Characterization of Cytochrome P4502E1 Turnover in Transfected HepG2 Cells Expressing Human CYP2E1

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 341, No. 1, May 1, pp. 25–33, 1997 Article No. BB979907

Characterization of Cytochrome P4502E1 Turnover in Transfected HepG2 Cells Expressing Human CYP2E1 Ming-Xue Yang and Arthur I. Cederbaum1 Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029

Received October 22, 1996, and in revised form January 10, 1997

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible proteolytic pathways responsible for the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Two methods were used to study the CYP2E1 turnover; after addition of cycloheximide, the half-life of the CYP2E1 in the intact cells was about 6 h as detected by PNP catalytic activity assay and immunoblot analysis of apoprotein content. CYP2E1 substrates or ligands such as 4-methylpyrazole, ethanol, glycerol, and dimethyl sulfoxide protected CYP2E1 against this rapid degradation, whereas CCl4 accelerated this process. The second procedure involved pulse–chase experiments after labeling CYP2E1 with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG. The half-life of CYP2E1 was about 2.5 h, and the various substrates or ligands modified the turnover process within intact cells as described for the cycloheximide experiments. More than 20 different reagents including antioxidants, physiological metabolites, lysosomal inhibitors, and protease inhibitors were screened for possible effects on CYP2E1 proteolytic degradation. Dibutyryl cAMP had no effect on CYP2E1 activity or turnover. Among those reagents tested so far, the serine protease inhibitor 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride exhibited some protection against CYP2E1 degradation. To demonstrate whether the proteasome complex is involved in this process, CzbIle-Glu(OtBu)-Ala-leucinal (PSI) as a cell penetrating aldehydic proteasome inhibitor and Czb-Leu-norleucinal (calpeptin inhibitor) as an aldehydic nonproteosomal protease inhibitor were used to examine their effect on both the normal and the CCl4-stimulated CYP2E1 proteolytic degradation pathways. Treatment with PSI at concentrations ranging from 5 to 80 mM resulted in a dose-dependent protection against the 1 To whom correspondence should be addressed at Department of Biochemistry, Mount Sinai School of Medicine, Box 1020, One Gustave L. Levy Place, New York, NY 10029. Fax: 212-996-7214.

loss of both the normal CYP2E1 and the CCl4-modified CYP2E1. The maximum protection by PSI at a concentration of 80 mM after a 12-h chase period was about 60% in cells treated with 2 mM CCl4 or 75% in cells without CCl4 treatment. Calpeptin inhibitor afforded little or no protection against CYP2E1 degradation in the absence or presence of CCl4 . PSI did not inhibit CYP2E1 catalytic activity, suggesting that it was not a ligand for CYP2E1. These results indicate that human CYP2E1 has a short half-life span and that substrates can significantly modify its turnover rate in intact HepG2 cells. The proteasome proteolytic pathway may be involved in the degradation process of both the normal and the CCl4-modified human CYP2E1 in this model. q 1997 Academic Press

Cytochrome P4502E1 (CYP2E1)2 is induced by ethanol and several other low-molecular-weight agents and is of special interest because of its ability to metabolize and activate numerous hepatotoxicants in the liver as well as carcinogens and fatty acids (1–6). Oxidation of ethanol by CYP2E1 produces acetaldehyde, a highly reactive compound which may contribute to the toxic effects of ethanol. CYP2E1 has been shown to be a loosely coupled enzyme that produces reactive oxygen species such as superoxide radical and H2O2 in high amounts relative to other P450 isoforms (7, 8). CYP2E1-derived reactive oxygen species have been implicated as playing a role in the hepatotoxic effects of 2 Abbreviations used: CYP2E1, cytochrome P4502E1; PNP, p-nitrophenol; 4-MP, 4-methylpyrazole; PMA, phorbol myristate acetate; MV2E1-9, HepG2 cells infected with retrovirus containing human CYP2E1; DTT, dithiothreitol; calpeptin inhibitor, Czb-Leunorleucinal; DTNB, dithionitrobenzoic acid; PSI, Czb-Ile-Glu(OtBu)-Ala-leucinal; MEM, minimal essential medium; TLCK, 1chloro-3-tosylamido-7-amino-2-heptanone hydrochloride; PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol bis(b-aminoethyl ether)-N*,N*-tetraacetic acid; LTRs, long-terminal repeats; TPCK, N-2-p-tosyl-L-phenylalanine chloromethyl ketone.

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0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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ethanol. CYP2E1 is also reactive in promoting breakdown of hydroperoxides (9). Regulation of CYP2E1 expression has been extensively studied and shown to occur at various levels, including gene transcription, posttranscriptional mRNA stabilization, translational, and posttranslational enzyme stabilization (10–16). A major level of regulation of CYP2E1 from rat or rabbit liver appears to be posttranscriptional, which is reflected by the balance between enzyme stabilization and degradation. The presence of substrate increases the content of CYP2E1 as the ligand-bound enzyme complexes appear to be protected from a rapid degradation by uncharacterized intracellular proteolytic pathways specific for CYP2E1 (12, 14, 17). What triggers CYP2E1 turnover and the nature of the proteases responsible for degradation are not clear. One suggested pathway involves a cAMPdependent phosphorylation of CYP2E1, followed by heme loss and subsequent degradation by serine proteases present in the endoplasmic reticulum (18–21). Tierney et al. (22), using an in vivo mouse model, showed that 2E1 inactivated by CCl4 and 3-aminotriazole was rapidly removed from the endoplasmic reticulum. In this model they detected production of highmolecular-weight ubiquitin-conjugated microsomal proteins after CCl4 treatment. Roberts et al. (17) using an in vitro microsomal system plus a 105,000g supernatant fraction found that the loss of CYP2E1 was accompanied by the appearance of high-molecular-weight ubiquitin conjugates, suggesting that ubiquitin conjugates may target CYP2E1 for rapid proteolysis. It is now well established that the proteasome complex constitutes a major extra lysosomal proteolytic system which is responsible for ubiquitin-dependent and ubiquitin-independent pathways of intracellular proteolysis (23–26). These pathways are involved in diverse cellular functions such as cell growth and mitosis, antigen processing, and degradation of short-lived regulatory proteins such as oncogene products, transcription factors, and cyclins (27–30). A HepG2 cell model which stably and constitutively expresses human CYP2E1 has recently been established (31). The CYP2E1 was catalytically active with representative substrates such as p-nitrophenol (PNP), ethanol, and acetaminophen, and was inactivated by CCl4 treatment (31–33). Degradation of human CYP2E1 and modulation by substrates has not been reported. In the present study, we report that human CYP2E1 possesses a short half-life span, that substrates or ligands can modify this degradation process, and that the proteasome inhibitor Czb-Ile-Glu(OtBu)Ala-leucinal (PSI) significantly inhibited the degradation of both the normal and CCl4-modified CYP2E1 in intact cells; whereas other protease inhibitors such as Czb-Leu-norleucinal (calpeptin inhibitor) and leupeptin exhibited no effect on this process. These results

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extend the recent report (34) that CYP2E1 in microsomes is degraded by an ATP-dependent proteolytic system found in the cytosolic fraction of HepG2 cells, and that PSI nearly doubled steady-state levels of CYP2E1 in these cells. MATERIALS AND METHODS Materials. PNP, protein G–agarose, N 6-2*-O-dibutyryladenosine-3*-5*-cyclic monophosphate (cAMP), cycloheximide, phorbol myristate acetate (PMA), the synthetic peptide Boc-Leu-Ser-Thr-ArgAMC, and fetal calf serum were purchased from Sigma Chemical Co.; CCl4 and 4-methylpyrazole (4-MP) were from Aldrich Chemical Co.; ethanol was from Fisher Scientific; NADPH was from Boehringer-Mannheim; minimal essential medium (MEM), dialyzed fetal bovine serum, penicillin-streptomycin antibiotics mixture, and L-methionine were from GIBCO/BRL; and L-[35S]methionine protein labeling mixture was from Dupont NEN. PSI and calpeptin inhibitor were kindly provided by Dr. Sherwin Wilk (Department of Pharmacology, Mount Sinai School of Medicine). Cell culture. HepG2 cell lines (clone MV2E1-9 was used for most experiments) expressing human CYP2E1 were grown in MEM supplemented with 1% penicillin–streptomycin antibiotic mixture and 10% fetal bovine serum (31). For treatments with chemicals such as 4-MP, DMSO, cAMP, ethanol, and 1-chloro-3-tosylamido-7-amino-2heptanone hydrochloride (TLCK), cells were grown to confluence and refed with fresh medium containing the appropriate dose of the chemicals tested. In some experiments, for higher level of expression of the transduced CYP2E1 (which is under control of promoter elements found in the LTRs of the retrovirus), 0.1 mg/ml PMA was added in the culture medium overnight, before harvesting the cells (33). As shown under Results, the PMA treatment had no effect on the turnover of CYP2E1 nor on the stabilizing effect of substrates and ligands. Isolation of microsomal fractions and immunoblot analysis. Cells were harvested and sonicated in 10 mM phosphate buffer, pH 7.4, containing 150 mM KCl. Microsomes were prepared by differential centrifugation of the sonicated cell extracts and resuspended in phosphate-buffered saline. Western blot analysis was carried out with microsomes using an 8% running gel and a 4% stacking polyacrylamide gel. Electrophoresis and electrotransfer procedures were performed with the Bio-Rad PROTEAN II system. Nitrocellulose membranes with transferred proteins were incubated with anti-human CYP2E1 polyclonal antibody (kindly provided by Dr. J. M. Lasker, Mount Sinai Medical Center, New York, NY) as the first antibody and horseradish peroxidase-conjugated goat anti-rabbit antibody as the second antibody and developed by enhanced chemiluminescent detection (ECL Kit; Amersham, Arlington Heights, IL). Cell labeling and immunoprecipitation. Procedures are minor modifications from those recently reported by Barmada et al. (35). The medium from individual confluent cultures of HepG2 cells in 35mm dishes was replaced with methionine-free MEM supplemented with 10% dialyzed fetal bovine serum and incubated for 120 min at 377C. At the end of this time, the cells were pulse-labeled with 50 mM of [35S]methionine EXPRESS label mixture for 90 min. Following the pulse, the cells were washed and chased with complete MEM medium supplemented with 300 mg/ml methionine. At various times, cells were lysed with 600 ml of 10 mM Tris–HCl buffer, pH 7.4, containing 0.5% Triton X-100, 1 mM EDTA, 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (lysis buffer). CYP2E1 was immunoprecipitated with anti-human CYP2E1 IgG-protein G–agarose as follows. Lysates were first incubated with preimmune rabbit IgG followed by addition of 30 ml of a 50% (v/v) suspension of protein G–agarose. After centrifugation, the supernatant was incubated with anti-human CYP2E1 IgG at 47C overnight followed by addition of 30 ml of a 50% (v/v) suspension of protein G–agarose. After centrifugation, the pellets

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FIG. 1. Effect of different substrates on the loss of p-nitrophenol hydroxylase activity in MV2E1-9 cell. The cells expressing CYP2E1 were treated with or without 0.1 mg/ml PMA for 24 h and cycloheximide was added to the medium at a concentration of 40 mM to stop new protein synthesis. When present, final concentrations of substrates were 50 mM ethanol, 200 mM glycerol, 2 mM CCl4 , 2 mM 4MP, and 7 mM DMSO. Microsomes were prepared from the cells at the indicated times (0, 4, 8, and 24 h) following the addition of the cycloheximide and oxidation of PNP was determined. Typical rates of PNP oxidation were (pmol/min/mg microsomal protein) 50 for control MV2E1-9 microsomes and 300 for PMA-treated MV2E1-9 cells. were washed three times with lysis buffer, one time with lysis buffer plus 2% Emulgen 911, and three times with 0.1 M Tris–HCl buffer, pH 6.8. CYP2E1 was eluted by boiling for 3 min in electrophoresis sample loading buffer and analyzed on 10% SDS–polyacrylamide gels. After electrophoresis, the gel was dried and exposed for several days to XAR film (Kodak). The fluorographs were analyzed with phosphor image software (Molecular Dynamics). Several exposure times were analyzed to validate that blots were quantified within the linear range. PNP oxidation was carried out as described previously (31). Protein content was determined by the method of Lowry et al. (36).

RESULTS

Characterization of human CYP2E1 turnover in transfected HepG2 cells. In order to characterize the time course of CYP2E1 degradation, two methods were used in this model; the first was to use cycloheximide, a protein synthesis inhibitor, to stop new protein synthesis followed by examination of the degradation rates of the remaining CYP2E1 protein within the intact cells. The MV2E1-9 cells treated with or without PMA were harvested at 4-, 8-, and 24-h intervals after the addition of cycloheximide, and microsomes were prepared by ultracentrifugation. The catalytic activity of CYP2E1 was determined by assaying oxidation of PNP, while the content of CYP2E1 was determined from immunoblots. Oxidation of PNP rapidly declined as a function of time after addition of cycloheximide (Fig. 1).

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Treatment with PMA elevated oxidation of PNP about sixfold, and this enhanced activity also rapidly declined after addition of cycloheximide. The half-life of CYP2E1 catalytic activity was about 5 to 6 h. Addition of CCl4 accelerated the loss of PNP oxidation as less than 20% activity remained 4 h after addition of cycloheximide plus 2 mM CCl4 , whereas substrates and ligands for CYP2E1 protected against the loss of PNP oxidation as 60 to 80% activity remained 24 h after addition of cycloheximide plus 4-MP, ethanol, DMSO, or glycerol (Fig. 1). Analogous to control incubations, 4-MP also protected against the loss of PNP oxidation in the PMAtreated samples (Fig. 1). Similar results were found with respect to CYP2E1 apoprotein content. CYP2E1 levels rapidly declined after addition of cycloheximide (Fig. 2A, control lanes 1 to 4 or PMA-treated lanes 1 to 4), with half-lives of about 6.5 h in the absence of PMA and 5.5 h in the presence of PMA (Fig. 2B). Treatment with CCl4 accelerated the degradation of CYP2E1 (Fig. 2A, CCl4-treated lanes 1 to 4), decreasing the halflife to less than 3 h. Treatment with 4-MP, ethanol, glycerol, and DMSO stabilized the CYP2E1 against degradation as little or no loss of CYP2E1 protein was observed even 24 h after addition of cycloheximide plus these agents (Figs. 2A and 2B). 4-MP could partially protect the CCl4-modified CYP2E1 against the rapid loss in both enzyme activity and apoprotein content (Fig. 2A, 4-MP / CCl4 lanes 1 to 4); protection by 4MP probably reflects inhibition of CCl4 metabolism. 4MP also protected against the rapid loss of the elevated levels of CYP2E1 found in PMA-treated MV2E1-9 cells (Fig. 2A, PMA / 4-MP lanes 1 to 4, compared to PMAtreated). CYP2E1 degradation may involve several consecutive steps such as loss of enzyme activity due to enzyme labilization, conformational changes in the enzyme, removal of the heme moiety, and sufficient marking or modification of the enzyme to subject it to proteolysis. A highly correlated linear regression plot (r 2 Å 0.92) was observed between the remaining CYP2E1 catalytic activity (PNP assay), and the remaining apoprotein contents detected on Western blots after the addition of cycloheximide, suggesting that there is little or no time lag or secondary effect between the inactivation of enzyme activity and loss of apoprotein; i.e., inactivated enzyme appears to be rapidly degraded and doesn’t accumulate. Since cycloheximide treatment may have effects on numerous proteins, including synthesis of proteases involved in CYP2E1 degradation, classical pulse–chase experiments with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG were carried out to confirm the above results. The cells were incubated with 50 mCi/ml [35S]methionine for 1.5 h and chased with 300 mg/ml cold methionine for varying periods of time (0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 h). As shown

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FIG. 2. Effects of different substrates on the loss of CYP2E1 apoprotein content in MV2E1-9 cells. Microsomes were prepared from MV2E19 cells at the indicated times (0, 4, 8, and 24 h) following the addition of cycloheximide in the absence or presence of various substrates. (A) Western blot analysis of the content of CYP2E1. 40 mg microsomal protein was separated by size with 8% SDS–PAGE, transferred to nitrocellulose, and immunoblotted with an anti-human CYP2E1 polyclonal antibody as the first antibody and goat anti-rabbit IgG conjugated to horseradish peroxidase as the second antibody. For all treatments, lanes correspond to the following: the cells were harvested at 0 (lane 1), 4 (lane 2), 8 (lane 3), and 24 (lane 4) h, respectively, following addition of cycloheximide. Final concentrations of substrates were CCl4 , 2 mM; 4-MP, 2 mM; ethanol, 50 mM; glycerol, 200 mM, and DMSO, 7 mM. (B) Quantitative analysis of CYP2E1 apoprotein levels. Specific CYP2E1 hybridization signals were quantitatively analyzed using Image Quant software. Results are from the immunoblots shown in (A).

in Fig. 3A, lanes 1 to 3, autoradiograms indicated the presence of a major band with a molecular weight in the 54-kDa region, prior to initiation of the chase. This band rapidly disappeared upon addition of cold methionine. The degradation of human CYP2E1 was biphasic, with the rapid phase having a half-life of about 2.5 h and the slower phase showing a half-life of about 6 h (Fig. 3B). Degradation of rabbit CYP2E1 in COS cells was recently shown (37) to be monophasic (half-life of 4.8 h), while turnover of rat CYP2E1 was biphasic, with half-lives of about 6 and 38 h (12, 17). It is not clear whether the varying half-lives reflect species differences or the different reaction systems or models utilized. CYP2E1 substrates and ligands significantly protected the CYP2E1 against this rapid turnover as the half-life of CYP2E1 was extended from about 3 h to more than 12 h in the presence of glycerol, 4-MP, and ethanol, whereas CCl4 accelerated this process, lowering the half-life of CYP2E1 to less than 2 h (data not shown). Effect of cAMP on CYP2E1 turnover in the transfected HepG2 cells. Previous reports (18–20) suggested that cAMP-dependent phosphorylation is involved in the process of rapid CYP2E1 protein degradation in rat hepatocytes. To study this possibility in the HepG2 cell model, experiments with dibutyryl cAMP, a cell-pene-

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trating cAMP analogue, were carried out. As shown in Figs. 4A and 4B, treatment of the cells with 2 mM dibutyryl cAMP for 24 h produced a small increase in both PNP oxidation and apoprotein content. When cycloheximide was added to the cell culture media, loss in PNP oxidation and degradation of the CYP2E1 were the same in cells pretreated with or without cAMP. Similar results showing a lack of effect of cAMP on human CYP2E1 degradation in the MV2E1-9 cells were also obtained with pulse–chase immunoprecipitation methods as the loss in labeled CYP2E1 was identical in cells treated with dibutyryl cAMP as with cells not treated (data not shown). Screening of various reagents for their effect on degradation of human CYP2E1. In order to evaluate which proteinases contribute towards the degradation of control CYP2E1, we screened a variety of reagents or inhibitors, including physiological metabolites, lysosomal inhibitors, protease inhibitors, and antioxidants, assessing their ability to maintain PNP oxidation rates after the addition of cycloheximide. In these experiments, MV2E1-9 cells were first treated with 40 mM cycloheximide, in the absence or presence of various additions. After 8 h, microsomal oxidation of PNP was determined. The cycloheximide treatment caused a 76% loss in PNP oxidation after 8 h incubation in the ab-

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FIG. 3. Determination of CYP2E1 turnover in the MV2E1-9 cells. The cells were metabolically labeled with [35S]methionine at a concentration of 50 mCi/ml for 1.5 h and chased with 300 mg/ml cold methionine. The cells were lysed with lysis buffer at the indicated times (0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 h) following the addition of the cold methionine. CYP2E1 was immunoprecipitated with anti-human CYP2E1 IgG–protein G–agarose and analyzed as described under Materials and Methods. (A) SDS–PAGE analysis of the content of the labeled CYP2E1. Lanes correspond to the following: 1–3, 4–6, 7–9, 10–12, 13–15, 16–18, 19–21, 22–24, 25–27, 28–30, the cells chased at 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 h, respectively. (B) Quantitative analysis of the labeled CYP2E1 protein. Following autoradiography, the signals shown in (A) were analyzed using Image Quant software.

sence of any additions. The following additions had no protective effect against this loss of PNP oxidation: 0.2 mM TPCK, 10 mM NH4Cl, 5 mM leucine, 100 mM glycine, 2 mM clotrimazole, 5 mM lactic acid, 15 mM Nacetylcysteine, 0.4 mM diamide. The following additions gave a small protective effect, changing the 76% loss in PNP oxidation to about a 50 to 60% loss: 2 mM EDTA, 2 mM EGTA, 210 units/ml aprotinin, 800 mM APMSF, 1 mM trolox (synthetic vitamin E analogue), 5 mM glutamine, 5 mM histidine, 333 mM leupeptin, 30 mM acetonitrile, 2 mM biotin. Full protection was afforded by 5 mM nicotinamide, while 200 mM TLCK changed the 76% loss in PNP oxidation to only a 21% loss. Effect of calpeptin inhibitor and PSI on CYP2E1 turnover. To evaluate a role for the proteasome complex in the degradation of CYP2E1 and CCl4-modified CYP2E1, the effect of the substrate analogue peptidyl aldehyde, PSI was determined. Calpeptin inhibitor, a peptidyl aldehydic nonproteosomal cytosolic protease inhibitor, was also evaluated to try to minimize the possibility of nonspecific effects by PSI. PSI and calpeptin inhibitor were dissolved in methanol, which at the final concentration used in these experiments (5 mM) did not significantly affect CYP2E1 turnover. Initial experiments validated that PSI inhibited the proteosome complex in the transduced HepG2 cells; addi-

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tion of 80 mM PSI to cell extracts resulted in 95% inhibition of proteolysis of the synthetic peptide Boc-LeuSer-Thr-Arg-AMC, a relatively specific substrate for the proteosome (37). Similarly, extracts from cells treated with 40 mM PSI showed decreased activity with this peptide substrate compared to control extracts, whereas treatment with calpeptin inhibitor had no effect on hydrolysis of the synthetic peptide. PSI proved to be effective in preventing CYP2E1 degradation. Results in Fig. 5 show that compared with the initial labeling intensity, the remaining labeled CYP2E1 signal after a 12-h chase was about 10% (lanes 3 and 4 compared to lanes 1 and 2; Fig. 7). PSI produced a concentration-dependent protection against CYP2E1 degradation over the range of 5 to 80 mM PSI (Fig. 5, lanes 11 to 20; Fig. 7). Significant protection could be observed at 10 mM PSI; at 40 to 80 mM PSI, about 70 to 75% of the labeled CYP2E1 remained after the 12h chase. Calpeptin inhibitor, over the range of 20 to 80 mM, provided little or no protection (Fig. 5, lanes 5 to 10; Fig. 7). TLCK provided protection against CYP2E1 degradation, but was not as effective as identical concentrations of PSI (Fig. 5, lanes 21 to 30; Fig. 7). At 80 mM TLCK, about 40% of the labeled CYP2E1 remained after the 12-h chase compared to 75% remaining in the presence of 80 mM PSI.

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Similar results were obtained with the CCl4-modified CYP2E1. Compared to the initial labeling intensity, the remaining labeled CYP2E1 signal after a 12-h chase in the presence of 2 mM CCl4 was less than 5% (Fig. 6, lanes 3 and 4 compared to lanes 1 and 2; Fig. 7). PSI produced a concentration-dependent protection against degradation of the CCl4-modified CYP2E1 (Fig. 6, lanes 11 to 20; Fig. 7). At 80 mM PSI, about 65% of the labeled CYP2E1 remained after the 12-h chase. Calpeptin inhibitor had no effect on the turnover of CYP2E1 in the presence of CCl4 (Fig. 6, lanes 5 to 10; Fig. 7). TLCK over the concentration range of 5 to 80 mM, did not protect against degradation of the CCl4-modified CYP2E1 (Fig. 6, lanes 21 to 30; Fig. 7).

FIG. 5. Dose-dependent effects of PSI, calpeptin inhibitor, and TLCK on the loss of labeled CYP2E1 in MV2E1-9 cells. The cells were labeled with [35S]methionine at a concentration of 50 mCi/ml for 1.5 h and chased with 300 mg/ml cold methionine. The cells were lysed at 12 h following the addition of the cold methionine. CYP2E1 was immunoprecipitated with anti-human CYP2E1 IgG–protein G– agarose and analyzed as described under Materials and Methods. Lanes correspond to the following: lanes 1 to 4, cells treated without chemicals and chased at 0 (lanes 1 and 2) and 12 (lanes 3 and 4) h; lanes 5 to 10, cells treated with calpeptin inhibitor at concentrations of 20 (lanes 5 and 6), 40 (lanes 7 and 8), and 80 (lanes 9 and 10) mM and chased for 12 h; lanes 11 to 20, cells treated with PSI at concentrations of 5 (lanes 11 and 12), 10 (lanes 13 and 14), 20 (lanes 15 and 16), 40 (lanes 17 and 18), and 80 (lanes 19 and 20) mM and chased for 12 h; lanes 21 to 30, cells treated with TLCK at concentrations of 5 (21 and 22), 10 (23 and 24), 20 (25 and 26), 40 (27 and 28), and 80 (29 and 30) mM, respectively, and chased for 12 h.

FIG. 4. Effect of cAMP on the loss of PNP catalytic activity and apoprotein content in MV2E1-9 cells. For CYP2E1 induction experiments, the microsomes were prepared from cells treated with or without 2 mM dibutyryl cAMP for 8 h. For CYP2E1 degradation experiments, cycloheximide was added to the cell culture media at a concentration of 40 mM to stop new protein synthesis and the microsomes were prepared from the cells at 8 h following the addition of the cycloheximide in the presence or absence of 2 mM dibutyryl cAMP. (A) PNP catalytic activity assay in MV2E1-9 cells. Experiments are from three separate preparations of cells. (B) Quantitative analysis of CYP2E1 apoprotein on Western blot. 40 mg microsomal protein was used for immunoblot analysis as described under Materials and Methods. Specific CYP2E1 hybridization signals were quantitatively analyzed using Image Quant software. Experiments are from three separate preparations of cells.

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PSI could theoretically prevent CYP2E1 degradation if this compound was a ligand for CYP2E1, analogous to results with 4-MP, DMSO, and glycerol. Inability of calpeptin inhibitor to protect suggests some specificity for the actions of PSI. PSI, added in vitro at concentrations up to 300 mM, had no effect on oxidation of PNP by microsomes, suggesting that PSI was not acting as a ligand or substrate for CYP2E1. DISCUSSION

Experiments using two different methods were carried out to characterize CYP2E1 turnover in HepG2 cells transfected with human CYP2E1 cDNA. One method used cycloheximide to stop new protein synthesis, and the remaining CYP2E1 in the cells was analyzed by both PNP catalytic activity and immunoblots to estimate the apoprotein content. In this model, the

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half-life of human CYP2E1 was found to be about 5 to 6 h as calculated by PNP activity and by Western blots. CYP2E1 substrates such as ethanol, 4-MP, and DMSO protected CYP2E1 against loss of activity or degradation, whereas CCl4 accelerated these processes. The second method, which involved pulse–chase experiments with [35S]methionine and immunoprecipitation with anti-human CYP2E1 antibody, supported the above results; the half-life of CYP2E1 was about 2.5 h, and ethanol and 4-MP protected against this rapid loss of labeled CYP2E1, whereas CCl4 accelerated this process. Thus, human CYP2E1 is similar to rodent and rabbit CYP2E1 with respect to rapid turnover and modulation of this turnover by ligands and substrates. The proteolytic degradation pathways responsible

FIG. 7. Quantitative analysis of the effects of PSI, calpeptin inhibitor, and TLCK on the loss of normal and CCl4-modified labeled CYP2E1. Following autoradiography, specific CYP2E1 hybridization signals were quantitatively analyzed using Image Quant software. Results are from the autoradiograms shown in Figs. 5 and 6.

FIG. 6. Dose-dependent effects of PSI, calpeptin inhibitor, and TLCK on the loss of CCl4-modified labeled CYP2E1 in MV2E1-9 cells. The cells were labeled with [35S]methionine at a concentration of 50 mCi/ml for 1.5 h and chased with 300 mg/ml cold methionine plus 2 mM CCl4 . The cells were lysed at 12 h following the addition of the cold methionine. CYP2E1 was immunoprecipitated with anti-human CYP2E1 IgG–protein G–agarose and analyzed as described under Materials and Methods. Lanes correspond to the following: lanes 1 to 4, cells treated with 2 mM CCl4 and chased at 0 (lanes 1 and 2) and 12 (lanes 3 and 4) h; lanes 5 to 10, the cells were treated with 2 mM CCl4 and calpeptin inhibitor at concentrations of 20 (lanes 5 and 6), 40 (lanes 7 and 8), and 80 (lanes 9 and 10) mM and chased for 12 h; lanes 11 to 20, cells treated with 2 mM CCl4 plus PSI at concentrations of 5 (lanes 11 and 12), 10 (lanes 13 and 14), 20 (lanes 15 and 16), 40 (lanes 17 and 18), and 80 (lanes 19 and 20) mM and chased for 12 h; lanes 21 to 30, cells were treated with 2 mM CCl4 plus TLCK at concentrations of 5 (21 and 22), 10 (23 and 24), 20 (25 and 26), 40 (27 and 28), and 80 (29 and 30) mM, respectively, and chased for 12 h.

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for CYP2E1 rapid degradation have received considerable attention and two different mechanisms have been postulated. One involves a cAMP-dependent phosphorylation of CYP2E1 followed by heme loss and subsequent apoprotein degradation by serine proteinases present in the endoplasmic reticulum which exhibit proteolytic activities in vitro toward detergent-solubilized rat liver CYP2E1 (21). Substrates or ligand are postulated to prevent CYP2E1 from rapid degradation by blocking the recognition sites of phosphorylation. CCl4 can also directly trigger this rapid degradation process in the absence of phosphorylation by covalent binding to the heme of CYP2E1. A recent study using a Ser-129 site-directed mutant suggested that such phosphorylation may have little if any role in regulating CYP2E1 expression (38). In order to determine whether cAMP can modulate human CYP2E1 turnover in the HepG2 cell model, the effect of dibutyryl cAMP on CYP2E1 degradation and turnover was evaluated. Dibutyryl cAMP slightly increased both CYP2E1 catalytic activity and apoprotein content when added to the MV2E1-9 cells. No obvious effect of the cAMP was observed on the rapid degradation of CYP2E1 as determined using the cycloheximide method and by pulse– chase experiments with [35S]methionine. These results suggest that in the HepG2 cell model, cAMP-dependent phosphorylation may not be involved in the proteolytic degradation of human CYP2E1. Additional results which do not support a general phosphorylation-linked hypothesis is that, although addition of the protein ki-

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nase c activator PMA to the cell culture media resulted in a sixfold increase in both catalytic activity and content of CYP2E1, there is no apparent effect on the degradation rate of CYP2E1 in the PMA-treated cells compared with the non-PMA-treated cells or on the ability of substrates and ligands to protect against degradation (Fig. 2). In other experiments, treatment of the transduced HepG2 cells with inhibitors of PKC and PKA, such as 20 mM H7 or 20 mM H8, for 12 to 24 h, had no effect on oxidation of PNP or on the degradation of CYP2E1 (data not shown). Another mechanism for the rapid proteolysis of CYP2E1 proposed by Koop and co-workers and Roberts et al. (12, 17, 22, 35) appears to involve ubiquitin proteolytic pathways. Experiments in vitro and in vivo have demonstrated that disappearance of the rat or mouse CYP2E1 apoprotein band on Western blots is accompanied by the appearance of ubiquitin conjugates at higher molecular weight ranging from 80 to 200 kDa, suggesting that proteinases within the cytosol are responsible for the rapid degradation of CYP2E1. Ubiquitin conjugates appear to be especially prominent after CCl4 treatment (17, 22). Although an ATP-dependent cytosolic protease system has been shown to catalyze the degradation of rat and human CYP2E1 (17, 34), a specific role of the proteasome in this degradation has not yet been established. The 20 S proteasome is a cylinder-shaped protein complex consisting of 28 subunits with molecular masses of 21–32 kDa which constitute a major extra lysosomal proteolytic system responsible (as the ‘‘catalytic core’’ of a larger 26 S complex) for ubiquitin-dependent and ubiquitin-independent pathways of intracellular proteolysis. The mammalian proteasome exhibits at least five distinctive endopeptidase activities; chymotrypsin-like (ChT-L), trypsin-like, peptidylglutamyl-peptide hydrolyzing, branched chain amino acid preferring, and small neutral amino acid preferring. A role for proteasomes has been implicated in degradation of abnormal proteins, degradation of short-lived regulatory proteins, and antigen presentation (27–30). Reagents that inhibit the proteasome proteolytic pathway in intact cells, such as the substrate-related peptidyl aldehydes Z-IE(OtBu)AL-CHO, can specifically inhibit ChT-L activities by binding at the active site of the proteasome to form a hemiacetal with the putative serine of the catalytic center. In the current studies, we used PSI as a cell-penetrating proteosome inhibitor, calpeptin inhibitor as an inhibitor of cytosolic calcium activated proteases, leupeptin as a lysosomal inhibitor, and TLCK as a serine-protease inhibitor to examine their effects on both normal and CCl4-stimulated degradation of the human CYP2E1. PSI at concentrations of 5 to 80 mM exhibited a dose-dependent protection of both normal and CCl4-modified human CYP2E1 against rapid degradation within the cells, whereas cal-

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FIG. 8. Hypothetical model for the degradation of human CYP2E1.

peptin inhibitor and leupeptin had no effect on this degradation process. TLCK gave some protection. The above results suggest that proteasomes may be involved in both the normal and CCl4-modified human CYP2E1 degradation process. One consequence of diminishing the proteolytic degradation rate of CYP2E1 should be accumulation of this enzyme within the cells. Treatment with 40 mM PSI for 24 h was found to produce a twofold accumulation of CYP2E1 within the cells under steady state conditions (34). A suggested working model for CYP2E1 degradation (modified from Ref. 40) is shown in Fig. 8. cAMPdependent phosphorylation does not appear to be involved in human CYP2E1 degradation in the transfected HepG2 cells. Proteasomes appear to be involved in the regulation of both normal and CCl4stimulated human CYP2E1 turnover in the HepG2 cell model although it is recognized that other inhibitors, e.g., lactacystin (39), should be evaluated. Substrates may protect CYP2E1 from proteasome degradation perhaps by causing conformational changes which prevent interaction with the proteasome or with ubiquitin-e3 complex; substrates may divert CYP2E1 degradation to a lysosomal pathway (40), preventing the rapid degradation process catalyzed by the proteasome. Recent studies show that much of the CYP2E1 is localized on the endoplasmic reticulum – cytosolic surface (41, 42), hence, making the enzyme available for cytosolic proteolysis. Whether the proteosome complex is involved in degradation of other cytochrome P450 isoforms remains to be studied. Further experiments with PSI, other proteosome inhibitors, and with cell-free systems to characterize the regulation of human CYP2E1 turnover are currently under investigation.

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TURNOVER OF HUMAN CYP2E1 IN HepG2 CELLS

ACKNOWLEDGMENTS These studies were supported by USPHS Grant AA-06610 from The National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Pilar Visco Cenizal for typing the manuscript, Dr. Sherwin Wilk for his generous gift of PSI and calpeptin inhibitor, and Dr. J. Lasker for providing the anti-human CYP2E1 IgG.

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