accumulation of protein cross-linked, detergent-insoluble aggregates was increased with AA/Fe-NTA treatment in E47 cells. Thus, reactive oxygen species generated upon CYP2E1-dependent oxidative stress mediated a decline in T-L proteasome function, increased carbonyl adducts in proteasomes, and promoted protein aggregate formation; this may alter the balance among protein oxidation, ubiquitination, and degradation.
Available from: Victor Zgoda
- "). 4 In contrast, CYP2B1 and CYP2E1 stably expressed in HeLa cells exhibit considerably shorter half-lives (t 1/2 Ϸ 8.7 and 3.7 h, respectively) and are degraded in a ubiquitin (Ub)-independent process, blocked by specific proteasomal but not lysosomal inhibitors (Huan et al., 2004). The basis for this accelerated P450 protein turnover and altered degradation route is unclear. "
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ABSTRACT: Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins with highly variable half-lives. CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin (Ub)-dependent 26S proteasomal degradation after suicide inactivation or after heterologous expression in Saccharomyces cerevisiae. In contrast, rat liver CYP2C11 is degraded by the vacuolar "lysosomal" pathway when similarly expressed in yeast. The structural determinants that commit P450s to proteasomal or lysosomal degradation are unknown. To further validate S. cerevisiae as a model for exploring mammalian P450 turnover, the degradation of phenobarbital-inducible liver CYP2B1, an enzyme reportedly degraded via the rat hepatic autophagic-lysosomal pathway, was examined in a yeast strain (pep4delta) deficient in vacuolar degradation and its isogenic wild-type control (PEP4). Although CYP2B1 was equivalently expressed in both strains during early logarithmic growth, its degradation was retarded in pep4delta strain, remaining at a level 5-fold higher than that in PEP4 yeast when monitored at the stationary phase. No comparable CYP2B1 stabilization was detected in yeast genetically deficient in the ER Ub-conjugating enzyme Ubc6p or Ubc7p or defective in 19S proteasomal subunit Hrd2p. Thus, as in the rat liver, CYP2B1 is a target of vacuolar/lysosomal rather than proteasomal degradation in yeast, thereby further validating this model for mammalian P450 turnover. It is intriguing that a chimeric protein, CYP2B1-3A4CT, with the CYP3A4 C-terminal heptapeptide grafted onto the CYP2B1 C terminus, was proteasomally degraded after similar expression. Such diversion of CYP2B1 from its predominantly vacuolar degradation suggests that the CYP3A4 heptapeptide could either actively signal its proteasomal degradation or block its vacuolar proteolysis.
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