![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Pacific Northwest National Laboratory, Richland, Washington (R.C.Z., S.S., N.B., D.W.L.); Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (T.A.K.); and School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri (M.S.D.)
Received September 19, 2002; accepted February 19, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
55-kDa
CYP3A band, opposite to what would be expected if the ubiquitin-proteasome
pathway degraded CYP3A. Only hemin treatment caused an increase in high
molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels
of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in
response to hemin treatment clearly were not due to proteasome inhibition.
Rather, because hemin treatment also caused an increase in HMM CYP3A in the
detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A
seems to represent a large protein complex that is unlikely to primarily
represent ubiquitination.
). CYP3A
protein has a relatively short half-life compared with most microsomal
proteins, including other P450s. Substrates stabilize CYP3A in a process that
may be dependent upon protein conformational changes associated with substrate
binding (Watkins et al.,
1986). In contrast, treatment with "suicide
substrates" accelerates CYP3A degradation
(Correia et al., 1992). The
evidence to date has suggested that the ubiquitin-proteasome system may be
responsible for the accelerated degradation of CYP3A observed after treatment
with suicide substrates. In one study, animals were cotreated with
3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-1,4-dihydropyridine (DDEP), a CYP3A
suicide substrate, and hemin, a proteasome inhibitor
(Correia et al., 1992).
Treatment with only DDEP increased the amount of ubiquitin detected in hepatic
microsomes and in immunoprecipitates of CYP3A. Cotreatment with hemin further
increased the amount of polyubiquitinated proteins detected in microsomes from
DDEP animals, although the effects of hemin treatment on ubiquitin conjugated
to immunoprecipitated CYP3A were not reported. More recently, the proteasome
inhibitors aclarubicin and MG132 were used to examine the effects of DDEP on
CYP3A degradation in primary cultured rat hepatocytes and were found to
decrease the DDEP-mediated loss of both the 55-kDa and HMM CYP3A forms
(Wang et al., 1999). Studies
examining the degradation of CYP3A expressed in yeast have provided strong
evidence that CYP3A is degraded by the proteasome in this system, although it
is unclear whether the expressed CYP3A was folded normally or was
catalytically active (Murray and Correia,
2001).
We recently developed an in vitro model of CYP3A degradation that involves
the conversion of CYP3A to high molecular mass (HMM) conjugates in hepatic
microsomes incubated at 37°C (Zangar
et al., 2002). Because CYP3A substrates prevented the formation of
the HMM CYP3A conjugates in the incubated microsomes
(Zangar et al., 2002), this in
vitro system provides a mechanistic model for studying substrate-mediated
stabilization of CYP3A. Although HMM ubiquitin could be detected in CYP3A
immunoprecipitates, it was clear that ubiquitin was conjugated to CYP3A in a
process that was distinct from the "classical" ubiquitination
process. That is, this reaction did not require cytosol, ubiquitin-activating
enzyme E1, ATP, Mg2+, or free monoubiquitin. In
addition, CYP3A was apparently in large molar excess to ubiquitin units in
these complexes, and the cytosol-mediated degradation of these HMM bands was
not affected by proteasome inhibitors. These results indicated that CYP3A was
conjugated to a pool of microsomal proteins that are already
polyubiquitinated, rather than CYP3A itself being directly ubiquitinated
(Zangar et al., 2002). In
turn, this conclusion suggests that the presence of ubiquitin in CYP3A
immunoprecipitates provides insufficient evidence to determine that classical
polyubiquitination of CYP3A has occurred and that processes other than
proteasome-mediated degradation may be important in the relatively short
half-life of CYP3A.
To further evaluate the role of proteasome-mediated degradation in defining CYP3A protein stability, we examined the effects of proteasome inhibitors on CYP3A protein levels in primary cultured rat hepatocytes. These inhibitors consistently decreased CYP3A protein in microsomal fractions, a result opposite of what would be predicted if CYP3A were degraded by the 26S proteasome. In addition, we found that treatment of cells with hemin resulted in the formation of HMM CYP3A bands in a process that seemed to be unrelated to proteasome inhibition. These HMM CYP3A bands were found to be concentrated in the "detergent insoluble" portion of the 10,000g (S10) pellet, as is typical of large protein aggregates. This last result suggests that the CYP3A complexes potentially may be degraded by a mechanism that is independent of the classical ubiquitin-proteasome system.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Primary Hepatocytes. Primary rat hepatocytes were isolated and
cultured on collagen-coated dishes as described previously
(Zangar et al., 1995). Three
100-mm dishes were pooled for each sample. Primary human hepatocytes were
prepared essentially as described previously
(Duanmu et al., 2002), except
that they were plated onto Matrigel-coated dishes (1.5 mg of Matrigel/60-mm
dish). Nine 60-mm dishes were pooled for each protein sample for the human
hepatocyte study. Hepatocytes were cultured without treatment for 3 days
before initiation of a 30-h treatment with 2 mM phenobarbital. For the last 6
h of phenobarbital treatment, some cells were cotreated with 20 µM
lactacystin, 200 µM MG132, 200 µM proteasome inhibitor 1, 100 µM
hemin, or 0.2% ethanol (vehicle for MG132 and proteasome inhibitor 1). The
proteasome inhibitors, lactacystin, MG132, and proteasome inhibitor 1, were
prepared from a previously unopened manufacturer's vial immediately before
cell treatment. This step seemed to be necessary to achieve a reproducibly
high level of proteasome inhibition.
Subcellular Fractionation and Western and Northern Blots.
Subcellular fractions were prepared essentially as described previously
(Zangar et al., 1992,
1995). Briefly, cultured cells
were harvested, washed, and homogenized in 50 mM KPO4, 250 mM
sucrose, 1 mM EDTA, 100 µg of phenylmethylsulfonyl fluoride/ml, 0.2 units
of aprotinin/ml, 10 µg of leupeptin/ml, and 10 mM N-ethylmaleimide
(homogenization buffer). Cell homogenates were centrifuged at 10,000g
for 10 min at 4°C to prepare the S10 pellet and supernatant. The S10
pellet was washed by suspending in 1 ml of homogenization buffer and repeating
the centrifugation step. The washed pellet was suspended in 200 µl of
homogenization buffer containing 0.5% NP40 and vortexed vigorously at 4°C
for 30 min. The samples were then centrifuged at 10,000g for 10 min,
4°C and the supernatants saved as the "NP40-soluble" fraction.
The remaining pellets were washed with 500 µl of homogenization buffer
containing 0.5% NP40, collected by centrifugation, and suspended by sonication
in 1% lauryl sulfate, 62.5 mM Tris-Cl, 1.5 M glycerol, and saved as the
"NP40-insoluble" S10 pellet fraction. Samples were stored at
–80° and were typically analyzed by Western blot analysis within 1
to 7 days of cell harvest.
The S10 supernatant was spun at 100,000g for 1 h. The supernatant
was saved as the cytosol fraction. The pellet was first gently washed and then
resuspended by sonication in 50 mM KPO4, 1 mM EDTA. Samples were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and
analyzed by Western blotting, as described previously
(Kim et al., 2001;
Zangar et al., 2002).
Typically, samples were diluted in an equal volume of loading buffer [62.5 mM
Tris, pH 6.8, 1% lauryl sulfate, 11% glycerol, 370 µM bromphenol blue, and
700 mM (5% by volume) 2-mercaptoethanol] and then heated to 65°C for 5
min. Alternatively, to determine whether increased detergent might alter the
levels of HMM CYP3A, samples were also diluted in 62.5 mM Tris, pH 6.8, 11%
glycerol, 370 µM bromphenol blue, 10% lauryl sulfate, 40 mM dithiothreitol,
and 560 mM (4%) 2-mercaptoethanol before heating. Microsomal and
NP40-insoluble fractions were loaded at 20 µg of protein/lane, whereas
other fractions were loaded at 30 µg/lane. Protein was then transferred to
nitrocellulose membrane (Bio-Rad, Hercules, CA) using a Bio-Rad Transblot
apparatus operated at 50 V for 2 h, with 4°C circulating water for
cooling. Blots were blocked in 5% milk powder and probed with 1:10,000
dilution of primary antibody in buffered saline (pH 7), 0.1% bovine serum
albumin, and 0.05% Tween 20. Blots were then probed with a 1:5000 dilution of
goat anti-mouse secondary antibody in saline (pH 7), 0.05% Tween 20, and 5%
powdered milk. Protein levels were determined using SuperSignal
chemiluminescent reagents and were imaged using a Lumi-Imager F1 (Roche
Diagnostics, Indianapolis, IN).
Total RNA was isolated from individual 60-mm dishes of human hepatocytes
(two dishes per treatment group) as described previously
(Xie and Rothblum, 1991).
Northern blot analyses for CYP3A and 7S RNA (used to control for RNA loading)
were performed as described previously
(Kocarek et al., 2002).
Microsomal Incubations. Hepatic microsomes were prepared from rats
that had been treated with 100 mg of nicardipine/kg/day for 7 days, as
described previously (Zangar et al.,
1999) and were stored as aliquots at –70°C for 1 year or
more before use. Treatment of rats with nicardipine induces high levels of
microsomal CYP3A, and microsomes from these animals spontaneously form HMM
CYP3A conjugates when incubated at 37°C (Zangar et al.,
1999,
2002). Microsomes were
incubated at 37°C using 75 µg of microsomal protein, 50 mM Tris, pH
7.5, 25 mM sucrose, 0.154 mM KCl, 2 mM CaCl2, and 3 µM
ZnCl2 in a total volume of 50 µl. Reactions were terminated by
addition of 50 µl of loading buffer [62.5 mM Tris, pH 6.8, 1% lauryl
sulfate, 11% glycerol, 370 µM bromphenol blue, and 700 mM (5% by volume)
2-mercaptoethanol] and heating to 65°C for 5 min. Samples were then
analyzed by Western blotting, as described above. Twelve micrograms of protein
was loaded per lane for these samples.
CYP3A catalytic activity was measured using erythromycin
N-demethylase activity, as described previously
(Wrighton et al., 1985).
Proteasome inhibitors were added to the microsomes at concentrations that were
found to stimulate an increase in polyubiquitinated proteins in primary
cultured rat hepatocytes in this study (see below) or, in the case of
aclarubicin, were used by others for proteasome inhibition
(Wang et al., 1999).
CYP3A Immunoprecipitation Analyses. The immunoprecipitation studies
were undertaken essentially as described previously
(Zangar et al., 2002). Each
microsome sample (100 µg of protein) was suspended in 400 µl of
immunoprecipitation buffer (RIPA; final concentration was 0.9% NaCl, 0.1 M
sodium phosphate, pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium
lauryl sulfate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.07 mg/ml aprotinin,
1 mM orthovanadate, and 100 µM butylated hydroxytoluene) and added to 100
µl of beads containing covalently attached anti-CYP3A2. Samples were gently
mixed at 4°C for 4 h. The beads were collected in Micro Bio-Spin columns
(Bio-Rad), drained by gravity flow, and washed with 2 ml of RIPA. CYP3A was
gently eluted at room temperature using 1 ml of 0.1 M glycine, 1% NP40, and 1%
CHAPS, pH 2.2. The protein was precipitated by adding trichloroacetic acid to
a final concentration of 20% (w/v), pelleted by centrifugation, washed in 70%
ethanol, and repelleted. The pellets were suspended directly in loading buffer
and analyzed by Western procedures, as described above.
Statistics. Data were initially analyzed using a one-way analysis of variance. Multiple comparisons were undertaken using Tukey's test. P < 0.05 was used to define significant differences on all tests.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), but the
effects of these agents on CYP3A levels in the absence of treatment with
suicide substrates have not been reported. Therefore, we treated cells with
lactacystin, hemin, and MG132 for 6 h. Lactacystin and MG132 inhibited the
proteasome in primary cultured rat hepatocytes, as demonstrated by respective
122 and 79% increases in the amounts of polyubiquitinated proteins
presence in microsome samples (Fig.
1). However, treatment with hemin did not significantly alter
levels of ubiquitinated proteins.
|
Opposite to their effects on ubiquitinated protein levels, treatment with
the proteasome inhibitors consistently reduced microsomal CYP3A protein levels
(Fig. 1). Specifically, mean
levels of the 55-kDa CYP3A band were decreased 37, 30, or 53% in cells
treated with lactacystin, hemin, or MG132, respectively, relative to control
levels. CFTR is known to be degraded by the proteasome when improperly folded
after synthesis (Gelman et al.,
2002) and was included as a control here. Western blot analysis of
CFTR indicated that levels of this protein were not altered after treatment
with lactacystin, hemin, or MG132 (Fig.
1), suggesting that CFTR is efficiently folded in the primary
cultured hepatocytes and therefore not degraded by the proteasome. More
importantly, this result suggests that the down-regulation of CYP3A observed
after treatment with proteasome inhibitors is selective for CYP3A. Additional
studies were undertaken using proteasome inhibitor 1 and results were similar
to those observed with lactacystin and MG132. That is, treatment with
proteasome inhibitor 1 significantly increased microsomal levels of
ubiquitinated proteins by 63%, decreased levels of the 55-kDa CYP3A band
by 52%, but did not alter HMM CYP3A levels (proteasome inhibitor 1 results are
from two separate primary hepatocyte preparations, with two individual
microsomal samples prepared for each cell preparation, for four samples
total). Overall, these data demonstrate that proteasome inhibition decreased
microsomal CYP3A levels in primary cultured rat hepatocytes and that this
effect was selective for CYP3A.
To determine whether there was an inverse correlation between microsomal CYP3A levels and inhibition of the proteasome, the concentration response of lactacystin was determined. We found that 20 µM lactacystin caused a marked increase in ubiquitinated proteins and a clear suppression of microsomal CYP3A levels (Fig. 2). Lower concentrations of lactacystin had negligible effects on either CYP3A levels or ubiquitinated protein levels.
|
If CYP3A is degraded by the proteasome in primary cultured hepatocytes, then treatment with proteasome inhibitors would be predicted to increase the amount of HMM CYP3A. Hemin was the only agent we studied that resulted in an increase in HMM CYP3A bands (Fig. 1). To determine whether these HMM CYP3A bands might be disrupted by increasing amount of detergent, microsomes from control, lactacystin-treated, or hemin-treated cells were diluted 1:1 in loading buffer containing either 1% (our usual concentration) or 10% lauryl sulfate. After heating, paired samples were then loaded side by side on a single gel, and CYP3A levels were analyzed by Western blot analysis. Paired samples seemed identical regardless of the loading buffer used (data not shown), indicating that the HMM CYP3A could not be dissociated with increased amounts of detergent. Such a result is consistent with other studies on HMM CYP3A bands we have undertaken with the microsome samples, where samples that were prepared in loading buffer containing a final concentration of 8 M urea or 1 M dithiothreitol did not alter the levels of HMM CYP3A detected in Western blot analyses (A. L. Kimzey, N. Bollinger, K. K. Weitz, and R. C. Zangar, unpublished data).
We were unable to detect any CYP3A in cytosolic fractions by Western blot analysis (data not shown). Analysis of the NP40-soluble portion of the S10 pellet only demonstrated a weak signal that otherwise looked identical to the strong signal in the microsome fraction (data not shown), suggesting there may be a low-level contamination of microsomal protein in the NP40-soluble fraction. In contrast, in the NP40-insoluble fraction obtained from the S10 pellet, significant amounts of CYP3A were detected (Fig. 3). Similar to microsomes, hemin treatment resulted in an increase in the amount of HMM CYP3A present in the NP40-insoluble fraction.
|
Immunoprecipitation studies were performed to examine the role of
ubiquitination in the formation of HMM CYP3A bands. As we have noted
previously, levels of HMM CYP3A do not immunoprecipitate as efficiently as the
55-kDa band, presumably due to steric hindrance
(Zangar et al., 2002). Even
so, consistent with results observed in microsomes, levels of HMM CYP3A were
increased over 50% in immunoprecipitates from microsomes from hemin-treated
cells relative to control cells or cells treated with lactacystin
(Fig. 4). Low levels of HMM
ubiquitin were also detected by Western blot analysis in the CYP3A
immunoprecipitates, although no increase of ubiquitinated proteins was
observed in the microsomes from hemin-treated cells compared with controls
(Fig. 4). In contrast, there
was a clear increase in HMM ubiquitin in the immunoprecipitated sample from
the lactacystin-treated cells (Fig.
4).
|
To determine whether proteasome inhibitors had similar effects on CYP3A levels in human cells, we exposed primary cultured human hepatocytes to lactacystin and hemin. Six-hour treatment with lactacystin or hemin reduced microsomal CYP3A protein levels by 26 or 41%, respectively (Fig. 5). In contrast to rat hepatocytes, hemin treatment did not result in an increase in detectable levels of HMM CYP3A in any subcellular fraction. Similar to changes in microsomal protein levels, lactacystin and hemin treatment decreased CYP3A mRNA levels by 20 and 24%, respectively (Fig. 5).
|
CYP3A substrates are known to stabilize CYP3A protein in vivo, in primary
cultured rat hepatocytes and in vitro
(Watkins et al., 1986;
Eliasson et al., 1994;
Zangar et al., 2002). Given
that approximately half of all prescribed drugs are believed to be metabolized
by CYP3A (Guengerich, 1999),
care must be taken when selecting pharmacological tools to ensure that these
agents are not CYP3A substrates that may affect protein levels by
substrate-mediated stabilization. For this reason, we examined the effects of
a series of proteasome inhibitors, used in this study or by others
(Wang et al., 1999), on a
model CYP3A-catalyzed reaction, erythromycin N-demethylase activity.
Clotrimazole, a high-affinity CYP3A substrate, was also included in this
analysis as a positive control. Clotrimazole nearly completely blocked CYP3A
catalytic activity (Fig. 6).
When at the same concentration used to inhibit proteasome activity, we found
that aclarubicin decreased CYP3A activity by about 20%
(Fig. 6). Because erythromycin
is present at a 20-fold greater concentration than aclarubicin, these results
suggest that CYP3A may have a significant affinity for aclarubicin when there
is not an excess of competitive substrate. MG132 and lactacystin seemed to
have no effect on CYP3A activity.
|
We have previously described an in vitro incubation system in which CYP3A
is converted to HMM bands that are degraded in the presence of cytosol
(Zangar et al., 2002). Because
CYP3A substrates block this process, it is likely that this system is a
mechanistic model of the processes associated with substrate-mediated
stabilization of CYP3A. Therefore, the ability of drugs to block the formation
of HMM CYP3A in this system is likely to be a good predictor of
substrate-mediated stabilization effects in living cells. Consistent with
previous results, HMM CYP3A was formed in hepatic microsomes from
nicardipine-treated rats when the microsomes were incubated for 2 h at
37°C (Fig. 7). Addition of
acetonitrile, a solvent control, was without effect on this reaction, whereas
clotrimazole effectively blocked the formation of HMM CYP3A. Aclarubicin was
also effective in blocking this reaction. MG132 and lactacystin did not seem
to have any effect. None of the agents examined blocked the upward migration
of ubiquitin that is also observed in the incubated microsomes
(Zangar et al., 2002),
suggesting that aclarubicin's inhibition of the formation of the HMM CYP3A
bands were due to specific interactions with the CYP3A protein.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Wang et al., 1999). However,
in this study, we found that treatment with proteasome inhibitors resulted in
the loss of microsomal CYP3A in primary cultured rat hepatocytes, a result
opposite of what would be expected if the ubiquitin-proteasome system
contributed to the relatively short half-life of CYP3A protein that is
observed even in the absence of suicide substrates. Because proteasome
inhibitors also decreased CYP3A mRNA levels, it seems that these agents act at
a pretranslational level to suppress CYP3A expression. Possibly the likeliest
explanation for this effect is that there is a protein that inhibits CYP3A
transcription that is degraded by the proteasome. When the proteasome is
inhibited, levels of this suppressor protein increase, resulting in decreased
expression of CYP3A. However, we cannot rule out that changes in protein
stability may have occurred in this study. We observe an increase in HMM
ubiquitin in CYP3A immunoprecipitates from lactacystin-treated cells
(Fig. 4), suggesting that some
CYP3A may be degraded by the proteasome. In this light, it may be that the
reason that lactacystin treatment does not increase HMM CYP3A is due to
suppression of the precursor 55-kDa CYP3A. However, we have previously
observed that CYP3A can cross-link to other ubiquitinated proteins in
incubated microsomes independent of classical ubiquitination processes
(Zangar et al., 2002).
Therefore, an alternative explanation is that the increased levels of
ubiquitin in the immunoprecipitated CYP3A samples simply reflected an increase
in the pool of ubiquitinated microsomal proteins available for cross-linking
and was not related to classical ubiquitination of CYP3A.
We examined several proteasome inhibitors that had previously been reported
to affect CYP3A degradation. Notably, a recent study reported that CYP3A was
stabilized in the presence of aclarubicin
(Wang et al., 1999).
Aclarubicin is an antineoplastic agent with a mass of 812 Da, too big for most
P450s to metabolize but typical of CYP3A substrates. We found that aclarubicin
inhibited CYP3A catalytic activity, as would be expected of a CYP3A substrate.
More importantly, we found that aclarubicin blocked the formation of HMM CYP3A
conjugates in incubated microsomes. Because the formation of these HMM CYP3A
conjugates seems to precede CYP3A proteolysis but is independent of classical
ubiquitination processes (Zangar et al.,
2002), the ability of aclarubicin to interfere with this process
suggests that it has an effect on CYP3A stability that is independent of
proteasomal inhibition. That is, the inhibition of the CYP3A conjugation in
incubated microsomes suggests that aclarubicin is likely to stabilize CYP3A in
living cells at concentrations used for proteasome inhibition.
Wang et al. (1999) found
that MG132 and lactacystin were ineffective in freshly isolated primary rat
hepatocytes unless glutathione levels were suppressed. However, we found these
agents to be effective proteasome inhibitors in cultured primary rat
hepatocytes without suppression of glutathione. The differences between these
two studies may reflect differences between cultured and freshly isolated
hepatocytes. However, because primary rat hepatocytes cultured for 1 to 6 days
have levels of intracellular glutathione that are at least as great as those
in freshly isolated cells (Lii et al.,
1996), the differences between these studies are unlikely to be
due to higher glutathione levels in freshly isolated cells.
Increases in HMM CYP3A that were observed after hemin and DDEP cotreatment
have been used as evidence that inactivated CYP3A is degraded by the
proteasome (Correia et al.,
1992). We also found that hemin treatment of primary cultured
hepatocytes increased HMM CYP3A. However, it seems unlikely that this effect
was the result of proteasome inhibition, because hemin did not seem to inhibit
the proteasome in this study and because the three active proteasome
inhibitors we examined did not increase HMM CYP3A. This conclusion is further
supported by evidence that levels of ubiquitin were not increased in CYP3A
samples immunoprecipitated from hemin-treated cells
(Fig. 4). Although hemin has
been reported to inhibit proteasomal activity by 50% at 25 µM in in
vitro experiments, hemin concentrations of 500 µM typically have been used
in studies on living cells (Etlinger and
Goldberg, 1980; Vierstra and
Sullivan, 1988). Therefore, it seems likely that the 100 µM
hemin used in this study was insufficient to block proteasome activity in the
cultured hepatocytes. We have observed that oxidative stress is likely to be
an important factor in the formation of the HMM CYP3A conjugates (A. L.
Kimzey, N. Bollinger, K. K. Weitz, and R. C. Zangar, unpublished
observations). Therefore, one possible explanation for the formation of the
HMM CYP3A bands is that hemin released free iron, which could have acted as a
pro-oxidant.
Overall, our data suggest that previous studies examining CYP3A degradation
have suffered from the use of proteasome inhibitors with nonspecific
properties. That is, these studies have relied primarily on aclarubicin and
hemin, which seem to have effects on CYP3A protein independent of proteasome
inhibition. Furthermore, because proteasome inhibitors may suppress CYP3A mRNA
levels, it seems that any data on CYP3A protein degradation obtained with
these inhibitors must be interpreted with care. Based on these conclusions and
previous evidence that CYP3A-ubiquitin conjugates can be formed independent of
classical ubiquitination processes and are not degraded by the proteasome
(Zangar et al., 2002),
mechanisms in addition to the ubiquitin-proteasome pathway must be considered
as potentially contributing to the relatively short half-life of CYP3A.
Aggregates of endoplasmic reticulum proteins that are not degraded by the
proteasome have been described previously. A mutant form of urate oxidase was
found to form large aggregates that contained ubiquitin but seemed to be
degraded by processes other than the proteasome
(Yokota et al., 2000). Similar
complexes called "aggresomes" have been reported
(Kopito, 2000). These
aggresomes may contain ubiquitin but are potentially degraded by an autophagic
route as well as by the proteasome (Kopito,
2000). One of the characteristics of aggresomes is that they tend
to be dense particles that are poorly soluble in detergents
(Kopito, 2000). For this
reason, we examined the S10 pellet for NP40-insoluble CYP3A complexes. In
contrast to the decrease in the 55-kDa CYP3A band observed in microsomes
(Fig. 1), there was no change
in the 55-kDa CYP3A band in the NP40-insoluble fraction with hemin
treatment (Fig. 3) or in
response to lactacystin or MG132 (data not shown). In addition, CFTR was
readily detectable in microsome samples
(Fig. 1) but was undetectable
in the NP40 insoluble fraction (data not shown). Therefore, it is clear that
the NP40-insoluble and microsomal fractions represent distinct pools of
protein. We found that hemin treatment of primary hepatocytes not only
stimulated the formation of HMM CYP3A bands in microsomes but also in the
NP40-insoluble fraction of the S10 pellet
(Fig. 3). However, in the
NP40-insoluble fraction, the amount of HMM CYP3A was greater than the
55-kDa CYP3A band that represents the intact apoprotein. In contrast,
levels of the 55-kDa CYP3A band were in large excess to the HMM CYP3A in
microsomes. Even across the range of HMM CYP3A bands, the HMM CYP3A were
predominately of a greater mass in the NP40-insoluble fraction than in
microsomes (Fig. 3). That is,
the proportion of the HMM CYP3A that is above 200 kDa compared with the
proportion that is in the 80- to 200-kDa range is greater in the
NP40-insoluble fraction compared with the microsomes. These results suggest
that hemin treatment not only stimulated the formation of HMM CYP3A but that,
once formed, the HMM CYP3A was primarily recovered in the NP40-insoluble
fraction.
Although we cannot rule out a role for the proteasome in degradation of the
HMM CYP3A complexes from the current studies, the results of this study
suggest that alternative routes of degradation may be important in the absence
of exposure to CYP3A suicide substrates. Taken together, the results presented
here and in a previous report (Zangar et
al., 2002) suggest a novel hypothesis about how CYP3A protein may
be degraded. That is, CYP3A is initially cross-linked to ubiquitinated
proteins and forms a large protein complex in a process that is increased by
hemin treatment. This complex may be degraded by either an autophagic
mechanism or by the proteasome. The presence of these HMM complexes has
apparently been overlooked in previous studies on P450 degradation, because
previous studies did not examine the detergent-insoluble, 10,000g
pellet that contains most of the HMM CYP3A complexes.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: P450, cytochrome P450; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; HMM, high molecular mass; PI1, proteasome inhibitor 1 or Z-Ile-Glu(OtBu)-Ala-Leu-CHO; CFTR, cystic fibrosis transmembrane conductance regulator; S10, pellet and supernatant fractions formed from the 10,000g centrifugation of hepatocyte homogenates; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; RIPA, radioimmunoprecipitation assay.
Address correspondence to: Richard C. Zangar, Pacific Northwest National Laboratory, 902 Battelle Blvd., MS P7-56, Richland, WA 99352. E-mail: richard.zangar{at}pnl.gov
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Correia MA, Davoll SH, Wrighton SA, and Thomas PE (1992) Degradation of rat liver cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: characterization of the proteolytic system. Arch Biochem Biophys 297: 228–238.[CrossRef][Medline]
Duanmu Z, Locke D, Smigelski J, Wu W, Dahn MS, Falany CN, Kocarek
TA, and Runge-Morris M (2002) Effects of dexamethasone on aryl
(SULT1A1)- and hydroxysteroid (SULT2A1)-sulfotransferase gene expression in
primary cultured human hepatocytes. Drug Metab Dispos
30:
997–1004.
Eliasson E, Mkrtchian S, Halpert JR, and Ingelman-Sundberg M
(1994) Substrate-regulated, cAMP-dependent phosphorylation,
denaturation and degradation of glucocorticoid-inducible rat liver cytochrome
P450 3A1. J Biol Chem
269:
18378–18383.
Etlinger JD and Goldberg AL (1980) Control of protein
degradation in reticulocytes and reticulocyte extracts by hemin. J
Biol Chem 255:
4563–4568.
Gelman MS, Kannegaard ES, and Kopito RR (2002) A
principal role for the proteasome in endoplasmic reticulum-associated
degradation of misfolded intracellular cystic fibrosis transmembrane
conductance regulator. J Biol Chem
277:
11709–11714.
Guengerich FP (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39: 1–17.[CrossRef][Medline]
Kim H, Putt DA, Zangar RC, Wolf CR, Guengerich FP, Edwards RJ,
Hollenberg PF, and Novak RF (2001) Differential induction of rat
hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2 and 2E1 in response to pyridine
treatment. Drug Metab Dispos
29:
353–360.
Kocarek TA, Dahn MS, Cai H, Strom SC, and Mercer-Haines NA
(2002) Regulation of CYP2B6 and CYP3A expression by
hydroxymethylglutaryl coenzyme A inhibitors in primary cultured human
hepatocytes. Drug Metab Dispos
30:
1400–1405.
Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10: 524–530.[CrossRef][Medline]
Lii CK, Wang ST, Chen HW, and Sheen LY (1996) Glutathione and glutathione-related enzyme activities of male and female rat hepatocytes under various culture conditions. Arch Toxicol 70: 822–829.[CrossRef][Medline]
Murray BP and Correia MA (2001) Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae? Arch Biochem Biophys 393: 106–116.[CrossRef][Medline]
Vierstra RD and Sullivan ML (1988) Hemin inhibits ubiquitin-dependent proteolysis in both a higher plant and yeast. Biochemistry 27: 3290–3295.[CrossRef][Medline]
Wang HF, Figueiredo Pereira ME, and Correia MA (1999) Cytochrome P450 3A degradation in isolated rat hepatocytes: 26S proteasome inhibitors as probes. Arch Biochem Biophys 365: 45–53.[CrossRef][Medline]
Watkins PB, Wrighton SA, Schuetz EG, Maurel P, and Guzelian PS
(1986) Macrolide antibiotics inhibit the degradation of the
glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in
primary monolayer culture. J Biol Chem
261:
6264–6271.
Wrighton SA, Schuetz EG, Watkins PB, Maurel P, Barwick J, Bailey BS, Hartle HT, Young B, and Guzelian P (1985) Demonstration in multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cytochrome P-450 of the rat. Mol Pharmacol 28: 312–321.[Abstract]
Xie WQ and Rothblum LI (1991) Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques 11: 324, 326–327.[Medline]
Yokota S, Kamijo K, and Oda T (2000) Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum. Histochem Cell Biol 114: 433–446.[Medline]
Zangar RC, Kimzey AL, Okita JR, Wunschel DS, Edwards RJ, Kim H, and
Okita RT (2002) Cytochrome P450 3A conjugation to ubiquitin in a
process distinct from classical ubiquitination pathway. Mol
Pharmacol 61:
892–904.
Zangar RC, Okita JR, Kim H, Thomas PE, Anderson A, Edwards RJ,
Springer DL, and Okita RT (1999) Effect of calcium channel
antagonists nifedipine and nicardipine on rat cytochrome P-450 2B and 3A
forms. J Pharmacol Exp Ther
290:
1436–1441.
Zangar RC, Springer DL, McCrary JA, Novak RF, Primiano T, and
Buhler DR (1992) Changes in adult metabolism of aflatoxin B1 in
rats neonatally exposed to diethylstilbestrol. Alterations in 
-class
glutathione S-transferases. Carcinogenesis
13:
2375–2379.
Zangar RC, Woodcroft KJ, Kocarek TA, and Novak RF
(1995) Xenobiotic-enhanced expression of cytochrome P450 2E1 and
2B1/2B2 in primary cultured rat hepatocytes. Drug Metab
Dispos 23:
681–687.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  C.-M. Lee, J. Pohl, and E. T. Morgan Dual Mechanisms of CYP3A Protein Regulation by Proinflammatory Cytokine Stimulation in Primary Hepatocyte Cultures Drug Metab. Dispos., April 1, 2009; 37(4): 865 - 872. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Zangar, N. Bollinger, S. Verma, N. J. Karin, and Y. Lu The Nuclear Factor-{kappa}B Pathway Regulates Cytochrome P450 3A4 Protein Stability Mol. Pharmacol., June 1, 2008; 73(6): 1652 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lu, R. Gallegos, P. Li, C. Q. Xia, S. Pusalkar, V. Uttamsingh, D. Nix, G. T. Miwa, and L.-S. Gan INVESTIGATION OF DRUG-DRUG INTERACTION POTENTIAL OF BORTEZOMIB IN VIVO IN FEMALE SPRAGUE-DAWLEY RATS AND IN VITRO IN HUMAN LIVER MICROSOMES Drug Metab. Dispos., April 1, 2006; 34(4): 702 - 708. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
