(Received for publication, July 19, 1996, and in revised form, December 9, 1996)
From the Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892-1256
The cytochrome P-450 family of enzymes performs an incredibly diverse range of detoxification and oxidation reactions within the cell and constitutes between 5 and 10% of protein in hepatic endoplasmic reticulum. In this report it is demonstrated that constitutively expressed membranous P-450s are targeted for destruction by the proteasome, in a process which is ubiquitin-independent and is demonstrated in vitro to require prior labilization of the enzyme. This process was specific for P-450s CYP1A2, CYP2E1, CYP3A, and CYP4A and was not demonstrated to be involved in the turnover of CYP1A1, CYP2B1/2, or NADPH reductase. In reconstitution experiments using purified proteasomes and microsomal fractions, labilized P-450 conformations are protected from 20 S proteasome degradation by substrate addition, with proteolysis occurring while P-450s are still attached to the endoplasmic reticulum.
The cytochrome P-450 multigene family encodes a broad variety of membranous proteins that generally serve to render exogenous or endogenous compounds hydrophilic. They are inducible, share similar topology (1), and constitute between 5 and 10% of endoplasmic reticulum (ER)1 protein in hepatocytes. The P-450s possess rapid to medium half-lives in the constitutive state (for a review, see Correia (2)), in contrast to NADPH cytochrome c reductase (NADPH reductase) and cytochrome b5, both of which exhibit half-lives on the order of several days (2). These observations suggest a series of specific intracellular events may target the P-450s for rapid proteolysis by an as yet unidentified pathway. Recently, ubiquitination has been associated with membranous proteins, apparently acting as a means of tagging or translocating these proteins for destruction either in the lysosomal vacuole (3) or 26 S proteasome (4, 5). The discrete 20 S proteasome has been implicated in these interactions (4), but unlike the 26 S proteasome it does not possess ATPases capable of unfolding complex globular proteins. Intracellular distribution of the 26 and 20 S proteasomes suggests one third of the 20 S associates with the ER, while the 26 S is found predominantly in the cytosol (6, 7). In this report it is suggested that a major biological role of the 20 S proteasome is to remove cytochrome P-450s from the ER, in a mechanism which is shown in vitro to require prior labilization of the enzyme.
Lactacystin was the generous gift of S. Omura (The Kitasato Institute) and E. Corey (Harvard University). ALLN was purchased from Sigma. All other other chemicals and consumables were of the highest grade commercially available.
Cell Culture, Microsome Preparation, and ImmunoblottingIsolated hepatocytes were obtained from untreated
male Harlan Sprague Dawley rats by the collagenase perfusion method
(95% viable) and seeded on a collagen matrix at a density of 1.5 × 106 cells/well (6 × wells/plate). Cells exhibited
95% attachment prior to the commencement of experiments. Plated cells
were washed thrice with 2 ml of Waymouth's MB 752/1 medium at pH 7.4, containing 2.2 g/liter NaHCO3 supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 5% fetal bovine serum,
and 5 ng/ml dexamethasone. The final wash was conducted with the
addition of lactacystin (25 µM per culture well,
dissolved in Tris-HCl, pH 7.4, and prefiltered through a 0.22-µm
Millipore membrane), ALLN (200 µM per well, dissolved and
prefiltered in dimethyl sulfoxide and added such that the final
concentration of Me2SO was no more than 0.4% v/v). Controls received culture medium only. Primary cultures were incubated for 24 or 30 h in 5% CO2 at 37 °C. Following this
period, culture medium was aspirated off, and cells were washed with 2 ml of ice-cold Tris-HCl, pH 7.4, containing 0.25 M sucrose
and 20 µg of leupeptin (buffer 1). After washing, cells were scraped
and resuspended in 0.3 ml of the Tris buffer. To obtain sufficient
microsomes for analysis, cells from three wells were pooled and used as
starting material. Cells were homogenized in Sorvall 2-ml
ultracentrifuge tubes using a Potter-Elvehjem Teflon pestle in
approximately 1 ml of Tris buffer. Homogenates were centrifuged at
9000 × g for 20 min, and the supernatant was removed
and centrifuged at 105,000 × g for 1 h. All centrifugation
steps were carried out at 4 °C in a Sorvall model TFT-80.2 rotor.
The resultant pellet from the 105,000 × g step
(microsomal) was resuspended in 200 µl of Tris-HCl, pH 7.4, 0.25 M sucrose, 20% v/v glycerol and stored at 70 °C for
further use. Approximately 150 µg of microsomal protein were obtained
using this method. Antibodies to CYP1A2, CYP4A, and CYP3A were
purchased from Amersham Life Science. Polyclonal antisera to CYP2E1 was
the gift of B. J. Song (National Institute of Alcohol Abuse and
Alcoholism) and CYP1A1/2 and CYP2B1/2, J. Hardwick (Northeastern Ohio
Medical College). Bands corresponding to CYP1A1, CYP1A2, CYP2B1, and
CYP2B2 have previously been authenticated by the supplier of these
antibodies. A polyclonal antibody to NADPH reductase was obtained from
Oxford Biomedical Research (Oxford, MI). Anti-ubiquitin IgG was
purchased from Sigma. In all cases 5-20 µg of
microsomal protein were used to determine P-450 levels by
chemiluminescent immunoblotting (Amersham).
The isolation buffer used for dose-response curves was essentially buffer 1 in the absence of leupeptin. All procedures were conducted at 4 °C. Cells were preincubated with lactacystin for 4 h, isolated by scraping, and lysed by homogenization, and the 9000 × g supernatant was used for analysis of chymotryptic activity. Because it is a reversible proteasome inhibitor, ALLN was added to the extracts 10 min prior to the start of the experiment. Preliminary experiments using purified proteasome revealed ALLN to inhibit >95% of chymotryptic activity following this preincubation step. Activity was determined by the cleavage of fluorogenic free 7-amino-4-methylcoumarin/min from the peptide, succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin (SLLVT-AMC). Incubations were carried out at 37 °C in a shaking water bath for 2 h using 5 mM MgCl2, 50 µM SLLVT-AMC, 80 µg of extract, and 0.1 M Tris-HCl, pH 8.0, in a total volume of 1 ml. Absorbance was measured at 380-nm excitation, 440-nm emission, on a Perkin-Elmer model LS-50B fluorimeter.
Ubiquitin conjugates were detected in the 9000 × g
supernatants of 24-h ALLN-treated cells (200 µM) after
extracts were quenched with SDS-PAGE loading buffer. Prior to the this,
cells were isolated either in buffer 1 or buffer 2. Buffer 2 consisted
of buffer 1 plus 5 mM EDTA, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 µg/ml
2-macroglobulin, and 20 µM ALLN.
Generally, 1 ml of each buffer was used to resuspend and homogenize 1 mg of cellular protein. The loss of high molecular mass conjugates
using either buffer 1 or 2 was calculated by ubiquitin IgG staining and
densitometry of the 80-200-kDa region. To enhance detection of
putative P-450 microsomal ubiquitin conjugates, samples were run at 50 µg/lane and transferred on 6% acrylamide gels.
Studies using CYP2E1 were conducted using microsomes from ethanol-treated animals using an administration protocol previously described (8). Experiments with CYP3A utilized microsomes from dexamethasone-treated animals (100 mg/kg/d by oral gavage, for 3 days). Male Harlan Sprague Dawley rats (200-300 g, Taconic Farms, Germantown, NY) were used for all protein and organelle isolates. Animals were housed, maintained, and treated in accordance with National Institutes of Health guidelines. 3,5-Dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) used in these experiments inactivates CYP3A by alkylating its prosthetic heme group and was the generous gift of M. A. Correia (University of California, San Francisco). The rapid destruction of CYP3A by DDEP and CYP2E1 by CCl4 is well documented in vivo (9, 10). 20 and 26 S proteasomes were purified according to the method described by S. Ugai et al. (11). One unit of proteasome activity is defined as the cleavage of 1 nmol of fluorogenic free 7-amino-4-methylcoumarin/min from the peptide, SLLVT-AMC. Based on crude preparations from whole liver it was estimated that the ratio of 20 S proteasome:microsomes used in this study was in the order of 2:1, found normally in the hepatocyte.
Proteasome inhibitors lactacystin and ALLN were incubated with
primary hepatocyte cultures at concentrations of 25 µM
and 200 µM respectively and levels of several
constitutive P-450s determined by immunoblotting. Fig. 1
shows representative blots of CYP2E1 (Fig. 1A) and CYP4A
(Fig. 1B) in the microsomal fraction. Marked loss of CYP2E1
and CYP4A was observed after 24 h of primary culture, with
lactacystin and ALLN conferring a substantial protective effect on the
loss of the two isoforms shown. No staining was observed in the high
molecular mass region of Fig. 1, A and B, following incubation with proteasome inhibitors, suggesting an absence
of P-450 ubiquitin conjugation. Proteins identified as P-450s following
incubation with proteasome inhibitors appeared to migrate identically
on SDS-PAGE as those isolated at t0 and t24 h, and were therefore not considered to
arise via the cleavage action of ubiquitin isopeptidases or other
artefactual mechanisms. When the ER fraction was probed with ubiquitin
IgG (Fig. 1C) no significant changes were observed in higher
molecular mass ubiquitin staining (>80 kDa) either in the absence or
presence of ALLN. It is conceivable that some of the staining observed
with the ubiquitin IgG represents free ubiquitin chains, instead of
ubiquitin-protein conjugates, and therefore would not be expected to
change in the presence of proteasome inhibitors such as ALLN. In the
same experiments, levels of CYP1A2 and CYP3A were also determined
following 24 h of culture with and without proteasome inhibitors.
Again, proteasome inhibitors conferred a significant protective effect
on the degradation of these isoforms without high molecular mass
staining,2 suggesting the involvement of a
proteolytic pathway which is proteasome but not ubiquitin dependent.
Using CYP2E1 as an example, several methods were used to determine
whether P-450 ubiquitin conjugates were present in these cells, but
below the levels of detection used in this study (Fig.
2). First, using a methodology similar to Haas and
Bright (12), a comparison of differing buffer systems was employed to
establish whether loss of ubiquitin conjugates occurred during
preparation. The use of a buffer with a mixture of chelating agents and
protease inhibitors had little effect on ubiquitin conjugate loss when
compared with that of ice-cold standard buffer containing leupeptin
(Fig. 2A). In the time taken for isolation of microsomal
fractions (150 min), some of the very high molecular mass (>200 kDa)
ubiquitin conjugates were "trimmed" to a slightly lower molecular
mass using the standard buffer. Considering a previous report by
Wilkinson et al. (13), it is possible that some of this
activity represents free polyubiquitin chain disassembly by
isopeptidase T or variants thereof. Overall, there was a 20% decrease
in the amount of ubiquitin conjugates between 80 and 200 kDa, the
region where we have conventionally found most P-450-ubiquitin
conjugates to occur. Buffer B appeared to protect against the trimming
of higher molecular mass conjugates during this time. A second approach
was to take cells extracts immediately quenched with SDS-PAGE buffer
and probe with CYP2E1 IgG before the isolation of microsomal fractions
(Fig. 2B). Unfortunately, cytosolic extracts contain several
other proteins that cross-react with polyclonal CYP2E1 antisera.
Despite this cross-reactivity, there is no observable ubiquitin-CYP2E1
in the 100-200-kDa region of the gel upon fresh isolation. Third,
immunoblots were overexposed using chemiluminescence on low percentage
acrylamide gels (Fig. 2C) containing approximately 10-fold
higher protein concentrations than shown in Fig. 1. This procedure
allowed an estimate of detection sensitivity. Using immunoreactive
CYP2E1 as a measure of 100% chemiluminescent density, this procedure
would have allowed conjugates to be detected if they were greater than
5% of total staining. Considering that of the 78% of CYP2E1 lost
after 24 h, lactacystin and ALLN restored 54 and 66%,
respectively, then 90% of the ubiquitin conjugates must have been
degraded or lost during isolation, assuming ubiquitination is the
principal mechanism of P-450 degradation. To discount the possibility
that ubiquitin conjugates were removed by residual proteasome activity,
dose-response curves were constructed using lactacystin and ALLN (Fig.
3). Because lactacystin is an irreversible proteasome
inhibitor, it was possible to preincubate the cells for 4 h with
this chemical, prepare extracts, and assess the level of proteasome
inhibition using chymotryptic cleavage as a prototypical activity.
Under these conditions the Ki was established to be
6 µM with maximal inhibiton achieved at 10 µM (Fig. 3A). Because ALLN is a reversible
inhibitor and washed out during cell isolation, it was added back into
the extracts to determine inhibitory potency (Fig. 3B).
Under these conditions the Ki was estimated to be 30 µM with maximal inhibition at 100 µM. Given
the fact that experiments were conducted at twice the concentration
required for maximal proteasome inhibition, it is reasonable to
conclude that its activity was abolished in these cells. The immunoblot
data from several separate sets of cultured hepatocytes were scanned
densitometrically and are summarized in Table I. CYP2E1
and CYP3A are turned over quite rapidly in cultured hepatocytes, with
CYP1A2 and CYP4A displaying comparatively smaller losses over a 24-h
period. Generally, ALLN was found to have a slightly more potent
inhibitory effect on P-450 turnover in hepatocytes than did
lactacystin, possibly because it affects other proteases in addition to
the proteasome. In the case of CYP2E1 and CYP3A the loss from culture
was not fully blocked by lactacystin and ALLN, suggesting other
proteases may contribute in part to the proteolysis of these forms.
With CYP1A2 and CYP4A, lactacystin and ALLN also significantly
inhibited protein turnover, with these forms intrinsically more stable
than either CYP2E1 or CYP3A in culture. Overall, the amount of
constitutive P-450 recovered after 24 h of culture was 2-4-fold
higher in the presence of proteasome inhibitors.
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Although proteasome-mediated degradation was clearly involved in the
destruction of several P-450s, it was necessary to establish negative
controls in hepatocyte cultures to interpret these findings in the
context of in vivo turnover data, and confirm that
proteasome degradation is a specific event. CYP1A1, CYP1A2, CYP2B1,
CYP2B2, and NADPH reductase were evaluated in primary cultures
following extended incubation (30 h) with ALLN (Fig. 4).
As anticipated from Table II, CYP1A2 was rapidly
degraded in an ALLN-sensitive manner (Fig. 4A). In contrast,
CYP1A1 (Fig. 4A), CYP2B1/2 (Fig. 4B), and NADPH
reductase (Fig. 4C) were relatively stable in culture, and
levels were unaffected by ALLN. Approximately 40-60% of the initial
protein levels were lost over 30 h, suggesting a
proteasome-independent pathway degrades these isoforms.
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The findings from the first part of this study were compared against existing data on the half-lives of several hemoproteins resident in the ER (Table II). Of those investigated CYP2B1/2 and NADPH reductase have been reported to be the most stable in the ER. CYP2E1, CYP3A, and CYP1A2 possess shorter half-lives in the order of 12 h or less, with CYP1A1 falling in between with a value of approximately 15-16 h. No data were available for CYP4A. In this study, the presence of proteasomal degradation was coincident with P-450s exhibiting reported half-lives of 12 h or less and was not observed in ER proteins exhibiting longer half-lives.
To confirm that the proteasome can directly degrade P-450s in the ER, a
reconstituted system was used in which purified proteasomes were
co-incubated with microsomal fractions derived from rat livers (Figs.
5 and 6). Using purified 26 and 20 S
proteasomes, together with suicide inhibitors known to accelerate P-450
proteolysis in vivo, it was possible to provide evidence on
the mechanism of P-450 destruction by the cytosolic proteasome. CYP2E1
and CYP3A were used as representative P-450s because specific
labilizing agents were available, and they are both turned over by the
proteasome in cultured cells. Both CYP2E1 and CYP3A were resistant to
proteolysis using purified preparations of rat liver proteasome (Fig.
5), suggesting P-450s must undergo conformational changes in
vivo to explain the results presented in Table I. The 26 S
proteasome was found to have little effect on deactivated forms of
P-4503; however, the 20 S proteasome
exhibited substantial proteolytic activity toward membranous P-450s
independently of ATP (Fig. 5, A and B). From the
data presented it is clear that CYP2E1 and CYP3A are marked for 20 S
proteasome destruction by the NADPH-dependent activation of
reactive metabolites. In the case of CYP3A, it was found that prolonged
storage of microsomes (>4 months) or repeated cycles of freeze/thawing
labilized this isoform to such an extent that it could be degraded by
the 20 S proteasome. The partial labilization these procedures cause
could be attenuated by the presence of the CYP3A substrate
troleandomycin (TAO, Fig. 5B), suggesting there
is an allosteric modification in the ligand bound state that inhibits
20 S proteolysis of CYP3A.
Using carbon tetrachloride (CCl4) it was possible to track the pattern of CYP2E1 fragmentation by the 20 S proteasome (Fig. 6A). After treatment with this suicide substrate, CYP2E1 is degraded by the 20 S proteasome, with the immediate appearance of a fragment of CYP2E1 at around 32 kDa. This process was blocked in the presence of lactacystin (Fig. 6B). Notably, there is little evidence of any intermediate breakdown products on the blot, suggesting the 20 S proteasome rapidly cleaves a large unfolded segment of CYP2E1 oligopeptide. Reisolation of the microsomal fraction (Fig. 6C) suggests the fragment of CYP2E1 left after initial cleavage is still attached to the ER, awaiting further destruction by the 20 S proteasome. Similar findings were obtained with DDEP-inactivated CYP3A (Fig. 6, B and D); however, no discrete degradation products were reproducibly observed in this study.
Recent findings suggest a number of ER proteins are targeted for destruction, either by ubiquitin conjugation (4, 5, 9, 10, 14) or possibly by the proteasome itself (4, 15). Evidence for the latter is less rigorous, because ubiquitinated proteins can be identified by a variety of techniques, whereas the direct action of the proteasome can only be inferred in whole cells by the recent advent of specific proteasome inhibitors such as lactacystin (16). The cytochrome P-450 family of enzymes is particularly intriguing from the perspective of protein turnover as there is considerable heterogeneity in the turnover rates of different isoforms, despite apparently similar topologies in the ER membrane (1).
Primary hepatocyte cultures from untreated rats exhibit a
characteristic loss of cytochrome P-450s within the first 24 h of plating due to the inability of hepatocytes to maintain steady state
levels of P-450 mRNA (17). The expressed proteins are subsequently
degraded at varying levels via a pathway that is unknown. This property
of primary cultures is somewhat fortuitous as it allows the proteolytic
pathway of several P-450s to be investigated within one study, an issue
which is difficult to address in artificial expression systems. The
role of the cytosolic proteasome in P-450 turnover was investigated
using lactacystin and ALLN. Reports to date indicate that lactacystin
(active metabolite, clasto-lactacystin -lactone) (18) is
a specific inhibitor of the 20 S proteasome (19) and therefore a useful
tool to investigate the role of this protease in various metabolic
processes (4, 5). ALLN (also known as LLnL) is also a potent inhibitor,
although its effects are not entirely confined to the proteasome (19).
Because the 20 S proteasome is the catalytic core of the larger 26 S
proteasome, it would be expected that these inhibitors will prevent the
removal of both ubiquitinated and nonubiquitinated proteins. After
24 h of culture, proteolysis of P-450s from the four major gene
families was evaluated and was found to be blocked by lactacystin and
ALLN (Fig. 1 and Table I). CYP2E1 and CYP3A were most rapidly removed, with CYP1A2 and CYP4A exhibiting greater stability in cultured cells.
Despite these differences in turnover rates, it appears that a feature
common to all is their destruction by a mechanism involving the
proteasome. Surprisingly, there was no indication of P-450
ubiquitination in these cells (Figs. 1 and 2), contrasting with
previous studies in which ubiquitination of P-450s has been shown to
occur following exogenous chemical damage (9, 10). Previously, our
laboratory and others (9, 10, 14) have observed P-450-ubiquitin
conjugates in microsomal preparations as high molecular mass species of
immunoreactive bands detectable by Western blotting. How then, are the
present observations of ubiquitin-independent P-450 degradation to be
reconciled with previous reports? Notably, prior studies on P-450
ubiquitination used highly induced levels of CYP2E1 (10, 14) and CYP3A
(9), such that the concentrations were 5-10-fold higher than the
control levels employed in this study. Because these two isoforms
constitute such a large percentage of ER protein in the induced state
(2-5%) it is conceivable that secondary proteolytic pathways may
become invoked to dispose of them following acute damage.
Ubiquitination may therefore constitute an alternative mechanism by
which P-450 s can be degraded. Furthermore, the relative
contribution of proteasome- versus
ubiquitin-dependent pathways had not been established in previous studies, thus in no way discounting a direct proteolytic event. To date, we do not know whether the previously observed P-450-ubiquitin conjugates arise from ubiquitin tagging of the whole
protein or fragments thereof. If the latter is the case, then initial
cleavage of the protein would be rate-limiting in allowing exposure of
lysine residues and subsequent ubiquitination. Given the many
permutations in which the processing of these enzymes might occur, the
data presented in this study suggest the proteasome, either in the 20 S
form, or contiguous with PA28 or 19 S subunits, degrades CYP1A2,
CYP2E1, CYP3A, and CYP4A.
To confirm that these findings were not the result of nonspecific P-450 proteolysis, or labilization by heme depletion, the culture time was extended, and several other ER hemoproteins known to exhibit long half-lives were examined under otherwise identical conditions (Fig. 4). Out of those tested, CYP1A1, CYP2B1, CYP2B2, and NADPH reductase were lost slowly from culture, in comparison to CYP1A2. Notably, the turnover of these isoforms was unaffected by the addition of ALLN, suggesting these enzymes are not degraded by the proteasome. When the data generated in this study were compared with previous studies investigating P-450 turnover in vivo (Table II), it is apparent that isoforms with a half-life of approximately 12 h or less (14, 20-23) are degraded to a significant extent by a proteasome-mediated pathway, whereas those with longer half-lives (>16 h) (23, 24) are unaffected by the addition of proteasome inhibitors. Collectively, these findings suggest a pattern of P-450 proteolysis, in which rapid turnover is proteasome inhibitor-sensitive, and a slower component that acts independently of the proteasome and may conceivably be part of the lysosomal turnover of ER membrane described by other researchers (25, 26). Lactacystin did not completely block the rapid loss of CYP2E1 and CYP3A from culture (Table I). Approximately 24% of both isoforms is likely to be degraded by alternate pathways, some of which are sensitive to ALLN (Table I). It is conceivable that, if the labilizing events which target CYP2E1 and CYP3A for rapid degradation were allowed to continue unchecked due to inhibition of their disposal pathways, then the enzymes may eventually be formatted to a different proteolytic system (as alluded to earlier in this discussion). Whether proteasome-independent pathways act upon these isoforms under normal conditions in vivo is unknown and the subject of further investigation. In the case of CYP1A2 and CYP4A no significant extra-proteasomal degradation could be demonstrated (Table I), thus during constitutive expression the proteasome is the significant protease in rapid P-450 degradation.
The entrance to the 20 S proteasome is 13 Å in diameter (27) and cannot degrade a native P-450 that is roughly 50 Å across. Therefore the protein must be partially unfolded under its own auspices or molecular chaperones associated with the ER (28). A third possibility is a chaotropic effect exerted by components of the 19 S subunit of the 26 S proteasome; however, this normally requires ubiquitination or antizyme attachment (29). Data presented in Figs. 5 and 6 suggest membranous CYP2E1 and CYP3A are capable of unfolding in the ER to an extent sufficient to allow physical exposure to the 20 S proteasome and subsequent proteolysis. Labilized CYP3A is partially protected from degradation by the addition of the substrate troleandomycin. Although little data is available on the three-dimensional structure of mammalian P-450s, phosphorylation of CYP2E1 and CYP3A1 is blocked upon substrate binding (30, 31) with a concordant shift in enzyme spin-state (32). Based on the data in Fig. 5B, the net result is that ligand-bound P-450s are resistant to proteasome degradation and thereby likely to accumulate when substrates are present (14, 21, 22, 33). With CYP2E1 this scenario is easy to envisage, as CYP2E1 ligands are low molecular mass chemicals and are unlikely to be prevented access to the heme pocket even if changes have occurred in overall protein folding or topology. Whether efficient P-450 catalysis occurs when the enzyme is "induced" in this manner is unclear, and in this context it has been suggested that the coupling of electron transfer to oxygen insertion into the heme-bound substrate is impaired in ethanol-stabilized CYP2E1 (34). Such observations may account for the increase in free radicals and lipid peroxides observed after prolonged ethanol exposure (35). During ethanol withdrawal the ligand is rapidly removed from all tissues, and the protein is subsequently degraded with a half-life of 6 h or less (8). In this respect the 20 S proteasome may be providing a very valuable service by removing a protein that is not only damaged but also hazardous.
The P-450s may be unusually susceptible to destruction by the 20 S proteasome in vivo for several reasons. First, their catalytic mechanism involves oxygen transfer, a pathway by which the potential exists for auto-oxidation (a recurring theme in proteolysis) (36). The P-450 active site is hydrophobic and sequestered from the action of cytosolic antioxidants; thus, contrary to the surface of the protein, the heme and adjacent amino acids are chronically exposed to superoxide radicals and hydrogen peroxide that develop spontaneously from side reactions of normal catalysis (34, 37). Studies using hemoglobin (another hemoprotein) suggest artificial oxidation of this enzyme by hydrogen peroxide targets it for destruction by the 20 S proteasome (38). Within this context, it has been shown that individual P-450s do not exhibit the same propensity to "uncouple" their oxygen transfer in reconstituted systems. CYP2B1/2 has been demonstrated to be less susceptible to this side reaction than CYP2E1 (39), thus providing a possible basis for differential proteolytic susceptibility. Because the half-lives of P-450s are in the order of hours rather than minutes, it is anticipated that these changes are of a gradual nature and may therefore be difficult to reproduce in vitro. Clearly, the challenge of identifying the molecular mechanism/s of P-450 labilization in vivo is an important one.
In addition to biochemical factors, the microsomal P-450s are localized in an organelle that is intimately associated with many 20 S proteasomes (6, 7). Current literature suggests the 20 S proteasome association with the ER is related to antigen processing. The 20 S proteasome may function as a pore by which peptides are transferred to transporters associated with antigen processing or as a final pruning system for the selection of appropriate antigenic peptides (7, 40). Given the high constitutive expression and inducibility of many P-450s by environmental chemicals, it is conceivable that shuffling proteasomes around the ER to process damaged P-450 isoforms significantly influences major histocompatibility complex class 1 antigen presentation in the liver. Ethanol withdrawal is an excellent example of a process in which large quantities of proteasome are allocated to the destruction of a highly induced P-450 (CYP2E1). Considering the breadth of membranous proteins starting to emerge as proteasome and/or ubiquitin targets, it will be particularly interesting to study the dynamics of proteasome distribution and antigen processing in the ER.
I thank S. E. Shoaf for support and encouragement during these experiments, and also D. R. Koop and B. J. Song for their helpful discussions. Sterile culture facilities were generously provided by R. L. Eskay.