Degradation Of Cytochrome P450 2E1 Research Articles

Стресіндуковані зміни вмісту цитохрому Р450 2Е1 у печінці мишей при хронічному психоемоційному перенапруженні

In this work we investigated changes in the cytochrome P450 2E1 (CYP2E1) expression level in the liver of mice that have been exposed to psycho-emotional stress (PES). It was shown twofold relative to control reduction of the enzyme, which does not normalize after termination of the stressor. The changes in level of Cyp2e1 mRNA is not observed at any time point of the experiment. At the same time it was found a significant decrease in the expression level of hsp90--one of the factors determines of the level of CYP2E1 degradation in the cell. Also it was shown, the oxidative stress develops in the liver of experimental animals, and this is indicated by a 3-fold increase in the malondialdehyde level on the background of a 5-fold decrease in catalase activity. A decrease in the level of expression of cytochrome P450 2E1 in the liver of mice that have been exposed to psycho-emotional stress is due to the intensification of the peroxide process and the development of oxidative stress. Reduction of protein content of CYP2E1 is apparently not related to the hsp90-dependent processes of its degradation in the cell.

Regulation of Cytochrome P450 2E1 by Heat Shock Protein 90-Dependent Stabilization and CHIP-Dependent Proteasomal Degradation

Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any member of this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly by various substrates, such as ethanol and CCl(4). CYP2E1 is degraded by the ubiquitin-proteasome pathway, and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balance between protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulated by the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stably expressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited by the proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressing the truncated CYP2E1(Delta3-29) yields coadsorption of CYP2E1(Delta3-29). Cotransfection of HEK cells with both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(Delta3-29) degradation, and CYP2E1(Delta3-29) co-immunoadsorbs with myc-CHIP from cytosol of cotransfected cells. Purified, bacterially expressed CYP2E1(Delta3-29) is ubiquitylated in a CHIP-dependent manner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2, and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated by the hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates, such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundly alter enzyme turnover.

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

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.

CYP2E1 Degradation by in Vitro Reconstituted Systems: Role of the Molecular Chaperone hsp90

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-γS, 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.

NADPH-Dependent Microsomal Electron Transfer Increases Degradation of CYP2E1 by the Proteasome Complex: Role of Reactive Oxygen Species

Increased levels of cytochrome P450 2E1 (CYP2E1) produced by low-molecular-weight compounds is mostly due to stabilization of the enzyme against proteolytic degradation. CYP2E1, in the absence of substrate or ligand, normally has a short half-life, but the factors which regulate CYP2E1 turnover or trigger its rapid degradation are not known. Since CYP2E1 is active in producing reactive oxygen species, experiments were carried out to evaluate whether reactive oxygen species modulated the degradation of CYP2E1. CYP2E1 present in human liver microsomes was very stable. Addition of the cytosol fraction produced degradation of CYP2E1, and this was enhanced when NADPH was present in the reaction system. Antioxidants or iron chelators which prevent lipid peroxidation, prevented the degradation of CYP2E1 by the cytosolic fraction. Similarly, diphenyleneiodonium chloride, which inhibits NADPH-dependent electron transfer, prevented the degradation of CYP2E1, as did 4-methylpyrazole, a ligand which increases the level of CYP2E1. If microsomes were first incubated with NADPH for 30 min, followed by the addition of these agents, there was no protection against CYP2E1 degradation. Lactacystin, an inhibitor of the proteasome, decreased the degradation of CYP2E1. In intact HepG2 cells transduced to express CYP2E1, proteasome inhibitors elevated steady-state levels of CYP2E1. Steady-state levels of CYP2E1 were increased by about 50% when the cells were incubated with trolox. Trolox decreased the rate of loss of CYP2E1 protein when the cells were treated with cycloheximide. These results suggest that NADPH-dependent production of reactive oxygen species may result in oxidative modification of CYP2E1, followed by rapid degradation of the labilized CYP2E1 by the proteasome complex. It is interesting to speculate that one consequence of the high rates of production of reactive oxygen species by CYP2E1 is its own labilization and subsequent rapid degradation, and this may be a regulatory mechanism to prevent high levels of the enzyme from accumulating within the cell.

Selective fast degradation of cytochrome P-450 2E1 in serum-deprived hepatoma cells by a mechanism sensitive to inhibitors of vesicular transport.

Cytochrome P-450 2E1 (CYP2E1) is characterized by a rapid turnover in the liver and some cell lines and the ability of substrates and heme iron ligands to inhibit significantly enzyme degradation. In the Fao hepatoma cell line, CYP2E1 was found to be fairly stable (half-life of 26 h), but serum withdrawal resulted in its rapid disappearance from the microsomal fraction (half-life of about 7 h) as evaluated using cycloheximide chase. The effect of serum withdrawal could be partially reversed by the addition of albumin to the culture medium, whereas insulin and the insulin-like growth factor IGF-I had no additional effect. The effect of serum withdrawal was specific for CYP2E1 since (a) no concomitant fast degradation of CYP2B1 and NADPH-cytochrome P-450 reductase was observed and (b) the CYP2E1 ligands ethanol and imidazole prevented the fast degradation of the enzyme. The lysosomotropic agent ammonium chloride and the inhibitor of autophagocytosis 3-methyladenine slowed down CYP2E1 degradation by about 30%, while leupeptin had no effect. Under the same conditions, the degradation of total long-lived cell protein showed the same sensitivity to ammonium chloride, but was significantly less sensitive to 3-methyladenine and serum and not sensitive to ethanol and imidazole. CYP2E1 degradation was inhibited by combined treatment with brefeldin A and nocodazole, which blocks both anterograde and retrograde vesicular transport between endoplasmic reticulum and the Golgi apparatus. The data point to the existence of a selective mechanism for the degradation of membrane proteins in serum-deprived cells in addition to nonselective autophagocytosis. The selective degradation of CYP2E1 may be attained by means of its selective vesicular transport to an acidic post-endoplasmic reticulum compartment.

Rapid changes in cytochrome P4502E1 (CYP2E1) activity and other P450 isozymes following ethanol withdrawal in rats

This study describes the effects of chronic ethanol (ETOH) treatment and withdrawal on the rat hepatic mixed-function mono-oxygenase system. Male Sprague-Dawley rats (150–200 g, 10 per group) were administered ETOH as part of the Lieber-deCarli liquid diet for 3 weeks. Ethanol was removed, and the animals were euthanized at 0, 24, 48, 72 and 168 hr post-withdrawal. Microsomes were prepared, and ethanol-inducible cytochrome P4502E1 (CYP2E1) activity was measured using the enzyme markers N-nitrosodimethylamine demethylase (NDMAd), p-nitrophenol hydroxylase (PNPH) and aniline hydroxylase (AH). Activities were found to be induced significantly after chronic ETOH feeding using all three assays (NDMAd, 5-fold; PNPH, 3.5-fold; AH, 9-fold). Upon ETOH withdrawal, all three activities dropped markedly, with NDMAd and PNPH at control values at 24 hr and all subsequent time points. AH activity remained 3-fold higher than controls at 24, 48 and 72 hr. Western blot analyses showed that immunoreactive CYP2E1 returned to control at 24 hr, consonant with NDMAd and PNPH activities. The prolonged induction of AH activity following ETOH withdrawal indicates that it is not a specific marker of CYP2E1-catalyzed reactions. Collectively, these data are suggestive of a rapid mechanism of CYP2E1 degradation in the rat liver. Of the other parameters investigated in this study, total cytochrome P450 content was increased 2.5-fold after ETOH feeding, with levels dropping markedly 24 hr post-withdrawal. NADPH-dependent cytochrome c reductase activity was unchanged throughout the course of the study. CYP1A1, CYP2B1 and CYP3A activities were assessed by the substrate probes ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD) and erythromycin N-demethylase (ERNd). EROD and PROD were induced significantly by ETOH administration (2-fold) at 0 hr, with EROD remaining elevated over controls 24 hr post-withdrawal. Quantitative western blot analysis of CYP1A1 and CYP2B1 revealed a pattern of immunostaining generally consistent with but less variable than levels predicted by the respective substrate markers. Both proteins were induced significantly by chronic ethanol administration (CYP1A1, 1.9-fold; CYP2B1, 4-fold). Induction of these P450 isoforms persisted for several days following withdrawal. In contrast, immunoreactive CYP1A2 was found to decrease significantly (by 30–40%) during ethanol withdrawal (24, 48, 72, 168 hr). ERNd activity was induced significantly by chronic ETOH feeding (2.5-fold) and remained so for 24 hr into the withdrawal period (2-fold). Immunoreactive CYP3A1 was also induced significantly following ETOH administration (0 hr) and 24 hr following withdrawal. Collectively, the data presented suggest that chronic ethanol feeding induces in vivo at least four distinct P450 isoforms: CYP2E1, CYP1A1, CYP2B1 and CYP3A1. Ethanol withdrawal resulted in a 30–40% loss of CYP1A2. CYP1A1, CYP2B1 and CYP3A1 were elevated during ethanol withdrawal, unlike CYP2E1, a protein that undergoes rapid degradation following the removal of ethanol. Such findings are consistent with several distinct mechanisms of P450 isoform induction/ degradation by ethanol.

Purification and Characterization of Two Membrane-Bound Serine Proteinases from Rat Liver Microsomes Active in Degradation of Cytochrome P450

Two serine proteinases capable of digesting cytochrome P4502E1 (CYP2E1) have been purified from sodium cholate solubilized rat liver microsomal membranes. After chromatography on hydroxyapatite, DEAE-Sepharose chromatography resolved the CYP2E -degrading activity into two peaks, and the two proteinases were finally purified on benzamidine-Sepharose. Both have a Mr of 32,000 on SDS-PAGE, are optimally active at pH 8, and show a susceptibility to inhibitors typical of serine proteinases. CYP2E1 degradation patterns exhibited by the proteinases are identical to each other and similar to that observed during the proteolysis of endogenous CYP2E1 in the microsomal membranes, which indicates that the proteinases can degrade CYP2E1 in its native environment. We suggest a role of these proteinases in the rapid phase of cytochrome P450 degradation in the endoplasmic reticulum.

Hormone- and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes.

We have examined differences in post-translational regulation between rat liver ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible CYP2B1 using hepatocyte cultures and subcellular fractions, prepared from starved and acetone-treated rats. The intracellular degradation of CYP2E1 was rapid (approximate t1/2 = 9 h) and increased by glucagon treatment of the cells in an isozyme-specific manner, whereas CYP2B1 degradation in the same cells, was slower (t1/2 = 21 h). The glucagon effect on CYP2E1 degradation was abolished by either cycloheximide treatment of cells, indicating the involvement of protein components with rapid turnover, or by lowering of the culture temperature to 23 degrees C. The rapid phase of CYP2E1 degradation was not influenced by inhibitors of the autophagosomal/lysosomal pathway. In vitro experiments with isolated liver microsomes revealed the presence of a Mg(2+)-ATP-activated proteolytic system active on CYP2E1, previously modified by phosphorylation on Ser-129 or denatured by reactive metabolites formed from carbon tetrachloride. Imidazole, a CYP2E1 substrate, specifically inhibited the rapid intracellular degradation of CYP2E1 and also prevented phosphorylation and subsequent proteolysis in isolated microsomes. In contrast, no proteolysis of CYP2B1 occurred under the conditions used. The microsomal Mg(2+)-ATP-dependent CYP2E1 proteolysis could not be solubilized with high salt and 0.05% sodium cholate, indicating the action of membrane-integrated protease(s). Subfractionation of microsomes revealed that the Mg(2+)-ATP-dependent proteolytic system active on CYP2E1 was present in both rough and smooth endoplasmic reticulum. It is suggested that hepatic cytochromes P450 are degraded both in a bulk process, according to the autophagosomal/lysosomal pathway and more rapidly, in a hormone- and substrate-regulated fashion, by a specific proteolytic system in the endoplasmic reticulum, active on physiologically or exogenously modified molecules.

Degradation of cytochrome P450 2E1: Selective loss after labilization of the enzyme

Mechanism-based inactivation of cytochrome P450 can result in the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. In the present study we took advantage of different modes of inactivation of P450 2E1 by CCl 4, 1-aminobenzotriazole, or 3-amino-1,2,4-triazole to investigate parameters which target P450 2E1 for proteolysis from the microsomal membrane. Treatment of mice with CCl 4 at the point of maximal induction of P450 2E1 after a single oral dose of acetone resulted in the complete loss of P450 2E1-dependent p-nitrophenol hydroxylation and a 75% loss of immunochemically detectable protein within 1 h of administration. Treatment with 1-aminobenzotriazole at the point of maximal induction caused a complete loss of P450 2E1-dependent p-nitrophenol hydroxylation but only a 12% loss of immunochemically detectable protein 1 h after administration. Treatment of mice with 3-amino-1,2,4-triazole caused a rapid loss of both catalytic activity and microsomal p-nitrophenol hydroxylase activity. However, unlike CCl 4 treatment, the activity and enzyme level rebounded 5 and 9 h after treatment. The P450 2E1 ligand, 4-methylpyrazole, administered at the point of maximal induction maintained the acetone-induced catalytic and immunochemical level of P450 2E1. These results suggest that differentially modified forms of P450 2E1 show a characteristic susceptibility to degradation. While there are many potential pathways for protein degradation, the loss of P450 2E1 was associated with increased formation of hieh molecular weight microsomal ubiquitin conjugates. The formation of ubiquitin-conjugated microsomal protein which correlates with P450 2E1 loss suggests that ubiquitination may represent a proteolytic signal for the rapid and selective proteolysis of certain labilized conformations of P450 2E1 from the endoplasmic reticulum.