©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ethanol Induces CYP2E1 by Protein Stabilization
ROLE OF UBIQUITIN CONJUGATION IN THE RAPID DEGRADATION OF CYP2E1 (*)

(Received for publication, August 2, 1995)

Ben J. Roberts (1)(§) Byoung-Joon Song (2) Yunjo Soh (2) Sang S. Park (3) Susan E. Shoaf (1)

From the  (1)Laboratory of Clinical Studies and the (2)Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892-1256 and the (3)Laboratory of Comparative Carcinogenesis, NCI, National Institutes of Health, Frederick, Maryland 21702

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the present study, we demonstrate that ethanol induces CYP2E1 by protein stabilization in vivo. The control half-life of CYP2E1 was determined to be 6-7 h followed by a slower secondary phase. The half-life of ethanol-stabilized CYP2E1 was calculated to be 38 h. The mechanism underlying the rapid degradation of CYP2E1 was also investigated and appears to involve the ubiquitin-proteasome proteolytic pathway. An in vitro assay using the cytosolic fraction was developed to further characterize CYP2E1 degradation. Using this assay, 40-50% loss of CYP2E1 was observed in 1 h, coincident with the formation of high M(r) ubiquitin-CYP2E1 conjugates. At concentrations approximating those found in vivo, ethanol protects CYP2E1 from cytosolic degradation. No loss of CYP2B1/2 was observed under identical conditions, suggesting that this reaction is specific for certain P-450s which are rapidly turned over.


INTRODUCTION

A member of the microsomal P-450 family of proteins, P-450 (CYP2E1), (^1)is induced by a variety of chemicals and physiological states, some of which have been linked to the development of liver disease and carcinogenesis via oxidative stress(1) . The most significant chemical inducer of CYP2E1 is ethanol, a compound which is also a substrate for the protein. Debate still exists as to the precise mechanism of CYP2E1 induction by ethanol, with some reports indicating that ethanol stabilizes CYP2E1 in vitro(2) and another group suggesting that ethanol induces CYP2E1 by increasing protein synthesis(3) . In the present study, we have attempted to define the nature of CYP2E1 regulation by ethanol in vivo using controls, chronic ethanol-treated, and ethanol-withdrawing animals. Results from CYP2E1 pulse-labeling show that ethanol induces CYP2E1 predominantly by protein stabilization, with the administration of ethanol attenuating the rapid phase of CYP2E1 degradation. We have also characterized the pathway responsible for this degradation, and present data in vivo and in vitro that suggest ubiquitin conjugation may target CYP2E1 for rapid proteolysis.


MATERIALS AND METHODS

Reagents

NaH^14CO(3) (specific activity 55 mCi/mmol) was purchased from Amersham Corp. Cyanogen bromide (CNBr)-activated Sepharose 4B was obtained from Pharmacia Biotech Inc. (Uppsala, Sweden). Lieber-deCarli Shake and Pour Liquid Diet (LD`82) and Isocaloric Control Diet were obtained from Bio-Serv (Frenchtown, NJ), as were all feeding tubes and accessories. Ethanol (190 proof) was purchased from Midwest Grain Products Co. (Atchison, KS). N-Nitrosodimethylamine was purchased from Aldrich Chemical Co. Anti-ubiquitin IgG and NAD-alcohol dehydrogenase ethanol determination kits were purchased from Sigma. Immunoblot and protein determination kits were purchased from Pierce. Protease inhibitors (leupeptin, aprotinin, and alpha(2)-macroglobulin) were obtained from Boehringer Mannheim.

In Vivo Radiolabeling

Outbred male Sprague-Dawley rats (100-120 g) were obtained from Taconic Farms (Germantown, NY) and kept in temperature-controlled rooms with a 12-h light-dark cycle. Throughout the study, animals were housed, maintained, and treated in accordance with National Institutes of Health guidelines. Rats were administered ethanol (35% of total calories) as part of the Lieber-deCarli liquid diet. An additional group was fed isocaloric maltose-dextrin liquid control diet. Animals were allowed free access to ethanol for 2 weeks, in order to maximally induce CYP2E1, and euthanized at various time intervals during ethanol exposure and withdrawal as indicated. Following injection of a single pulse of NaH^14CO(3) (9 mCi/kg), groups of 2 animals were sacrificed at 1, 2, 3, 4, 5, 9, 12, 24, and 48 h in the ethanol-treated group. In controls, groups of 2 animals were sacrificed 1, 2, 3, 4, 5, 10, 12, 24, and 48 h following radiolabel injection. In the withdrawal group, ethanol was replaced with standard control liquid diet 2 h following radiolabel injection, in order to ensure that during withdrawal the starting specific activity of ^14C-labeled CYP2E1 was the same as that of the ethanol-treated animals. During ethanol withdrawal, individual animals were sacrificed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, and 48 h after the removal of ethanol diet.

Preparation of Microsomal and Plasma Fractions

After sacrifice of animals by decapitation, trunk blood was collected, spun down, and plasma was stored at -70 °C for subsequent blood ethanol analysis using the Sigma NAD-alcohol dehydrogenase kit. All animals exhibited blood ethanol concentrations between 0.15 and 0.25% w/v at the time of sacrifice, with the withdrawal group showing no detectable blood ethanol levels 3 h after ethanol removal (data not shown). Livers were removed, frozen in liquid nitrogen, and stored at -70 °C for subsequent microsomal preparation. Microsomes were isolated by differential centrifugation (4) and stored at -70 °C prior to enzyme analysis, immunoblotting, and immunopurification. Protein content was determined using the Pierce kit.

Immunopurification of CYP2E1

Mouse monoclonal antibody 1-98-1 raised against rat CYP2E1 was used throughout the study according to the immunopurification method described by Cheng et al.(5) with modifications as per Song et al.(6) . This antibody has previously been shown to recognize only CYP2E1. To obtain sufficient pure protein for specific activity determination in ethanol-treated and control animals, CYP2E1 was purified from pooled liver microsomes (n = 2) at the times described in the previous section. In the case of the ethanol withdrawal group, CYP2E1 was purified from the liver microsomes of individual animals 2, 3, 4, 5, 6, 8, 10, 12, 14, 24, and 48 h following withdrawal. After calculating the amount of purified CYP2E1 by gel electrophoresis, the radioactivity of each sample was determined by scintillation counting as described previously(6) . Specific activity is expressed as disintegrations/min/nmol of CYP2E1.

Immunoblot Analyses

Immunoblotting of CYP2E1 was carried out as described previously (4) using a rabbit polyclonal antibody specific to the rat 2E1 antigen. CYP2B1/2 was detected using an antibody which detects 3 bands in the 50-kDa range, the highest molecular mass of which corresponds to CYP2B1/2(4) . Bands were visualized using the Pierce NBT/BCIP substrate staining kit. The anti-ubiquitin IgG used in this study has been reported previously to detect high molecular weight ubiquitin conjugates in mouse liver endoplasmic reticulum(7) .

In Vitro Assay for CYP2E1 Degradation

The 105,000 times g supernatant (cytosol) was prepared by differential centrifugation from control livers previously frozen in liquid nitrogen and used in an assay with ethanol-induced microsomes. CYP2E1 degradation was determined in the presence of 0.75 ng of leupeptin, 6 mM MgCl(2), 0.6 mM ATP, 0.2 mM NADPH, 5 mg of microsomes, 50 mg of cytosol, and 0.1 M Tris, 0.25 M sucrose, pH 7.4, in a total assay volume of 3 ml. To a separate set of ethanol-induced microsomes was added CCl(4) (20 mM) as a positive control for CYP2E1 ubiquitination. Microsomal samples with and without CCl(4) were preincubated at 37 °C for 1 h in the presence of NADPH and MgCl(2) in a 1-ml volume, prior to the addition of cytosol, ATP, and leupeptin (total, 3 ml). ATP was added to start the reaction. Controls were treated in an identical manner except that 0.1 M Tris, 0.25 M sucrose, pH 7.4, was substituted for cytosol. The incubation was carried out in a shaking water bath at 37 °C for 1 h, and the tubes were placed on ice immediately to stop the reaction. Microsomes were reisolated and probed with the antibodies outlined under Immunoblot Analyses. Changes in CYP2E1 levels and ubiquitin-CYP2E1 conjugates were quantitated using a computing densitometer (Molecular Dynamics).

Statistical Analyses

Rates of CYP2E1 turnover were calculated from control and ethanol-treated animals by regression analysis. Control data were fitted to a 2-compartment model and ethanol data by simple linear regression.


RESULTS AND DISCUSSION

The regulation of CYP2E1 activity can be modulated at several levels, ranging from transcriptional to post-translational induction and inhibition. Diet, starvation, and chemical administration all facilitate changes in CYP2E1 activity, suggesting that together with its high level of constitutive expression it may play an important role in the oxidation of endogenous substrates(8) . It has been proposed that substrate/inducers of CYP2E1 such as acetone, 4-methylpyrazole, and ethanol bind to CYP2E1, producing a stable conformation that resists proteolytic degradation(6, 9, 10) . The net result is in fact not ``induction'' per se, but the accumulation of substrate-stabilized CYP2E1. Evidence from this laboratory and others supports this concept; however, direct proof that ethanol (the most clinically significant substrate) stabilizes CYP2E1 in vivo remains elusive. Surprisingly, the only other protein turnover study conducted with ethanol suggests that an increase in protein synthesis accounts for CYP2E1 induction(3) . The authors concluded that protein stabilization is not involved, with control and ethanol-treated animals exhibiting a monophasic half-life of 27 h.

In the present study we demonstrate that CYP2E1 is degraded in control rat liver smooth endoplasmic reticulum with a rapid half-life of 6-7 h, followed by a slower secondary phase (Fig. 1). Conversely, immunopurified CYP2E1 from ethanol-treated animals exhibits a much slower monophasic half-life of 38 h. Overall, 93% of radiolabeled CYP2E1 is lost from controls in the first 48 h and 50% from ethanol-induced microsomes. In ethanol-treated animals, the starting specific activity of CYP2E1 is approximately 5-fold lower than that of controls, a finding that reflects the dilution of specific activity by pre-existing ethanol-stabilized CYP2E1. This phenomenon has previously been reported with acetone(6) . If CYP2E1 induction were due to increases in translational efficiency, mRNA levels, or transcription rate, then the specific activity of CYP2E1 would start at a level approximately equal to that of controls. This was not observed in the present study, suggesting that together with an altered half-life, protein stabilization is the primary mechanism of CYP2E1 induction by ethanol. During ethanol withdrawal (Fig. 1, B and C), the specific activity of CYP2E1 decreases for the first 2-4 h and then rises gradually to a maximum 12 h later. At this and subsequent time points, the degradation curve of CYP2E1 is virtually indistinguishable from controls. This observation is explained by the differing steady state levels of unlabeled CYP2E1 prior to pulse injection. During withdrawal, unlabeled induced 2E1 is rapidly degraded (together with labeled) such that after the first few hours the specific activity of CYP2E1 begins to rise as it approaches control steady state. This process results in a specific activity comparable to controls after 12 h, suggesting that the degradation of induced CYP2E1 is virtually complete by this time(11) . The findings for the first phase of this study are in agreement with the concept of protein stabilization reported previously in the literature (6, 9, 10) but are directly in contrast to the prior turnover study reported by Tsutsumi et al.(3) . In the previous study(3) , methionine was used as the radioactive precursor; however, previous literature suggests that this label is highly reutilized in protein synthesis(12) , thus rendering it unsuitable for the study of proteins with fast turnover rates. This problem is largely abrogated with the use of NaH^14CO(3), a precursor exhibiting substantially lower levels of reutilization (12) .


Figure 1: Turnover of radiolabeled CYP2E1 during control conditions, ethanol treatment, and ethanol withdrawal. Animals were injected with NaH^14CO(3) (9 mCi/kg) and sacrificed at the time points indicated on the x axis in A, B, and C. In each figure, the y axis denotes the specific activity of CYP2E1 in disintegrations/min/nmol of purified CYP2E1. In B and C, the degradation curves for controls (B) and ethanol-treated animals (C) were replotted against CYP2E1 purified from animals in the ethanol withdrawal group. The y and x axes are the same as shown for A. A typical Coomassie Blue-stained gel of immunopurified CYP2E1 is shown in D. Relative migration of M(r) standards are shown on the left.



The rapid loss of CYP2E1 during ethanol withdrawal prompted us to investigate the proteolytic pathway responsible. Previously, Eliasson et al.(9, 10) have reported the existence of a microsomal degradation system responsible for the rapid degradation of CYP2E1. Alternatively, cytosolic ubiquitin (M(r) = 8,600) has been associated with the formation of high molecular weight ubiquitin-CYP2E1 conjugates following treatment with the CYP2E1 suicide substrate, carbon tetrachloride(7) . The contribution of cytosolic versus membranous proteolytic systems was resolved directly using an assay similar to that described by Correia et al. (13) with 105,000 times g supernatant (hepatic cytosol). The results from this assay are presented in Fig. 2and 3. Carbon tetrachloride treatment in vitro followed by cytosolic incubation (Fig. 2A) resulted in substantial and rapid CYP2E1 degradation. (^2)The loss of CYP2E1 was accompanied by the appearance of high M(r) ubiquitin conjugates (Fig. 2B) and is NADPH-dependent. When microsomes are incubated without carbon tetrachloride (Fig. 2C), ubiquitin conjugation occurs; however, the degradation of CYP2E1 and formation of Ub-2E1 conjugates are comparatively lower. The pattern of ubiquitin conjugates observed in this study is somewhat typical of that previously described in the literature, ranging from 80 to well over 200 kDa in molecular mass. In samples without cytosol, there is no significant loss of CYP2E1 from the microsomal fraction in the presence of ATP or carbon tetrachloride (Fig. 3A). This finding suggests that the microsomal fraction does not possess a self-contained proteolytic system capable of degrading CYP2E1 under nonsolubilized conditions, as previously proposed(9, 10) . In the presence of leupeptin and ATP, 40-50% of ethanol-induced CYP2E1 is degraded in a period of 1 h, compared to 25-30% without exogenous ATP (Fig. 3A). The observation that CYP2E1 is degraded by the cytosol in the absence of exogenous ATP or ubiquitin is not surprising, given previous reports that suggest substantial pools of free ubiquitin and ATP are present in crude lysates (14) and hepatic cytosol(15) . In this context, it should be stressed that under our conditions the proteasome itself could conceivably degrade CYP2E1 without the advent of ubiquitination. The relative contribution of proteasomal versus ubiquitin-mediated degradation of CYP2E1 awaits further clarification. When ethanol was added to the incubates at physiological concentrations (60 mM) CYP2E1 proteolysis was inhibited (Fig. 3A), concordant with the pulse label data generated in vivo. Constant maintenance of these ethanol concentrations in vivo is therefore likely to result in the progressive accumulation of CYP2E1. To verify that this system is selective in the degradation of rapid turnover P-450s, we also measured CYP2B1/2 under identical conditions described to remove 40-50% of CYP2E1. No loss of CYP2B1/2 (Fig.3B) was observed in any samples in direct contrast to the degradation of CYP2E1. Therefore, it appears that CYP2B1/2 is either a poor substrate for the ubiquitin-proteasome proteolytic pathway, or alternatively, that it is not recognized by the enzymes involved in effecting ubiquitin conjugation and is degraded in a slower lysosomal process (16) which may also be responsible for the removal of acetone-stabilized CYP2E1 (17) . These data are consistent with the slow turnover of CYP2B1/2 reported in vivo(18) and in vitro(10) in other studies and is in agreement with the data generated in the cytosolic assay.


Figure 2: Formation of high molecular weight ubiquitin-CYP2E1 conjugates. Following cytosolic incubation as described under ``Materials and Methods,'' microsomes (25 µg/well) were reisolated, electrophoresed, and immunoblotted with antibodies specific to CYP2E1 and ubiquitin. In A, a typical experiment shows the relative degradation of CYP2E1 after cytosolic incubation. In B, ubiquitin conjugation of CYP2E1 was studied over a high molecular weight range in the presence of a mixture of protease inhibitors, consisting of leupeptin (7 ng), aprotinin (8 ng) and alpha(2)-macroglobulin (4 times 10 units). Lane 1 corresponds to microsomes pretreated with carbon tetrachloride (20 mM); lane 2, control. In C, microsomes were probed with CYP2E1 and ubiquitin antibodies in the absence (lane 1) and presence of hepatic cytosol (lane 2 and 3). Assay conditions were otherwise identical. Ubiquitin-CYP2E1 (UB-CYP2E1) conjugates and M(r) standards are as shown.




Figure 3: Effect of various experimental conditions on the degradation of CYP2E1 and CYP2B1/2. In A, the results from 4-6 separate determinations of CYP2E1 degradation under various conditions are shown. Microsomes from ethanol-treated animals were used in each case. For ease of interpretation, statistical significance is calculated relative to the ATP + cytosol group designated on the figure. Ethanol and carbon tetrachloride were added at concentrations of 60 mM and 20 mM, respectively. In B, a typical immunoblot for CYP2B1/2 is shown, performed on microsomes havng lost 40-50% of CYP2E1. All bands were quantitated using laser densitometry. *, statistically significant (p <0.05).



The triggering mechanism responsible for CYP2E1 degradation has received considerable attention and is postulated by one group to be mediated by cAMP-dependent phosphorylation of CYP2E1 at Ser-129(9, 10) . This has been reported to result in heme loss and subsequent holoprotein degradation(9) . Conversely, a recent study using a Ser-129 site-directed mutant suggests this residue has little if any role in regulating CYP2E1 expression(19) . Preliminary data (^3)from our laboratory suggest there is no change in the isoelectric point of CYP2E1 under any of the conditions outlined in this study, although we cannot totally dismiss a role for phosphorylation in some component of this pathway (e.g. regulation of an E class protein(20) ). Our findings are suggestive of another explanation, namely that CYP2E1 is a substrate for ubiquitin- and/or proteasome-mediated proteolysis. CYP2E1 is ubiquitin-conjugated in vivo(7) and in vitro (Fig. 2, B and C), although clearly this process is more efficient in removing heme-damaged/stripped CYP2E1 pretreated with carbon tetrachloride (Fig. 3A). Given these observations, protein stabilization by ethanol and the subsequent degradation of CYP2E1 during ethanol withdrawal may eventuate in one of three ways. (i) Substrate binding prevents heme alkylation, thus removing one potential trigger of ubiquitin targeting. (ii) Allosteric modification of CYP2E1 by ligand binding directly inhibits ubiquitin conjugation or interferes with an E3 class protein involved in substrate recognition(21) . (iii) Removal of exogenous substrate/inducers switches the substrate load of CYP2E1 back to endogenous fatty acids, resulting in progressive oxidative damage to the protein. Treatment with the suicide substrate carbon tetrachloride may exaggerate this process by heme destruction (22) or electrophilic protein damage(7) . In these scenarios, a phenomenon such as phosphorylation might be coincident with rather than causative of CYP2E1 degradation. Of broader significance are the emerging data presented in this and previous studies (7, 13) that the cytosolic orientation (23) of the P-450 family of proteins is sufficient to render them substrates for ubiquitin conjugation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, 10 Center Dr. MSC-1256, Bethesda, MD 20892-1256. Tel.: 301-496-4936; Fax: 301-402-0445.

(^1)
The abbreviations used are: CYP2E1, ethanol-inducible cytochrome P-450; CYP2B1/2, the major P-450 isozymes induced by phenobarbital; Ub-2E1, ubiquitin-conjugated P-450.

(^2)
Time course experiments indicated that CCl(4)-treated 2E1 possessed a half-life in the order of 10-15 min in vitro.

(^3)
B. J. Roberts, B.-J. Song, Y. Soh, S. S. Park, and S. E. Shoaf, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Dennis R. Koop for helpful discussion concerning these experiments.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.