Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Cytochromes P450 (P450) constitute a superfamily of cysteine thiolate enzymes that catalyze a diverse array of biochemical reactions (Nelson et al., 1996). CYP3A is a subfamily of integral membrane proteins that are expressed at high levels in the endoplasmic reticulum (ER) of liver and intestine. In humans, CYP3A is important in the metabolism of approximately 50% of all prescribed drugs as well as lipophilic hormones such as testosterone (Guengerich, 1999). As a result of this broad substrate specificity and high level of expression, drug-drug interactions resulting from altered CYP3A activity are well documented (Thummel and Wilkinson, 1998).

After heme destruction and denaturation by reactive metabolites, CYP3A and CYP2E1 have been reported to be degraded by the ubiquitin-proteasome system (Correia et al., 1992; Tierney et al., 1992). Even in the absence of denaturation by reactive metabolites, these two P450s have relatively short half-lives (~7-10 h) compared with most other P450s or resident ER proteins (typical t_1/2 of 18-36 h) (Watkins et al., 1987; Koop and Tierney, 1990). Polyubiquitination commonly marks a protein for degradation by the 26S proteasome (Brodsky and McCracken, 1999; Schwartz and Ciechanover, 1999). Covalent linkage of ubiquitin typically requires the sequential action of a three-enzyme system. The first enzyme, ubiquitin-activating enzyme or E1, conjugates the ubiquitin C-terminal Gly to a Cys on the E1 enzyme. This thiolester-forming reaction is dependent upon ATP hydrolysis and Mg²⁺. The ubiquitin C-terminal glycine is then transferred to a ubiquitin carrier protein (E2). Transfer of the ubiquitin to a protein substrate typically requires recognition of the protein substrate by a ubiquitin protein ligase (E3). Addition of subsequent ubiquitins to a monoubiquitinated protein commonly proceeds on the initial ubiquitin side chain rather than on another Lys in the substrate protein. Methylated ubiquitin (MeUb), in which the lysines are blocked but the reactive C-terminal glycine is intact, will inhibit polyubiquitination reactions after one or a few MeUb molecules are attached to the substrate protein. Although it has not been shown whether the ubiquitin/proteasome system contributes to the relatively short half-life of these P450s, it is interesting that both CYP3A and CYP2E1 generate high levels of reactive oxygen species and that this NADPH-dependent oxidase activity is inhibited by the presence of P450 substrate (Persson et al., 1990; Puntarulo and Cederbaum, 1998). The presence of substrate also stabilizes these P450s such that their half-lives are comparable with other ER proteins (Watkins et al., 1987). As such, it has been postulated that generation of reactive oxygen species by CYP3A and CYP2E1 may result in heme modification and subsequent protein degradation by the ubiquitin/proteasome system in a manner analogous to inactivation by reactive metabolites (Korsmeyer et al., 1999).

In this study, we demonstrated that hepatic microsomes from nicardipine-treated rats generate HMM CYP3A that is complexed with polyubiquitin. Similar to in vivo, CYP3A substrates blocked the formation of the HMM CYP3A bands. The formation of the HMM CYP3A bands was independent of the addition of ATP, Mg²⁺, or cytosol and therefore was distinct from the classical E1-dependent ubiquitination process. Although addition of cytosol resulted in the loss of the HMM CYP3A, proteasome inhibitors did not affect this degradative process. Furthermore, peptide fingerprinting analysis of the HMM bands strongly suggested that CYP3A proteins were present in molar excess relative to ubiquitin conjugates, consistent with the concept that the shift of CYP3A into the HMM region was not simply the result of addition of a chain of ubiquitin molecules. Combined, these data suggest an alternative pathway for CYP3A degradation other than the classical ubiquitin/proteasome pathway and that this pathway may be important in the substrate-mediated stabilization of CYP3A.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Powdered rat chow was purchased from Harlan Teklad (Madison, WI). CYP2E1 antibody was purchased from Oxygene (Dallas, TX). Anti-peptide antibodies specific for CYP3A2 and CYP3A23 have been described previously (Debri et al., 1995; Zangar et al., 1999). Polyclonal antibodies against rat CYP4A or CYP3A were purchased from Gentest (Woburn, MA). A monoclonal antibody raised against ubiquitin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody raised against an N-terminal peptide of the ubiquitin-activating enzyme E1 was obtained from Calbiochem (San Diego, CA). Secondary antibodies, conjugated to horseradish peroxidase, were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). SuperSignal West Pico chemiluminescent reagent, GelCode Blue protein stain, and bis-acrylamide/azolactone "Ultralink" beads were obtained from Pierce Chemical (Rockford, IL). Novex 4 to 20% SDS-PAGE minigels were from Invitrogen (Carlsbad, CA). MeUb was from Affiniti Research Products (Mamhead, UK). Lactacystin, MG132, and protease inhibitor I were from Calbiochem. Isoelectric focusing (IEF) strips, Immobilized pH gradient buffer, urea, thiourea, and dithiothreitol used for two-dimensional (2D) gels were from Amersham Biosciences, Inc. (Piscataway, NJ). The Plus-One silver staining kit and Nonidet P-40 were from Amersham Biosciences, Inc. Micro Bio-Spin columns were from Bio-Rad (Hercules, CA). Centricon C-10s were from Amicon (Beverly, MA). Other reagents, including the polyclonal ubiquitin antibody and the calcium channel antagonists were from Sigma Chemical (St. Louis, MO).

Animals. Six-week-old male Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) were used in all studies. Nicardipine, nifedipine, diltiazem, clotrimazole, and pregnenolone 16-carbonitrile were typically mixed with unsweetened applesauce and the mixture was then added to powdered rat chow. Control rats were also fed the same powdered rat chow diet containing applesauce, but without the drugs. In one study, nicardipine (100 mg/kg/day) was diluted in 0.5% methylcellulose (1 ml/kg) and administered by gavage for 1, 2, or 3 days before sacrifice. For this study, control animals were gavaged with 0.5% methylcellulose alone. Food was removed from the rats 12 to 15 h before they were sacrificed. Induction of CYP3A by dexamethasone was undertaken using the treatment regimen described previously (Sherratt et al., 1989). This regimen calls for treating rats with intraperitoneal injections of 10 mg of dexamethasone/kg/day for 4 days and then sacrificing 24 h after the last treatment. In all studies, rats were sacrificed by administering sodium pentobarbital to induce anesthesia before removal of the livers. Microsomes and cytosols were prepared as described previously (Okita et al., 1993). Protein concentrations were determined as described previously (Lowry et al., 1951). Microsome aliquots were stored at 80°C for 1 year or more without previous thawing and refreezing.

Primary Cultured Rat Hepatocytes. Primary cultures of rat hepatocytes were isolated and cultured as described previously (Zangar et al., 1995). Cells were cultured without treatment for 3 days before the initiation of a 30-h treatment with 2 mM phenobarbital or 10 µM dexamethasone. The last 6 h of treatment with these CYP3A-inducing drugs, cells were cotreated with 10 µM cycloheximide with or without 330 µM nicardipine or 0.1% DMSO (v/v).

Western Blots. Western blots were prepared as described previously (Zangar et al., 1993). Protein bands were imaged and quantitated by chemiluminescence with a Lumi-Imager F1 (Roche Applied Science, Indianapolis, IN). Two ubiquitin antibodies were used, a polyclonal and a monoclonal antibody, to ensure that the HMM ubiquitin banding pattern was reproducible with different antibodies. These ubiquitin antibodies gave essentially identical results in side-by-side comparisons and were used interchangeably in these studies. Primary antibodies were diluted as follows: anti-CYP3A23, 1:40,000; anti-CYP3A2, 1:100,000; anti-CYP2E1, 1:60,000; anti-CYP4A, 1:60,000; polyclonal anti-ubiquitin, 1:200; monoclonal anti-ubiquitin, 1:750; and anti-E1 ubiquitin-activating enzyme, 1:2000. Secondary antibodies were used at 1:5000 dilutions.

Microsome Incubation Reactions. Incubations were undertaken 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, 3 µM ZnCl₂, 5 mM Na₂ATP, and 1 µM ubiquitin in a total volume of 50 µl unless noted otherwise. Reactions were terminated by addition of 50 µl of SDS-PAGE loading buffer (62.5 mM Tris, pH 6.8, 1% SDS, 11% glycerol, 370 µM bromphenol blue, and 0.5% 2-mercaptoethanol) and heating to 98°C for 5 min. To examine cytosol-dependent proteolysis, cytosol was added to the microsome incubates in some cases. In such cases, the amount of each reagent added to the incubation solution was adjusted to maintain the same concentration in the final reaction volume of 50 µl. After completion of the incubation, the microsomes were pelleted by ultracentrifugation before analysis by Western blotting.

Two-Dimensional Gels. Microsomal samples were incubated as described above except that the reaction was scaled up to 800 µl. To decrease salts that interfere with the IEF step, after incubation the microsomes were pelleted by ultracentrifugation and resuspended in 25 mM Tris, pH 7.5, 12.5 mM sucrose, 75 mM KCl. IEF strips, pH 4 to 7, were rehydrated overnight in a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% IEF buffer, pH 4 to 7, 65 mM dithiothreitol, 1 mM Tris, pH 7.5, 0.5 mM sucrose, 3 mM KCl, 0.01% bromphenol blue, and 125 µg of microsomal protein. IEF was carried out by linearly increasing from 0 to 500 V over the first 0.1 h, continuing at 500 V for 1 h, and then linearly increasing to 3500 V over 5 h and maintaining at 3500 V for 12 h. After focusing, the strips were incubated first in 2% lauryl sulfate, 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 0.01% bromphenol blue, and 65 mM dithiothreitol for 1 h, and then for 15 min in the same solution except that the dithiothreitol was replaced with 135 mM iodoacetamide. The strips were then placed on top of an 8% SDS-PAGE gels and the gels were run as described previously (Zangar et al., 1993). The gels were silver stained with the Plus One kit (Amersham Biosciences, Inc.), according to the manufacturer's instructions.

Peptide Fingerprinting and MS/MS Analyses. Microsomes from rats treated with 100 mg of nicardipine/kg/day were incubated as described above. Samples were denatured in SDS-PAGE loading buffer (63 mM Tris, pH 6.8, 1% SDS, 1.5 M glycerol, 370 µM bromphenol blue) by heating to 65°C for 5 min. Denatured samples were loaded onto a 4 to 20% acrylamide gel and electrophoresed for 1 h at 20 mA. Gels were rinsed in deionized water three times for 5 m each, stained in GelCode Blue for 1 h, and washed with water for 1 h. Protein bands were excised with a sterile scalpel, transferred to a 500-µl microcentrifuge tube, and crushed. To destain the protein, 400 µl of acetonitrile/25 mM aqueous ammonium bicarbonate (1:1, v/v) was added, the tubes were vortexed and then allowed to sit for 10 min. The liquid was removed using a gel-loading pipette tip. This destaining step was repeated once. The gel was dehydrated by addition of 400 µl of acetonitrile, quickly vortexed, and allowed to sit 5 min before removal of the solvent. Dehydrated gel fragments were further dried under vacuum for 20 min. Gel fragments were first rehydrated with 7.5 µl of 20 ng/µl trypsin solution suspended in 25 mM ammonium bicarbonate, covered with an additional 7.5 µl of 25 mM ammonium bicarbonate, and digested at 37°C for 2 h. Peptides were recovered from the digests by adding 30 µl of acetonitrile/aqueous 25 mM ammonium bicarbonate (50:50, v/v) and vortexing for 10 min, and transferring the liquid phase to a new tube. This step was then repeated, with a quick vortexing, and samples were pooled and lyophilized to dryness.

Immunoprecipitation of CYP3A. The polyclonal antibody against rat CYP3A was conjugated to Ultralink beads according to the manufacturer's instructions (Pierce Chemical) and used for immunoprecipitation of CYP3A as follows. Microsomal samples from nicardipine-treated rats were incubated for 0 or 2 h as described above, except that the reaction was scaled up to 250 µl. The microsomal membranes and proteins were suspended in an equal volume of 2× immunoprecipitation buffer (final concentration was 0.9% NaCl, 0.1 M sodium phosphate, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.07 mg/ml aprotinin, 1 mM orthovanadate). Samples were gently mixed at 4°C for 5 h. The beads were collected in Micro Bio-Spin columns, drained by gravity flow, and washed with 2 ml of immunoprecipitation buffer. CYP3A was gently eluted at room temperature by using 1 ml of 0.1 M glycine, 1% Nonidet P-40, and 1% CHAPS, pH 2.2, and immediately neutralized by adding 500 µl of 1 M Tris base, pH 7.4. Suspension of the beads in SDS-PAGE loading buffer and heating to 95°C for 5 min indicated that this elution procedure was sufficient to remove essentially all of the bound CYP3A. The eluant was transferred to a Centricon C-10 and concentrated to ~150 µl by centrifugation for 2000g for ~2.5 h. The equivalent of 15 µl of each concentrated sample was then analyzed by Western blot techniques as described above. In these analyses, the secondary antibody used for Western blot detection was raised in goat, the same host species as the antibody used for immunoprecipitation. This step combined with the gentle elution conditions, which eluted only trace amounts of the bound capture antibody from the column, eliminated any detectable interference by the capture antibody in the Western analyses.

Statistics. Statistical analysis of the density of the ubiquitin signal in Western blots was undertaken using a one-way analysis of variance followed by a Tukey's multiple comparison with SigmaStat 2.0 software (SPSS Science, Chicago, IL). A probability value of < 0.05 was used for both analyses.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

CYP3A and Ubiquitin-Conjugating Activity in Incubated Microsomes. We sought to develop a model system to study the molecular processes involved in the degradation of microsomal CYP3A proteins. Based on previous studies, we expected this system to require the addition of cytosolic proteins to the microsomal samples. Therefore, we undertook initial studies to characterize the loss of CYP3A protein in microsomes incubated in the presence or absence of cytosol. These microsomes were obtained from the livers of rats treated for 7 d with 100 mg/kg/day nicardipine, a calcium channel antagonist that is also an efficacious CYP3A inducer (Zangar et al., 1999). Western blot analyses with specific anti-peptide antibodies for CYP3A23 and CYP3A2 indicated the formation of HMM bands in the incubated microsomes (Fig. 1A). Although these HMM bands were characteristic of ubiquitinated proteins, we knew of no reports demonstrating ubiquitination of ER proteins in the absence of cytosol and therefore proceeded to more extensively characterize the formation of the HMM protein conjugates. To determine whether formation of these HMM bands was specific for proteins that are ubiquitinated, we also probed these blots for CYP2E1, which is known to be ubiquitinated, and CYP4A, which is not known to be ubiquitinated. CYP2E1 formed HMM bands in the incubated microsomes, although the amount of HMM CYP2E1 formed relative to the intact CYP2E1 protein was much less than observed for the CYP3A proteins (Fig. 1A). In contrast to CYP3A and CYP2E1, no HMM banding of CYP4A was observed in the incubated microsomes (Fig. 1A), suggesting that the conjugating reaction was selective for certain P450s. The addition of cytosol to these samples resulted in the loss of the HMM CYP3A23, CYP3A2, and CYP2E1 bands, consistent either with the degradation of HMM CYP3A by cytosolic proteases or with cytosol inhibiting the formation of the bands. (The ~80-kDa band detected with the CYP2E1 antibody in the lane containing cytosol is a cross-reactive cytosolic protein that is unaffected by incubation.)

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Incubation of microsomes from nicardipine-treated rats results in the formation of high molecular mass bands (~MW) that are degraded in the presence of cytosol. A, microsomes were incubated for 2 h with or without cytosol. Samples were then fractionated using SDS-PAGE, transferred to nitrocellulose, and probed using antibodies specific for CYP3A23, CYP3A2, CYP2E1, or CYP4A. Approximate molecular mass, shown on the left, were taken from prestained standards on the CYP3A23 blot. Because all blots were run under similar conditions, protein migration on the other blots is similar but may vary slightly. B, microsomes were incubated at 37°C for 0, 1, or 2 h without cytosol or were incubated for 2 h with 20, 30, or 50% cytosol (v/v) added at the start of the incubation (2 Hr Cytosol) or 1 h later (1 Hr Cytosol). The protein concentration of 100% cytosol was 26 mg/ml. After incubation, microsomes were pelleted by ultracentrifugation and analyzed by Western blot procedures with a CYP3A23-specific antibody.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Time-dependent changes in CYP3A23 and total ubiquitinated proteins in hepatic microsomes isolated from nicardipine-treated rats. Microsomes were incubated for 0 to 4 h at 37°C and then analyzed using Western blot techniques. A, Western blot analysis of CYP3A23. Each lane represents a separate microsomal incubation. B, graph of the time-dependent change in the density of the ~55-kDa CYP3A23 band in A. Each data point represents the average of two bands. C, Western blot analysis of total ubiquitinated proteins prepared using a polyclonal ubiquitin-specific antibody.

Detection of Ubiquitin in Immunoprecipitated HMM CYP3A. To determine whether ubiquitin is bound to the HMM CYP3A bands, we immunoprecipitated CYP3A and analyzed for ubiquitin by Western blot analysis. The HMM CYP3A immunoprecipitated with a decreased efficiency compared with the ~55-kDa CYP3A, presumably due to steric hindrance of antigenic sites in the HMM conjugates. Even so, sufficient HMM CYP3A was obtained for these analyses such that a clear increase in HMM CYP3A could be observed in a 2-h incubated sample compared with a control sample (Fig. 3). Probing of an identical blot with anti-ubiquitin showed that the HMM CYP3A band from the 2-h incubate had a marked increase in HMM ubiquitin conjugation. These data clearly demonstrated that CYP3A is complexed with ubiquitin in these microsomal samples without the addition of cytosol.

View larger version (80K): [in this window] [in a new window]   Fig. 3.   Presence of HMM ubiquitin in CYP3A immunoprecipitates. CYP3A was immunoprecipitated as described under Experimental Procedures. Samples were then analyzed using Western blot procedures and CYP3A23-specific polyclonal and ubiquitin-specific monoclonal antibodies. Approximate mass of CYP3A23 bands is indicated by arrows.

Formation of HMM Conjugates Is Largely Insensitive to Conditions Designed to Elute Weakly Bound Membrane Proteins. Studies were undertaken to determine whether conditions known to free weakly bound proteins from lipid membranes would prevent formation of the HMM conjugates in the microsomal membranes. In these studies, microsomes were incubated with high salt buffer, low salt buffer, high pH buffer, or exposed to mild detergent before repelleting, resuspension, and analysis for CYP3A-conjugating activity. In all cases, the relative loss of the ~55-kDa CYP3A protein band was nearly the same as (at least 90% of) that observed with microsomes incubated in physiologically buffered solution, and formation of the HMM CYP3A and ubiquitin bands was similar between all samples (data not shown). These data indicate that any factors catalyzing the formation of HMM microsomal proteins were tightly bound to the membranes.

Formation of HMM Proteins in Microsomal Samples Is Distinct from Classical Ubiquitin Ligation System. To further investigate whether the HMM CYP3A banding was the result of a ubiquitination reaction, we examined the concentration-dependent interactions of ubiquitin and MeUb, a known inhibitor of ubiquitin ligation reactions. We were surprised to see that formation of the HMM CYP3A or ubiquitin bands was not dependent upon addition of ubiquitin. As shown in Fig. 4, there was little effect on the loss of the ~55-kDa CYP3A band or the formation of HMM CYP3A bands after addition of ubiquitin to the reaction mixture. The presence of low levels of ubiquitin (1 and 10 µM) in the reaction solution had only modest effects at best on the formation of the HMM ubiquitin bands, but the HMM ubiquitin bands did increase sharply at 100 µM ubiquitin. Because the K_d value for E1 binding with ubiquitin is in the submicromolar range, E1-mediated ubiquitination reactions would be expected to be nearly saturated by 10 µM ubiquitin (Haas and Siepmann, 1997). Therefore, the increase in protein ubiquitination at 100 µM ubiquitin is unlikely to be mediated by E1 ubiquitin-activating enzyme.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects of ubiquitin and MeUb on the formation of HMM CYP3A23 or ubiquitin proteins. Microsomes were incubated for 2 h at 37°C in the presence of 0, 1, 10, or 100 µM added ubiquitin and/or MeUb. Samples were then analyzed by Western blot with polyclonal antibodies specific for CYP3A23 or ubiquitin.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Levels of E1 ubiquitin-activating enzyme are very low in microsomes compared with cytosol. Hepatic cytosol and microsome samples from rats treated with 100 mg of nicardipine/kg/day for 1 week were analyzed by Western blot with an antibody specific for E1 ubiquitin-activating enzyme.

Protein Fingerprinting Analysis of HMM Protein Bands. Although monoubiquitin is normally a cytosolic protein, Western analysis showed a pool of polyubiquitinated protein in the microsomes before incubation that did not seem to be associated with CYP3A (Fig. 2C, 0-h incubates). Conceivably, de-ubiquitinating enzymes could release ubiquitin from these microsomal proteins and provide free ubiquitin that could conjugate to microsomal CYP3A even in the absence of added ubiquitin. Much of the HMM CYP3A migrated above 200 kDa, indicating that if ubiquitin conjugation alone accounted for the increase in CYP3A mass, there must be a molar ratio of over 15 ubiquitin molecules per each CYP3A molecule. Trypsin digestion and tandem mass spectrometry can be used to confirm the presence of ubiquitin in conjugated proteins (W. Li, personnel communication). Therefore, to determine whether levels of ubiquitin in the HMM bands were sufficient to account for the upward migration of CYP3A, we identified the most abundant proteins present in the HMM bands using in-gel trypsin digestion and MS analysis. Due to competition between peptides for ionization energy within the MS, peptides derived from low-abundance proteins may be detected less frequently or not at all.

11

9

Substrate-Mediated Stabilization of CYP3A Protein in Incubated Microsomes. To determine whether CYP3A protein conjugation observed in microsomes from nicardipine-treated rats might be related to the molecular processes that stabilize CYP3A protein in living cells, we treated microsomes with agents previously shown to stabilize CYP3A protein in vivo and in primary cultured hepatocytes (Eliasson et al., 1994). These CYP3A substrates, clotrimazole, ketoconazole, and erythromycin, prevented the loss of the ~55-kDa CYP3A23 band and the formation of the HMM CYP3A23 bands (Fig. 6). In contrast to this effect, none of these CYP3A substrates decreased the upward migration of the pool of proteins detected using the ubiquitin antibody. These results suggested that CYP3A substrates acted directly on CYP3A rather than any potential conjugating enzymes and supported the hypothesis that substrate-mediated stabilization results from altered CYP3A conformation. These results also confirmed that the amount of HMM ubiquitin conjugates formed was independent of HMM CYP3A levels.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Substrate-mediated inhibition of the formation of HMM CYP3A23. Microsomes from rats treated with 100 mg of nicardipine/kg/day were incubated at 37°C for 2 h in the absence (CON) or presence of 0.1% acetonitrile (ACN; vehicle for CYP3A substrates), 10 µM clotrimazole (CTZ), 10 µM ketoconazole (KCZ), or 100 µM erythromycin (EM). Western blot analyses with polyclonal antibodies specific for CYP3A23 (A) or ubiquitinated (B) protein levels are shown.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Nicardipine stabilizes CYP3A protein in incubated microsomes and primary cultured rat hepatocytes. A, microsomes from rats treated with 100 mg of nicardipine/kg/day were incubated at 37°C for 2 h in the presence of 0.9% acetonitrile alone or in combination with 10, 30, 100, 300, or 1000 µM nicardipine and then analyzed using Western blot procedures and a specific CYP3A23 antibody. B, primary rat hepatocytes were cultured for 72 h, treated with phenobarbital, and dexamethasone for 24 h to induce CYP3A levels, and then cotreated with 10 µM cycloheximide (CHX) alone or with 330 µM nicardipine or 0.1% DMSO for 6 h. Cells were then harvested, and microsomes prepared and analyzed for CYP3A23 levels with Western blot procedures.

Formation of HMM Protein Conjugates Is Dependent upon Extended in Vivo Treatment with Agents That Transcriptionally Induce and Stabilize CYP3A. To determine whether the microsomal CYP3A-conjugating activity was dependent upon nicardipine treatment, we examined hepatic microsomes from rats treated with 0, 25, 50, or 100 mg nicardipine/kg/day for 7 days. CYP3A2 was measured in this study because, unlike CYP3A23, CYP3A2 can be readily detected in untreated rats. Microsomes from rats treated with either 50 or 100 mg of nicardipine/kg/day formed HMM CYP3A2 conjugates, although greater activity was observed in microsomes from the animals that received the higher dose (Fig. 8). In contrast, in microsomes from control rats or those treated with 25 mg nicardipine/kg/day, no upward shift in CYP3A2 was observed. Similarly, the incubation-dependent upward migration of the pool of ubiquitinated proteins was only observed at the two higher doses. These results indicated that the presence of the CYP3A protein conjugation reaction in the microsomal fractions was dependent upon in vivo treatment with nicardipine at doses of 50 mg/kg/day or higher and correlated with the ubiquitin conjugation reaction.

View larger version (85K): [in this window] [in a new window]   Fig. 8.   Dose-dependent effects of nicardipine on the formation of HMM CYP3A2 or ubiquitinated proteins. Rats were treated with 0, 25, 50, or 100 mg of nicardipine/kg/day for 1 week and hepatic microsomes were isolated. Microsomes were incubated for 0 or 2 h at 37°C and then analyzed with Western blot techniques that used polyclonal antibodies specific for CYP3A23 or ubiquitin. The results of three different rats per treatment are shown such that each 0- and 2-h pair of samples represents microsomes taken from an individual animal.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Induction of CYP3A proceeds induction of protein-conjugating activity in rats exposed to nicardipine. Three rats per treatment were exposed to 100 mg of nicardipine/kg/day for 1 to 3 days. Hepatic microsomes were isolated and then incubated at 37°C for 2 h. Western blot analysis was used to compare the time dependence of the induction of the CYP3A23 protein and the microsomal ubiquitin-conjugating activities. The results of three different rats per treatment are shown such that each 0- and 2-h pair of samples represents microsomes taken from an individual animal.

View larger version (71K): [in this window] [in a new window]   Fig. 10.   Induction of microsomal CYP3A-conjugating activity is not characteristic of CYP3A inducers or calcium channel blockers. Hepatic microsomes from rats treated with a prototypical CYP3A inducer, dexamethasone (A), or with one of two calcium channel blockers, nifedipine and diltiazem (B), were compared with microsomes from nicardipine-treated rats. Three different rats per treatment were examined. Microsomes were incubated in the presence of ATP and ubiquitin at 37°C for 0 or 2 h. Western blot analysis was used to assess the presence of a microsomal CYP3A-conjugating activity with CYP3A23- or CYP3A2-specific antibodies.

View larger version (42K): [in this window] [in a new window]   Fig. 11.   Sustained xenobiotic induction of CYP3A is sufficient to induce the microsomal conjugating activity. Rats were treated with 50 mg/kg/day of clotrimazole (CTZ) or pregnenolone 16-carbonitrile for 1 week. Samples were then incubated for 2 h at 37°C, and analyzed for CYP3A levels by Western blot analysis as described under Experimental Procedures.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We found that hepatic microsomes from nicardipine-treated rats catalyzed the formation of HMM protein conjugates. Although CYP3A2, CYP3A23, CYP2E1, and ubiquitin were substrates for this conjugation reaction, CYP4A and most other microsomal proteins were not. Proteolysis was minimal in the isolated microsomes because there was no detectable loss of the ~55-kDa CYP3A protein in incubated microsomes that lacked the protein-conjugating activities (i.e., microsomes from rats not treated with high doses of nicardipine) and the HMM CYP3A proteins were maintained out to 4 h of incubation (Fig. 2). Attempts to strip the protein-conjugating activity by using various buffer systems known to remove weakly bound proteins from lipid membranes were unsuccessful, indicating that any factors associated with this conjugating activity were tightly bound to the ER membrane and were not cytosolic contaminants. Immunoprecipitation studies with CYP3A demonstrated that the HMM CYP3A was conjugated to ubiquitin. However, because the reaction was not dependent on addition of ATP, Mg²⁺, or cytosol to the microsomes, this reaction seemed to be mechanistically distinct from the classical model of ubiquitin ligation. MS fingerprinting analysis of the HMM region of the gel also strongly suggested that CYP3A proteins were in molar excess to ubiquitin, a relationship opposite what would be expected if the HMM CYP3A bands were primarily due to polyubiquitination. It is possible that there is ubiquitin conjugated to microsomal E2 enzymes that could be transferred to CYP3A. Still, each E2 protein can bind only a single ubiquitin and, in the absence of E1 and ATP, this activated form of ubiquitin cannot be replenished. CYP3A protein is at very high levels in microsomes from nicardipine-treated rats, such that the darkest protein band observed in stained gels comigrated with the CYP3A protein and this band was primarily shifted to HMM proteins after incubation. Polyubiquitinated proteins are in low abundance in microsomes relative to cytosol (R. C. Zangar and A. L. Kimzey, unpublished observations) and in vivo nicardipine treatment decreased this limited pool of ubiquitinated microsomal proteins to ~35% of control levels. Therefore, it seems implausible that these microsomes contain enough ubiquitin, a cytosolic protein, in either free or bound forms to convert the majority of an abundant protein such as CYP3A into HMM protein conjugates by a classical ubiquitination reaction.

Overall, the data presented herein support a model in which protein conjugation in the microsomes can occur independent of additional polyubiquitination. Because addition of monoubiquitin to the incubation reaction was not required for the upward migration of the pool of ubiquitin proteins (Fig. 4, first two lanes), this upward migration was most probably due to ubiquitinated proteins forming larger complexes by conjugation to themselves or to other proteins in the microsomes rather than further polyubiquitination. Because the antibodies used in this study preferentially detected ubiquitin-protein conjugates over monoubiquitin, the increased signal intensity in incubated microsomes detected by the ubiquitin antibodies most probably reflects an increase in protein-protein linkages involving ubiquitin rather than an increase in the total pool of ubiquitin. The cross-linking of polyubiquitin chains has been reported to be catalyzed by E2-25K, suggesting that a similar process could be occurring in the microsomes used in this study (Yao and Cohen, 2000).

It is interesting that the time course analysis of the CYP3A23 conjugation process indicated the early formation of two bands containing CYP3A23 that are approximately 2 and 3 times the mass of the intact protein (Fig. 2, arrows). In immunoprecipitated CYP3A samples, no increase in signal was detected in the Western blot for ubiquitin in the regions corresponding to these bands (Fig. 3), even though a chain of 13 ubiquitin molecules would be expected for each CYP3A molecule present for a mass shift of this magnitude. We analyzed the upper CYP3A band (i.e., the potential trimer) in the immunoprecipitated samples using trypsin digestion and tandem mass spectrometry but only peptides from CYP3A23 were detectable (data not shown). These results raise the possibility that CYP3A23 may form homocomplexes before conjugation with other proteins. Because MeUb inhibits formation of these potential oligomers, it seems likely that some interaction with ubiquitin is required before any CYP3A23 conjugates are formed. Therefore, it may be that the CYP3A23 in these bands is monoubiquitinated. In contrast, MeUb did not inhibit proteins already conjugated to ubiquitin from forming higher molecular mass complexes. This result suggests that once a protein is conjugated to ubiquitin, further ubiquitination is unnecessary for forming complexes of greater mass.

The HMM CYP3A protein complexes were degraded by cytosolic proteases in a process that was not affected by proteasomal inhibitors. The formation of ER protein complexes in living cells that contain ubiquitin but are refractory to cytosolic protein degradation has been reported previously (Johnston et al., 1998; Yokota et al., 2000). In a particularly relevant study, Yokoto et al. (2000) overexpressed urate oxidase that contained an altered N terminal, causing this enzyme to be retained in the ER. The urate oxidase formed lipoprotein complexes that immunostained positive for ubiquitin. Pulse-chase experiments readily detected degradation of the ubiquitin-urate oxidase complexes, but this degradation could not be attributed to proteasomal activity.

We demonstrated that CYP3A substrates prevented the loss of the ~55-kDa CYP3A band, the formation of HMM CYP3A bands, and the cytosol-mediated degradation of HMM CYP3A. This is the first evidence that CYP3A substrates may prevent the loss of CYP3A protein by decreasing conjugation and subsequent proteolysis. CYP3A substrates did not alter the formation of the HMM ubiquitin conjugates, consistent with the hypothesis that substrate binding alters CYP3A conformation and thereby stabilizes the protein, as suggested previously (Watkins et al., 1986). The inactivation and denaturation of CYP3A with agents such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine, which destroys the central heme group, has been reported to accelerate the degradation of CYP3A by the ubiquitin/proteasome system (Korsmeyer et al., 1999). However, because CYP3A in the microsomes from nicardipine-treated rats was able to bind substrate in a manner that prevented subsequent conjugation, the nicardipine-induced CYP3A most probably contained a functional heme moiety that allowed substrate binding and associated conformational changes. This conclusion is consistent with our previous study that showed that in vivo nicardipine treatment increased CYP3A catalytic activity as well as CYP3A protein levels (Zangar et al., 1999). Therefore, it seems likely that in the absence of CYP3A substrate, even catalytically competent CYP3A is a substrate for this microsomal conjugation reaction. We also attempted to denature CYP3A by repeated freeze-thaw cycles, a process reported to increase microsomal CYP3A degradation by the 20S proteasome (Roberts, 1997). The repeated freeze-thaw cycles did not alter formation of the HMM CYP3A bands in microsomes from nicardipine-treated rats nor did it result in the formation of HMM CYP3A or ubiquitin conjugates in microsomes from nifedipine-, diltiazem-, dexamethasone-treated, or control rats (data not shown). These results further support that the microsomal-conjugating activity was dependent upon in vivo nicardipine treatment but was not dependent upon CYP3A denaturation.

Nicardipine doses of 50 and 100 mg/kg/day were required for induction of the microsomal ubiquitin ligase activity (Fig. 8). In contrast, nicardipine doses of between 5 and 15 mg/kg/day are sufficient for calcium receptor antagonism and antihypertensive effects in rats (Sorkin and Clissold, 1987). Furthermore, treatment with other calcium channel antagonists at comparably high doses for 2 weeks did not induce the microsomal ubiquitin ligase activity (Fig. 6B). Therefore, the mechanism by which nicardipine induced the microsomal conjugating activity seemed to be unrelated to calcium channel antagonism.

Nicardipine activates the pregnane X receptor (PXR) (Drocourt et al., 2001), which regulates CYP3A23 gene transcription and potentially could induce other proteins involved in a microsomal conjugating system. However, treatment of rats with dexamethasone, a prototypical CYP3A inducer that both induces and activates the PXR (Kliewer et al., 1998; Huss and Kasper, 2000), did not induce the formation of HMM CYP3A in incubated microsomes (Fig. 10A). Therefore, it seems that activation of PXR is not sufficient to induce the microsomal conjugating activity. Still, it is interesting that the short-term, direct effect of nicardipine on CYP3A in the incubated microsomes or primary cultured hepatocytes is to stabilize this protein (Fig. 7). This result suggests that the CYP3A conjugation reaction observed in microsomes from nicardipine-treated rats is not the result of direct interaction between nicardipine and CYP3A but is the result of sustained, high-level induction of CYP3A. This possibility was supported by studies that showed that microsomes from rats treated with the potent CYP3A-inducers clotrimazole and pregnenolone 16-carbonitrile also exhibited the CYP3A conjugation reaction (Fig. 11). Therefore, it seems likely that extended maintenance of high levels of microsomal CYP3A is sufficient to induce or activate the conjugation process in microsomes. The loss of CYP3A protein under these circumstances is probably inhibited by the inducing agents, which are also CYP3A substrates.

Thus, taken together these data suggest that extended treatment with potent CYP3A inducers resulted in the induction or activation of microsomal factors that catalyzed the formation of both the HMM CYP3A and ubiquitin conjugates. Even so, this process was distinct from the classical ubiquitin/proteasome pathway. CYP3A substrates blocked this pathway, providing the first insight into the molecular processes involved into substrate-mediated stabilization of CYP3A.