(Received for publication, August 2, 1995)
From the
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 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.
A member of the microsomal P-450 family of proteins,
P-450 (CYP2E1), (
)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.
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 NaHCO
, 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 NaHCO
(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
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 = 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
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. (
)The
loss of CYP2E1 was accompanied by the appearance of high M
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 -macroglobulin (4
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
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 ()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.