Advertisement
Research ArticleArticle

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

Yuu Miyauchi, Kiyoshi Nagata, Yasushi Yamazoe, Peter I. Mackenzie, Hideyuki Yamada and Yuji Ishii
Molecular Pharmacology October 2015, 88 (4) 800-812; DOI: https://doi.org/10.1124/mol.115.098582

Abstract

There is a large discrepancy between the interindividual difference in the hepatic expression level of cytochrome P450 3A4 (CYP3A4) and that of drug clearance mediated by this enzyme. However, the reason for this discrepancy remains largely unknown. Because CYP3A4 interacts with UDP-glucuronosyltransferase 2B7 (UGT2B7) to alter its function, the reverse regulation is expected to modulate CYP3A4-catalyzed activity. To address this issue, we investigated whether protein-protein interaction between CYP3A4 and UGT2B7 modulates CYP3A4 function. For this purpose, we coexpressed CYP3A4, NADPH-cytochrome P450 reductase, and UGT2B7 using a baculovirus-insect cell system. The activity of CYP3A4 was significantly suppressed by coexpressing UGT2B7, and this suppressive effect was lost when UGT2B7 was replaced with calnexin (CNX). These results strongly suggest that UGT2B7 negatively regulates CYP3A4 activity through a protein-protein interaction. To identify the UGT2B7 domain associated with CYP3A4 suppression we generated 12 mutants including chimeras with CNX. Mutations introduced into the UGT2B7 carboxyl-terminal transmembrane helix caused a loss of the suppressive effect on CYP3A4. Thus, this hydrophobic region is necessary for the suppression of CYP3A4 activity. Replacement of the hydrophilic end of UGT2B7 with that of CNX produced a similar suppressive effect as the native enzyme. The data using chimeric protein demonstrated that the internal membrane-anchoring region of UGT2B7 is also needed for the association with CYP3A4. These data suggest that 1) UGT2B7 suppresses CYP3A4 function, and 2) both hydrophobic domains located near the C terminus and within UGT2B7 are needed for interaction with CYP3A4.

Introduction

Cytochrome P450 (P450) 3A4 is one of the major drug-metabolizing enzymes involved in the metabolism of 50% of clinical drugs (Thummel and Wilkinson, 1998; Guengerich, 1999). There are marked interindividual differences in the expression level of hepatic CYP3A4, and the difference among individuals may be as much as 40-fold (Shimada et al., 1994; Lamba et al., 2002). In contrast, the variance in drug clearance catalyzed by CYP3A4 is less than 10-fold (Lamba et al., 2002). Thus, there is a large discrepancy between the expression level of CYP3A4 and drug clearance mediated by this P450. In many cases, such a discrepancy can be explained by a single-nucleotide polymorphism. However, the frequencies of CYP3A4 single-nucleotide polymorphisms, which are able to suppress enzyme function, are rare and seem to be unable to explain the aforementioned discrepancy (Hirota et al., 2004; Lakhman et al., 2009). It is also unconvincing that micro-RNA–dependent downregulation underlies the polymorphic function of CYP3A4 (Takagi et al., 2008; Pan et al., 2009; Singh et al., 2011). Because CYP3A4 plays a central role in drug metabolism, it is important to understand the reason why the variations in function of this enzyme cannot be simply explained by the expression level.

It is well established that P450 needs electrons supplied from redox partners, i.e., NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5). Therefore, it stands to reason that P450 function varies depending on the strength of P450-CPR/b5 coupling. In this context, early studies examined the conditions required for protein-protein interactions between P450 and CPR/b5 mainly in a reconstituted system (Lu et al., 1969; Hildebrandt and Estabrook, 1971; Miwa and Lu, 1984). However, to the best of our knowledge, the interindividual variation in P450 function has not been successfully explained by interactions with the redox partners. The catalytic function of P450s can be altered by homo- and hetero-oligomerization (Davydov, 2011; Reed and Backes, 2012). In addition, P450 can bind to different sorts of proteins: for instance, our laboratory has provided evidence that CYP1A1 associates with UDP-glucuronosyltransferase (UGT) and microsomal epoxide hydrolase (Taura et al., 2000; Ishii et al., 2005). Although many pairs of P450-UGT complexes can be formed (Ishii et al., 2007), we have particularly focused on a CYP3A4-UGT2B7 interaction, because these enzymes are involved in the metabolism of many drugs. The association of CYP3A4 and UGT2B7 has been confirmed by several biochemical techniques including coimmunoprecipitation and cross-linking (Takeda et al., 2005, 2009; Ishii et al., 2010, 2014). Because CYP3A4 alters the regio-selectivity of UGT2B7-catalyzed morphine glucuronidation (Takeda et al., 2005) the binding of CYP3A4 and UGT2B7 is undoubtedly a functional interaction. However, the reverse effect—whether UGT2B7 can modulate CYP3A4 activity—has not been investigated. If this interaction really takes place, such a post-translational regulation of P450 activity would be important for understanding why there is a large discrepancy in the magnitude of interindividual differences between the expression level of CYP3A4 and drug clearance catalyzed by this P450.

Our previous work has suggested that the J-helix of CYP3A4 is a candidate for the domain contributing to CYP3A4-UGT2B7 association (Takeda et al., 2009). However, the region of UGT2B7 involved in the interaction with CYP3A4 remains largely unknown. In the present study, we focused on this issue, and tried to identify the UGT2B7 domain capable of interacting with CYP3A4. To this end, we simultaneously expressed CYP3A4, CPR, and UGT2B7 using a baculovirus-insect cell system, and examined whether coexpression of UGT2B7 alters CYP3A4 activity. We also searched for UGT2B7 domains necessary for functional interaction with CYP3A4 by constructing a series of deletion mutants, substitution mutants, and chimeric proteins with calnexin (CNX), which is a nondrug-metabolizing protein having the same membrane topology as UGT.

Materials and Methods

Synthetic oligonucleotides were purchased from Life Technologies (Carlsbad, CA). Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Bio (Shiga, Japan). Emulgen 911 was kindly gifted by Kao Chemicals, Ltd. (Tokyo). A metabolite standard in high-performance liquid chromatography analysis, 6β-hydroxytestosterone, was purchased from Sigma-Aldrich (St. Louis, MO). Pooled human liver microsomes (HLMs) prepared from 50 donors were purchased from Corning Gentest (Woburm, MA). All other reagents were of the highest quality commercially available.

Subcloning of cDNAs into a Baculoviral Vector for Coexpression.

To prepare recombinant baculoviruses, a Bac-to-Bac Baculovirus Expression System (Life Technologies) was used. The open reading frame of UGT2B7 was amplified by polymerase chain reaction (PCR) using UGT2B7 cDNA (Jin et al., 1993) as the template. Pfu Turbo DNA polymerase (Agilent Technologies, Santa Clara, CA) was used for this amplification. The PCR product was digested with KpnI, and cloned into pFastBac1 restricted with the same enzyme. Human CPR cDNA was subcloned from a construct provided by us into the EcoRI site of pFastBac1. We also subcloned CNX cDNA into the pFastBacl vector. Because CNX is a chaperone protein expressing on the endoplasmic reticulum (ER) membrane with the same membrane topology as UGT (David et al., 1993; Bergeron et al., 1994), this study used CNX as a reference protein. Human CNX cDNA was purchased from OriGene Technologies (Rockville, MD), and the mRNA-coding region sandwiched between the PstI-cleaving sites was amplified. After PstI treatment, CNX cDNA was also cloned into pFastBac1. The construction of pFastBac1-CYP3A4 was carried out as previously described (Ishii et al., 2014). For the pull-down assay, CYP3A4 having a carboxyl terminus tagged with hexahistidine (His-CYP3A4) was constructed by PCR. All the primers used for subcloning are listed in the Supplemental Material (see Supplemental Tables 1–4). The nucleotide sequences of the constructs were confirmed by an ABI 3130xl Genetic analyzer, using a BigDye Terminator Cycle Sequencing Kit, version 3.1 (Life Technologies). Recombinant pFastBac1 vectors were transfected into the competent Escherichia coli DH10Bac strain (Life Technologies). After positive clones were selected according to the user’s manual, recombinant bacmids (i.e., baculoviral DNAs for transfection) were prepared.

Culture of Sf9 Cells and Expression of Recombinant Enzymes.

Sf9 insect cells were grown in a 500 ml plastic Erlenmeyer flask (screwed cap) containing Grace’s medium (Life Technologies) supplemented with 10% fetal bovine serum, 10 μg/ml gentamycin, 0.25 μg/ml Fungizone, and 1% CD Lipid Concentrate (Life Technologies). To obtain recombinant baculovirus, Sf9 cells (2 × 107 cell) were seeded once in a 175 cm2 culturing flask, and then transfected with recombinant bacmids in the presence of CellfectinII reagent (Life Technologies) diluted with Grace’s medium without any additives. After 5-hour incubation, the medium was replaced with fresh medium, and the cells continued to be cultured. The control bacmids, obtained from transfection of pFastBac1 carrying no passenger DNA (mock pFastBac1), were also used for transformation as the control. Primary virus was collected 1 week after the first transfection. The baculovirus DNA was purified by NucleoSpinBlood (Macherey-Nagel, Düren, Germany) from primary viruses in 200 µl culture medium, and their titer was determined using a BacPAK qPCR Titration Kit (Clontech, Mountain View, CA). The transfection of Sf9 cells with baculoviral DNA was repeated usually 4 to 5 times until a titer of over 1.0 × 107 plaque-forming units/ml was obtained. For expression of recombinant enzymes, Sf9 cells (2 × 106 cells/ml, 200 ml) were transfected with recombinant baculovirus in an Erlenmeyer flask, and collected after 72 hours by low-speed centrifugation. In the case of expression of CYP3A4, 1 mg/ml hemin/1.25% albumin complex was added to the medium at a concentration of 1.4 μM hemin 24 hours after transfection. Microsomes were prepared from the transfected cells according to the protocols described previously (Ishii et al., 2014).

Pull-Down Assay.

Microsomes were diluted to 2 mg protein/ml with 100 mM sodium phosphate (pH 7.4) containing 20% glycerol (PG buffer). Solubilization of the microsomes with sodium cholate was performed by a method described elsewhere (Takeda et al., 2005). The resulting solution was centrifuged at 105,000g for 1 hour, and the supernatant was collected as solubilized microsomes. An aliquot (125 μl) of the solubilized microsomes was diluted four times with PG buffer containing 200 mM sodium chloride, 40 mM imidazole, 0.05% Emulgen 911, and 0.2% bovine serum albumin. This solution (500 μl) was then gently mixed at 4°C for 1 hour with magnetic agarose beads conjugated with nickel-nitrilotriacetic acid (Qiagen, Hilden, Germany). The beads gathered by a magnet were washed three times with 20 mM Tris-HCl (pH 7.4 at 4°C) containing 200 mM sodium chloride, 40 mM imidazole, 20% glycerol, and 0.05% Emulgen 911. The proteins associated with His-CYP3A4 were eluted from the beads with 10 mM sodium phosphate (pH 7.4) containing 300 mM sodium chloride, 250 mM imidazole, 20% glycerol, and 0.05% Emulgen 911.

Immunoblotting.

Proteins separated by SDS-PAGE were electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). UGT2B7 was detected either by goat anti-mouse low pI form UGT antibody (Mackenzie et al., 1984) or rabbit anti-UGT2B7 antibody (Corning Gentest). A WB-MAB-3A Human CYP3A Western Blotting Kit (Corning Gentest) was used to detect CYP3A4. Rabbit anti-rat CPR (Enzo Life Sciences, Farmingdale, NY) and Rabbit anti-CNX (GeneTex, Irvine, CA) were also purchased from the sources indicated. They were diluted 2000-fold when used. Immunochemical detection was conducted either with horseradish peroxidase (HRP)–conjugated secondary antibodies, HRP-rabbit anti-goat IgG (MP Biomedicals, Santa Ana, CA), or HRP-donkey anti-rabbit IgG (GE Healthcare, Piscataway, NJ). These were diluted 10,000- and 40,000-fold before use, respectively. Clarity Western ECL Substrate (Bio-Rad, Hercules, CA) was used as the substrate of HRP, and the chemiluminescence emitted was analyzed by a ChemiDoc MP System (Bio-Rad).

Assessment of the UGT2B7 Effect on CYP3A4-Catalyzed Oxidation.

The activity of CYP3A4 was measured by two methods. In the first method, pentafluorobenzyl ether coupled with luciferin (Luciferin-PFBE, Promega, Madison, WI) was used as the substrate, and CYP3A4 activity was determined by measuring chemiluminescence according to the manufacturer’s protocol. In kinetics using the above substrate, the substrate concentration was varied from 6.25 to 100–200 μM. In the second method, CYP3A4 activity was measured using testosterone, a traditional CYP3A substrate, and an incubation mixture (250 μl) consisting of 100 mM potassium phosphate (pH 7.4), 200 μM testosterone, 1 mM NADPH, and 50 nM recombinant CYP3A4. After preincubation at 37°C for 10 minutes, the reaction was started by adding NADPH. The incubation was continued for 10 minutes and terminated with 100 μl of 1 M trichloroacetic acid. The solution was kept on ice for 1 hour, and then centrifuged at 15,000 rpm for 10 minutes. After the supernatant (300 μl) was collected, progesterone was added to the solution as an internal standard (final 130 μM), and a portion (20 µl) of the solution was subjected to high-performance liquid chromatography for the analysis of 6β-hydroxytestosterone. The instrument used for this analysis was a D-2000 Elite high-performance liquid chromatography system (Hitachi High-Technologies, Tokyo) consisting of an L-2200 autosampler, L-2130 pump, L-2300 column oven, and L-2400 UV detector. The operation conditions were as follows: 1) column, Nova-Pak C18 column (4 μm, 8 × 100 mm; Waters, Milford, MA); 2) column temperature, 25°C; 3) elution program (percentage of acetonitrile in water), 20% for 5.0 minutes, and then increased to 80% for 20 minutes, held at 80% for 5 minutes, and followed by a stepwise reduction to 20% for 0.1 minute (this condition was maintained for 5 minutes for the next sample); 4) flow rate, 1.2 ml/min; and 5) detection, UV absorbance at 240 nm. Under these conditions, 6β-hydroxytestosterone and progesterone were eluted at retention times of 14.0 and 27.0 minutes, respectively. Data were stored and processed using the D-2000 Elite System Manager software, version 3.0 (Hitachi High-Technologies).

CYP3A4-dependent NADPH consumption and H2O2 generation were analyzed according to the method used by Locuson et al. (2007) with slight modifications. The reaction mixture was the same as that used in testosterone hydroxylation except for the NADPH concentration (300 μM). The reaction was started by addition of NADPH, and a reduction in absorbance at 340 nm was measured for 20 minutes. The reaction rate was calculated using an extinction coefficient of 6.23 mM−1/cm−1. The NADPH consumption was measured in the presence and absence of substrate, 200 μM testosterone. The amount of H2O2 produced was measured by a Pierce Quantitative Peroxide Assay Kit obtained from Thermo Scientific (Rockford, IL) after incubating under the same conditions as the assay of CYP3A4-catalyzed testosterone hydroxylation. Peroxide-driven oxidation (shunt pathway) (Chefson et al., 2006; Omura, 2011) in the catalytic cycle of CYP3A4 was also conducted under the common conditions for testosterone hydroxylation, except that the reaction was initiated by adding cumene hydroperoxide (final 1 mM) instead of NADPH (Chefson et al., 2006).

Generation of UGT2B7 Mutants to Identify the Domains Interacting with CYP3A4.

A series of UGT2B7 mutants was designed to clarify the crucial domain(s) involved in the CYP3A4-UGT2B7 interaction (the sequences of mutants are listed in Tables 2–4). In the case of deletion mutants lacking C-terminal areas (Δ519-529, Δ511-529, and Δ493-529) (Table 2), the cDNA was prepared by PCR using a reverse primer in which the stop codon was set at the desired position. The KpnI sites were attached to both ends of the PCR product so as to clone into pFastBac1. In the case of the mutant containing a one-point alanine substitution (K518A) and a truncated mutant (Δ511-518), cDNAs were prepared by site-directed mutagenesis (Agilent Technologies) using the primers listed in Supplemental Table 2. In contrast, several mutants inserted with alanine substitutions (A7, 3CA, and A8), and chimeric mutants with CNX (chimera 1–3) were generated by the megaprimer method using the primers and templates listed in Supplemental Tables 3 and 4. In each PCR, pfu Turbo DNA polymerase was used and the primer concentration, if a synthetic oligonucleotide was used, was set at 300 nM. The PCR conditions for each step are described in Supplemental Tables 5 and 6. The summarized sequences of these chimeras are listed in Table 4. The sequences of all mutants were verified, and they were transfected in a similar fashion to the wild-type construct. To quantify microsomal UGT2B7, Sf9 microsomes expressing UGT2B7 alone were used as a standard and the relative expression level/mg of microsomal proteins was estimated by immunoblotting as a percentage of the standard. The ratio of UGT/P450 was calculated by dividing the UGT2B7 level (percentage of standard) by the CYP3A4 content (pmol/mg protein).

Analytical Methods.

The hydrophobic region of UGT2B7 buried in or attached to the ER membrane was predicted using an open software system, TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Möller et al., 2001). The secondary structure locations were estimated by Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008). Kinetic and statistical analyses were carried out using GraphPad Prism 5.04 software (GraphPad software, La Jolla, CA). More specifically, kinetic data were fitted to a sigmoidal model defined by the following equation:Embedded Imagewhere V is the reaction rate; S is the substrate concentration; Vmax is the maximum enzyme velocity; S50 is the substrate concentration giving half Vmax; and n is the Hill coefficient.

Other Methods.

Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. The microsomal content of P450 was quantitated by monitoring difference spectra between the reduced form and its CO complex (Omura and Sato, 1964). The activity of CPR was determined by measuring cytochrome c reduction (Imai, 1976). The substrate-binding differential spectrum of P450 was measured as described using testosterone as the substrate (Schenkman and Jansson, 2006). A 1 ml aliquot of reaction mixture containing 100 mM sodium phosphate (pH 7.4), 20% glycerol, and 1 μM microsomal CYP3A4 was placed in both the sample and reference cuvettes in a double-beam spectrophotometer. After 10 minutes of preincubation at 25°C, a baseline was calibrated. Then, testosterone and methanol (solvent) were added into the sample and reference cuvettes, respectively, and the absorbance from 300 to 500 nm was measured. Spectra were recorded at three different concentrations of testosterone (100, 200, and 400 μM). To quantify the degree of substrate binding to P450, the difference in absorbance at the maximal point (390 nm) and minimal point (417 nm) was calculated and defined as ΔAbsorbance390-417. To check the repeatability in the analysis, microsomes without UGT2B7 (expressing CYP3A4/CPR) and with UGT2B7 (expressing CYP3A4/CPR/UGT2B7) were independently prepared three times and six ΔAbsorbance390-417 values were obtained from spectra recordings under the condition of testosterone 200 μM. When we calculated the microsomal content of CPR, we used the reported value of specific activity, i.e., 3.0 μmol cytochrome c reduced/min per nmol CPR (Yasukochi and Masters, 1976).

Results

Detection of the Interaction between CYP3A4 and UGT2B7 by Pull-Down Assay.

A UGT2B7-CYP3A4 interaction was examined by pull-down assay in which UGT2B7 was simultaneously expressed with His-CYP3A4 capable of binding to Ni2+-coupled beads. As expected, UGT2B7 was detected in the solubilized microsomes of Sf9 cells expressing this UGT, independently of His-CYP3A4 coexpression (Fig. 1A). However, when we analyzed the imidazole-eluted fraction, UGT2B7 was detectable only in microsomes expressing both of UGT2B7 and His-CYP3A4 (Fig. 1A). When UGT2B7 was replaced with a negative control, CNX, it was not detected in the imidazole-eluted fraction even under the conditions of His-CYP3A4 coexpression (Fig. 1B). These results clearly demonstrated that CYP3A4 and UGT2B7 associate specifically with each other, and that this association seems to be fairly strong because the complex can be detected even in the presence of detergent.

Fig. 1.
Fig. 1.

Detection of an interaction between CYP3A4 and UGT2B7 by His-tag pull-down assay. UGT2B7 and CNX were expressed in the presence and absence of coexpressed His-CYP3A4. Microsomes were solubilized with sodium cholate and ultracentrifuged. The supernatants were collected, and an aliquot was gently mixed with magnetic agarose beads conjugated with nickel-nitrilotriacetic acid. After washing the beads three times, His-CYP3A4 and proteins trapped by this P450 were eluted with buffer containing imidazole at a high concentration. Each protein was detected by immunoblotting with a specific antibody: (A) UGT2B7; (B) CNX. Solubilized microsomes and His-CYP3A4–trapped samples are indicated as input and imidazole-eluted fraction, respectively. The lanes shown by D represent double expression, which indicates microsomes coexpressing UGT2B7 or CNX with His-CYP3A4. On the other hand, the lanes shown by S represent single expression, which indicates microsomes expressing UGT2B7 or CNX alone. Details are described in the section on Materials and Methods.

Suppression of CYP3A4 Activity by UGT2B7.

The effect of UGT2B7 on CYP3A4 function was examined in a ternary expression system carrying CPR as well as UGT/P450. Conceivably, CYP3A4 function varies depending on the expression level of CPR. Thus, it should be necessary to compare CYP3A4 activity between the constructs the P450/CPR ratios that are comparable. Keeping this in mind, we prepared a total of 12 batches of Sf9 cells coexpressing UGT2B7, CYP3A4, and CPR. Another 13 batches were constructed as the control expressing only CYP3A4 and CPR. The relationship involving the CYP3A4 activity, P450/CPR ratio, and UGT/P450 score is shown in Fig. 2. The activity of CYP3A4 toward the luciferin derivative in the absence of UGT2B7 coexpression (shown by the open bars in Fig. 2) was increased about 6-fold in a CPR content–dependent fashion. However, when coexpressed with UGT2B7 (shown by the shaded bars in Fig. 2), CYP3A4 activity became lower than that of UGT2B7-lacking microsomes, even though the paired constructs showing a close P450/CPR ratio were compared. Therefore, it is suggested that UGT2B7 modulates CYP3A4 activity negatively. This suppressive effect was scarcely affected by the UGT/P450 score: for instance, even samples showing a high score of 2.0–4.0 failed to exhibit augmented suppression compared with those exhibiting lower scores. The P450/CPR ratio and UGT/P450 score of pooled HLMs used in this study were estimated to be 3.19 and 1.36, respectively. These values were comparable to those observed in some of the ternary-expressed microsomes (Fig. 2). Therefore, it seems likely that the suppression of CYP3A4 by UGT2B7 can occur under physiologic conditions.

Fig. 2.
Fig. 2.

Batch-to-batch differences in the expression levels of CYP3A4, UGT2B7, and CPR, and their relationship to CYP3A4 activity. Two types of microsomes were prepared: one type [13 preparations (open bars)] expressed CYP3A4 and CPR, and the other [12 preparations (shaded bars)] expressed UGT2B7 as well as CYP3A4 and CPR. CYP3A4 and CPR were quantified by spectrophotometric methods and the relative expression level of UGT2B7 was determined by immunoblotting. Different levels of expression were achieved by transecting Sf9 cells with recombinant baculoviruses at different titers: for instance, the following ranges of multiplicity of infection were used for transfection: 0.5–1.0 (CYP3A4); 0.005–0.1 (CPR); and 0.0–3.0 (UGT2B7). For the calculation of the P450/CPR ratio and UGT/P450 score, refer to the section on Materials and Methods. CYP3A4 activity was measured using a luciferin-derivative as a substrate. The CYP3A4 activity in microsomes of each transfection is plotted along with the P450/CPR ratio and UGT/P450 score in this three-dimensional plot. The data for microsomes lacking UGT2B7 (UGT/P450 score = 0) are shown by open bars, and UGT2B7-carrying preparations are shaded. Each bar represents the mean of a triplicate assay.

We then carried out a kinetic analysis to investigate the suppressive mode of UGT2B7. The results of the Michaelis-Menten plots and the kinetic parameters obtained from these plots are listed in Fig. 3 and Table 1, respectively. UGT2B7 significantly reduced the Vmax value of CYP3A4, without affecting the S50 value and Hill coefficient. A reference protein, CNX (which is a chaperone protein having the same membrane topology as UGT toward the ER), did not exhibit any effect on the kinetic parameters. The coexpression of UGT2B7 also suppressed CYP3A4-catalyzed testosterone 6β-hydroxylation (Fig. 4A), although the activity was measured only at a single substrate concentration. In addition, UGT2B7 reduced other steps in the catalytic cycle of CYP3A4; for example, NADPH consumption, H2O2 generation through an uncoupling reaction, and cumene hydroperoxide–driven testosterone 6β-hydroxylation were all reduced to the same degree by this UGT (Fig. 4, B, D, and E). Interestingly, UGT2B7 did not affect NADPH consumption in the absence of a substrate (Fig. 4C). Because the effect of UGT2B7 on NADPH consumption was markedly different when the P450 substrate was present, we then compared the substrate-binding spectrum of P450 in the absence and presence of coexpressed UGT2B7. The spectra from three independent sets of microsomal preparations were compared and the degree of substrate-binding (ΔAbsorbance319-417) was quantified. A representative comparison of the spectra in all of the concentrations of testosterone shown in Fig. 5, A–C suggested that coexpression of UGT2B7 tended to inhibit the binding of substrate to CYP3A4; however, the quantified difference did not reach statistical significance (Fig. 5D). Although the spectral change at 390–417 nm was increased by adding testosterone in a concentration-dependent fashion (see the spectra depicted by the solid lines in Fig. 5, A–C), the percentage of alteration in a testosterone-induced spectral change between the absence and presence of UGT2B7 did not differ significantly (48, 58, and 65% at 100, 200, and 400 µM testosterone, respectively). Therefore, unlike the kinetics due to a change in substrate concentration, the effect of UGT2B7 on a substrate-induced spectral change seems to be saturated at the UGT2B7 concentrations used in the present study. In addition, UGT2B7 failed to alter the cytochrome c–reducing activity of CPR (Fig. 6). These results demonstrate that UGT2B7 most likely interacts directly with CYP3A4 and suppresses CYP3A4 activity through inhibiting entrance of substrate into the P450.

Fig. 3.
Fig. 3.

Effects of UGT2B7 and CNX on the kinetics of CYP3A4 expressed together with CPR in Sf9 cells. CYP3A4 activity was compared between double expression (CYP3A4/CPR), indicated as absence, and ternary expression [(A) CYP3A4/CPR/UGT2B7; (B) CYP3A4/CPR/CNX], indicated as presence. Each plot represents the mean ± S.D. of triplicate assays. Data were fitted to a sigmoidal model, and the kinetic parameters obtained are listed in Table 1. An asterisk represents statistical significance in the Vmax value (P < 0.017). See Table 1 for the criterion of significant difference.

View this table:
TABLE 1

Effect of coexpression of UGT2B7 and CNX on CYP3A4 activity

Kinetic parameters calculated from the results shown in Fig. 3 are listed. Statistical significance between parameters obtained in the absence and presence of UGT2B7 coexpression was determined by extra sum-of-squares F test. This test was repeated three times for each parameter. To keep the total error under 0.05, P values below 0.017 (= 0.05/3) were considered as statistically significant (*P < 0.017).

Fig. 4.
Fig. 4.

Effect of UGT2B7 on P450 catalytic cycle. The effect of UGT2B7 on CYP3A4-catalyzed reaction was examined using testosterone as a substrate. The comparison was carried out in the presence (black bar) or absence (white bar) of coexpression of UGT2B7. Each bar indicates the mean ± S.D. of triplicate assays in (A), (D), and (E). In (B) and (C), each bar represents the mean ± S.D. of three independent experiments. Statistical significance was calculated by Student’s t test (***P < 0.001; ns, not significant). In (A), CYP3A4-catalyzed testosterone 6β-hydroxylation is compared. Concentrations of CYP3A4, testosterone, and NADPH were fixed at 50 nM, 200 μM, and 1 mM in a final volume of 1 ml 100 mM potassium phosphate buffer (pH 7.4). In (B), CYP3A4-dependent NADPH consumption was measured by monitoring absorbance at 340 nm in the presence of substrate. The initial NADPH concentration was 300 μM and other conditions were the same as those described in (A). In (C), NADPH consumption was measured in the absence of substrate. In (D), generation of H2O2 though P450 uncoupling reaction was compared. The reaction conditions were the same as described previously. The amount of H2O2 in the reaction-quenched samples was determined with the Pierce Quantitative Peroxide Assay Kit (Thermo Scientific). In (E), Cumene hydroperoxide–driven CYP3A4-catalyzed oxidation was measured using testosterone as a substrate. The incubation conditions were the same as those described in (A), except that the reaction was started by addition of 1 mM cumene hydroperoxide instead of NADPH.

Fig. 5.
Fig. 5.

Effect of UGT2B7 on insertion of substrate into the catalytic pocket of CYP3A4. Substrate-binding difference spectra were compared in the absence and presence of coexpressed UGT2B7. Details are described in the Materials and Methods section. Three batches of microsomes expressing CYP3A4/CPR and CYP3A4/CPR/UGT2B7 were prepared and each spectrum was independently measured for each spectrum. Thus, we finally obtained six different spectra. In (A)–(C), the pairs of spectra that exhibited the most marked difference in each testosterone concentration are shown. To quantify the degree of CYP3A4 substrate binding, we subtracted the absorbance at the minimal point (417 nm) from that at the maximal point (390 nm) (ΔAbsorbance390-417) for every sample. Each value after ΔA denotes the degree. All spectra with different substrate concentrations were recorded using the same microsomal preparation. In (D), each symbol represents the difference in absorbance between 390 and 417 nm using each of the three different preparations under 200 μM of testosterone. Statistical significance was determined by Student’s t test (P < 0.05); however, there was no significant difference between microsomes with or without UGT2B7.

Fig. 6.
Fig. 6.

Effect of UGT2B7 on cytochrome c reduction mediated by CPR. CPR was expressed in the absence (−) and presence (++ or +) of UGT2B7 coexpression. In microsomes coexpressing UGT2B7, the expression levels of the UGT differed between preparations: high and low UGT levels are shown by (++) and (+), respectively. A reaction mixture (final volume 1 ml) contained the same amount of CPR determined by immunoblotting [0.95 mg cytochrome c, 1 mM KCN, and 3.3 mM MgCl2 in 300 mM potassium phosphate buffer (pH 7.5)]. Reaction was started by addition of an NADPH-generating system (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, and 0.4 U/ml glucose-6-phosphate dehydrogenase) and incubated at 25°C. CPR reactions were determined by measuring the absorbance at 550 nm. One of the representative examples for the alteration of absorbance at 550 nm is shown in (A). In (B), each bar represents the mean ± S.D. of three independent assays. No statistical significance (ns) was observed between CPR alone and coexpression by Dunnett’s test.

Role of the C-Terminal Area of UGT2B7 in the Suppression of CYP3A4 Function.

As described previously, CYP3A4 J-helix, a surface domain facing the cytosol, has been suggested to contribute to a CYP3A4-UGT2B7 interaction (Takeda et al., 2009). However, the main body of UGT is present in the luminal space of the ER, and only a short region at the C terminus, called the cytosolic tail, is extruded into the cytosol. Thus, we initially focused on the cytosolic tail (Lys510–Asp529) (Mackenzie, 1986; Radominska-Pandya et al., 1999) of UGT2B7 as a candidate region involved in the interaction with CYP3A4. To estimate the role of the C-terminal domains in the suppression of CYP3A4, several truncated mutants of UGT2B7 were prepared (see Table 2 for the constructs produced). As can be seen in the immunoblotting (Fig. 7A), a slight decrease in molecular weight was observed depending on the length of the deletion introduced. The effects on the Michaelis-Menten kinetics of CYP3A4 are shown in Fig. 7B and the kinetic parameters obtained are listed in Supplemental Table 7. A mutant, UGT2B7Δ519-529, which lacked 11 residues from the C terminus maintained the ability to reduce the Vmax value of CYP3A4 similar to wild-type enzyme. However, deletion of a further eight residues (Δ511-529) failed to suppress CYP3A4 activity. The UGT2B7Δ493-529 mutant lacking the whole transmembrane region also lost any suppressing potential. From these lines of evidence, the region between the 511 and 518th residues seemed to play an important role in CYP3A4 suppression. However, confusingly, a Δ511-518 mutant retained the suppressive effect similar to the wild type. Conceivably, this may come from the possibility that the membrane-spanning domain of UGT2B7 may actually differ from that predicted by traditional modeling.

View this table:
TABLE 2

Carboxyl-terminal sequences of deletion mutants of UGT2B7

Four mutants lacking parts of the C-terminal region were constructed. The postulated transmembrane region of UGT2B7 is underlined (Mackenzie, 1986; Radominska-Pandya et al., 1999), and the area adjacent to it is defined as the cytosolic tail.

Fig. 7.
Fig. 7.

Effect of the deletion of the C-terminal region of UGT2B7 on the repression of CYP3A4 activity. (A) The deletion mutants of UGT2B7 listed in Table 2 were expressed in Sf9 cells, and the microsomes (5 µg protein) were separated by 8.8% SDS-PAGE followed by immunobloting. HLM was used as a positive control. Mock represents control microsomes used as a negative control. The amount of protein amount in these controls was also 5 μg. Detection was carried out using goat anti-mouse UGT IgG (Mackenzie et al., 1984) and HRP-rabbit anti-goat IgG, as primary and secondary antibodies, respectively. (B) Four deletion mutants of UGT2B7 were expressed with CYP3A4, and their effects on the P450 activity were determined. The P450/CPR ratios of each pair of microsomes (double and ternary expressions, respectively) are shown below. The P450/CPR ratios of microsomes are shown (in the parentheses) without or with the UGT2B7 mutant, respectively: Δ519-529 (2.22, 2.16); Δ511-529 (4.51, 4.91); Δ493-529 (0.28, 0.36); and Δ511-518 (1.22, 0.73). Data were fitted to a sigmoidal model and are shown as Michaelis-Menten plots. Calculated parameters are listed in Supplemental Table 7. Each plot represents the mean ± S.D. of triplicate assays. An asterisk represents statistical significance in the Vmax value (P < 0.017).

In Silico Remodeling of the UGT2B7 C-Terminal Domain and Identification of the Region Involved in CYP3A4 Suppression.

To better understand the role of the UGT2B7 C-terminal domain on the suppression of CYP3A4 function, we simulated the secondary structures and hydrophobic region of UGT2B7 by in silico modeling (see the Materials and Methods for details). The model constructed suggests that the UGT2B7 C-terminal domain, including the membrane-anchoring region, can be divided into two parts: one is a hydrophilic region containing several charged residues, and the other is a hydrophobic region containing a transmembrane helix (Table 3). Regarding these regions, we initially focused on the charged residues in the hydrophilic area, making use of information that P450-CPR and P450/b5 associations are driven by electrostatic interactions (Bridges et al., 1998; Backes and Kelley, 2003; Laursen et al., 2011). While there are many hydrophilic amino acids near the C terminus of UGT2B7 (Table 3), we focused on Lys518 because this is the only charged residue located in the 511–518th region (Table 2), and the lines of results of deleted mutants suggested the region played some critical roles in the suppressive effect (see Fig. 7). Thus, a mutant (K518A) in which Lys518 was replaced with alanine was prepared to assess the role of the cationic site. In another mutant, A7, all the charged residues including Lys518 in the hydrophilic region were substituted with alanines. The expression of K518A and A7 was confirmed by immunoblotting (Fig. 8A). With K518A, a reduction in the Vmax value of CYP3A4 still took place (Fig. 8B; Supplemental Table 8), suggesting that the positive charge of Lys518 is not essential for suppression. Although the Vmax value did not exhibit a significant difference with and without A7 coexpression (Fig. 8B; Supplemental Table 8), the CYP3A4 activity appeared to be markedly lower in microsomes coexpressing A7 than in the control preparation [see the Michaelis-Menten plot (Fig. 8B)]. Therefore, we judged that the A7 mutant retains the suppressive effect, and hydrophilic amino acids play only a minor role in CYP3A4 suppression. Regarding the role of the hydrophobic region, we designed two mutants, 3CA and A8. In 3CA, three cysteines at the 511, 512, and 515th positions were substituted with alanines. On the other hand, in A8, the eight residues located at the 511–518th positions were substituted with alanines (see Table 3). Both of these mutants had no suppressive effect on CYP3A4 activity (Fig. 8B; Supplemental Table 8). This observation demonstrates that UGT2B7 needs part of the C-terminal region, especially the hydrophobic region forming a transmembrane helix.

View this table:
TABLE 3

Alanine substitution introduced into the C-terminal region of UGT2B7

The C-terminal position where the residue(s) was substituted with alanine(s) is shown in italics. This study predicted transmembrane or membrane-bound hydrophobic regions of all mutants by TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and these regions are shown in underline. The secondary structures of mutants were also predicted by Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008), and the residues constituting a helix are shown by brackets. The subscript numbers represent the residue number counted from the N terminus of wild-type UGT2B7.

Fig. 8.
Fig. 8.

Effect of alanine substitutions introduced into the C-terminal area of UGT2B7 on the suppression of CYP3A4. (A) Alanine-substituted mutants, the sequences of which are listed in Table 3, were expressed, and 5 μg of the microsomal protein was separated by 8.8% SDS-PAGE. HLM was used as a positive control. Mock represents control microsomes applied as a negative control. The amount of protein in these controls was also 5 μg. (B) Substitution with alanine was introduced into the C-terminal region of UGT2B7 as shown in Table 3. The mutants were tested to see if they worked as a CYP3A4 suppressor, similar to wild-type UGT2B7. Each P450/CPR ratio of the microsomes evaluated is shown as described in Fig. 7: K518A (1.22, 1.26); A7 (4.51, 3.73); 3CA (2.22, 2.41); and A8 (1.39, 1.38). The results of the kinetic analyses are shown as Michaelis-Menten plots. Data were fitted to a sigmoidal model, and the calculated parameters are listed in Supplemental Table 8. Each plot represents the mean ± S.D. of triplicate assays. An asterisk represents statistical significance in the Vmax value (P < 0.017).

UGT2B7 Domain Involved in the Suppression of CYP3A4: Prediction Using Chimeras.

For a better understanding of the role of the lipophilic region in the transmembrane helix near the C terminus in terms of a suppressive interaction with CYP3A4, we generated two chimeric proteins of UGT2B7 and CNX by replacing their hydrophilic ends. For instance, one (chimera 1: CNX-UGT tail) was constructed so as to have the transmembrane helix of CNX, followed by the hydrophilic C-terminal region of UGT2B7. In contrast, another chimera (chimera 2: UGT-CNX tail) was prepared as a fusion protein consisting of the transmembrane helix of UGT2B7 and the hydrophilic end of CNX. Their sequences are listed in Table 4. The expression of these chimeras was confirmed by immunoblotting with the antibodies recognizing the luminal domains of UGT2B7 and CNX (Fig. 9A). As expected, chimeras 1 and 2 were detectable with anti-CNX antibody and anti-UGT antibody, respectively. Since the hydrophilic end of CNX was longer than that of UGT2B7, chimera 1 had a lower molecular weight than the wild-type CNX. In contrast, the molecular mass of chimera 2 was greater than that of the wild-type UGT2B7. The predicted molecular weights of chimeras 1 and 2 were 67,000 and 78,000, respectively, and the chimeras were detected at the expected positions. The effects of these chimeras on CYP3A4 activity were quite different. While chimera 1 failed to reduce the CYP3A4 activity, chimera 2 showed a suppressive effect in the kinetic analysis (Fig. 9B; Supplemental Table 9). This observation strongly supports our hypothesis that the hydrophobic region in the UGT2B7 transmembrane helix plays a crucial role in the suppression of CYP3A4 activity.

View this table:
TABLE 4

Composition of UGT2B7/CNX chimeric proteins used in this study: replacement of hydrophilic C-terminal and internal membrane-binding regions between UGT2B7 and CNX

The sequences of the C-terminal (upper three) and internal membrane-binding (lower two) regions of the chimeric proteins are listed together with that of wild-type UGT2B7. The hydrophobic region and secondary structure were predicted using TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008), respectively. In the C-terminal region, the hydrophobic region and helix are shown in underline and by brackets, respectively. Chimera 1: the hydrophilic end of CNX was replaced with that of UGT2B7. Chimera 2: the hydrophilic end of UGT2B7 was replaced with that of CNX. Chimera 3: a part of the internal membrane-anchoring region of UGT2B7 was replaced with CNX amphiphilic helix spanning the 402–422nd residues (David et al., 1993; Bergeron et al., 1994; Ouzzine et al., 1999; Lewis et al., 2011). The superscript and subscript numbers represent the location of the residues of wild-type CNX and UGT2B7, respectively, counted from the N terminus. In addition, plain and italic characters represent UGT2B7 and CNX sequences, respectively.

Fig. 9.
Fig. 9.

Effect of the chimeric proteins on CYP3A4 activity. (A) Three chimeric proteins, i.e., CNX-UGT-tail (chimera 1), UGT-CNX-tail (chimera 2), and UGT-CNX-UGT-tail (chimera 3) were designed as shown in Table 4, and expressed in insect cells. The cellular microsomes (5 μg protein) were separated by SDS-PAGE followed by immunoblotting (10 and 7.5% gels for upper and lower panels, respectively). Specific antibodies to recognize luminal bodies of UGT2B7 and CNX were used as primary antibodies. HLMs and control microsomes indicated as Mock were loaded as positive and negative controls, respectively. (B) Effect of chimeric proteins on CYP3A4 activity is shown. The P450/CPR ratios of microsomes evaluated are shown below as described in Fig. 7: chimera 1 (1.11, 1.23); chimera 2 (1.40, 1.58); and chimera 3 (0.68, 0.71). Each plot represents the mean ± S.D. of triplicate assays. Kinetic parameters were calculated by fitting the curve to a sigmoidal model and are listed in Supplemental Table 9. An asterisk represents statistical significance in the Vmax value (P < 0.017).

The present study generated another chimera (chimera 3: UGT-CNX-UGT tail) to examine the role of the internal domain of UGT2B7 on CYP3A4 suppression. As reported previously (Meech and Mackenzie, 1998; Ouzzine et al., 1999; Lewis et al., 2011), the internal membrane-anchoring region of UGT2B7 seems to be located at the 183–200th residues. This domain was replaced with the luminal amphiphilic helix of CNX to produce chimera 3. Unlike other chimeras, this substitution showed little alteration in molecular weight compared with wild-type UGT2B7 (Fig. 9A). Although chimera 3 contained the whole C-terminal segment of UGT2B7, this chimera had no ability to suppress CYP3A4 activity (Fig. 9B; Supplemental Table 9). This result suggests that in order to have a suppressive effect on CYP3A4 activity two regions of UGT2B7 are required, i.e., both hydrophobic residues in the transmembrane helix and the internal membrane-anchoring domain are needed.

Discussion

This study provides evidence that UGT modulates P450 activity. In the pull-down assay employed in this study, UGT2B7, but not CNX, was trapped with His-CYP3A4 (Fig. 1). Such specific association between P450 and UGT agrees with our previous reports (Taura et al., 2004; Takeda et al., 2005; Ishii et al., 2007). Although the P450/CPR and UGT/P450 ratios varied among the microsomal preparations, coexpression of UGT2B7 lowered CYP3A4 function in every preparation. Pooled HLMs showed P450/CPR and UGT/P450 ratios that are within the range of variance in transformed Sf9 cells. These results strongly suggest that UGT2B7-dependent suppression of CYP3A4 function occurs under physiologic conditions. Early studies have reported that the activity of liver microsomal P450 is lower than that of reconstituted systems, even though their P450 contents are comparable (West and Lu, 1972; Kaminsky et al., 1983; Wood et al., 1983; Sonderfan et al., 1987). This may be due to the competition for CPR by different P450s and/or P450-P450 interactions (Reed and Backes, 2012). Moreover, it was also found that the CYP3A4/CPR system prepared in a baculovirus-insect cell system showed a higher turnover of CYP3A4 than that of HLMs (Crespi and Miller, 1999). The data reported here are apparently consistent with the aforementioned phenomena: that is, the lower function of CYP3A4 in HLMs than in artificial expression systems could be explained, at least partially, by coexisting UGTs. However, the degree of suppression was independent of the UGT/P450 score. As mentioned in the introduction, P450 can form homo- and hetero-oligomers (Davydov, 2011; Reed and Backes, 2012), and the same is true for UGTs (Ishii et al., 2001, 2010; Fujiwara et al., 2007; Lewis et al., 2011). If UGT-UGT and P450-UGT interactions take place at the same domain of UGT, UGT2B7 homo-oligomerization would compete with the CYP3A4-UGT2B7 interaction. Such competition may produce a situation under which the suppression of CYP3A4 activity takes place independently of the expression level of UGT2B7. The glycosylation status of UGT may be another factor affecting P450 function. Most UGTs including UGT2B7 have potential N-glycosylation sites in their sequences, and N-glycosylation of UGT has been reported to influence UGT conformations and functions (Mackenzie, 1990; Barbier et al., 2000; Nakajima et al., 2010). It is conceivable that defective UGT2B7 lacking correct modification with a sugar chain was expressed to some extent in our system, and such an enzyme failed to interact with CYP3A4. In this regard, it has been reported that, in a baculovirus-insect cell system, inactive UGT was expressed at a significant level when many viruses were used for transfection (Zhang et al., 2012).

In the kinetic analysis, UGT2B7 markedly reduced the Vmax value of CYP3A4 (Fig. 3; Table 1). Previous studies have suggested that the conformation and function of P450s vary according to how they interact with the membrane (Ahn et al., 1998; Kim et al., 2003). Thus, UGT2B7 may affect CYP3A4 topology in the membrane to repress CYP3A4 activity. On the other hand, the present study also suggests that UGT2B7 suppresses the whole catalytic cycle of P450. This is because coexpression of UGT2B7 reduced not only the oxidation of the substrate but also NADPH consumption and the uncoupling reaction to the same degree (Fig. 4). Moreover, UGT2B7 failed to alter NADPH consumption in the absence of substrate and did not affect the activity of CPR (Fig. 4C and 6). Additionally, coexpression of UGT2B7 tended to suppress the substrate binding of CYP3A4 with every concentration of testosterone we used (Fig. 5, A–C). Although no significant difference was observed in the quantified result (Fig. 5D), even a slight difference may equate to a large difference in the degree of substrate binding (Schenkman et al., 1967). Interestingly, coexpression of UGT2B7 reduced CYP3A4 sensitivity toward the testosterone quarter. From these results, it would be reasonable to consider that UGT2B7 suppresses CYP3A4 activity by inhibiting the insertion of substrates into the catalytic pocket of CYP3A4. The binding of substrate to P450 causes a change in its electrical potential, and this change drives the catalytic cycle of P450 (Isin and Guengerich, 2008). If UGT2B7 inhibits this first step, CYP3A4 function would be suppressed in all the later catalytic steps as we observed. Concerning the shunt pathway, P450 catalysis driven by cumene hydroperoxide, the observation that UGT2B7 reduced the reaction, also supports the above assumption (Fig. 4E).

To identify the UGT2B7 domain involved in the suppressive interaction with CYP3A4, we generated a series of mutants. In our previous study, the J-helix, one of the CYP3A4 helices facing the cytosol, was suggested as the domain that is involved in the interaction with UGT2B7 (Takeda et al., 2009). Thus, initially, we designed mutants lacking the C-terminal region called the cytosolic tail, which is the sole region extruded into the cytosol. A deletion mutant, Δ519-529, which lacked half of the tail, retained its suppressive potential on CYP3A4; however, further truncation led to a loss of effect (Fig. 7B; Supplemental Table 7). However, the analysis using deletion mutants failed to identify the essential segment in the C-terminal region. To overcome this, we made a further attempt to reconstruct a model for the secondary structure by in silico modeling. The in silico modeling suggested that the C-terminal transmembrane helix consisting of many hydrophobic residues is longer than the classic one, and passes from the luminal side to the cytosolic side. In contrast, the subsequent hydrophilic region of the C terminus is shorter than that considered before. Then, we introduced alanine substitutions into both the hydrophobic and hydrophilic areas. As expected, alanine substitutions following the in silico modeling enabled us to find important regions for the suppression of CYP3A4 activity. Even if mutations were introduced into the hydrophilic end, this mutant (A7) retained the ability to suppress CYP3A4 activity. In contrast, the suppressive potential declined when some residues in the hydrophobic region were replaced with alanines (Fig. 8B; Supplemental Table 8; Table 3). These results strongly suggest the important role of the hydrophobic region constituting part of the transmembrane helix of UGT2B7 in the suppression of CYP3A4 activity. For further investigation of the role of this region, some chimeric proteins were generated. In chimera 1 (CNX-UGT-tail) the CNX main body, linked to its own transmembrane helix, was fused to the hydrophilic end of UGT2B7 (Fig. 9B; Supplemental Table 9). The disappearance of a suppressive effect in chimera 1 agreed with the importance of the hydrophobic region in the transmembrane helix. Conversely, in chimera 2 (UGT-CNX-tail), the UGT2B7 main body, linked to its own transmembrane helix, was fused to the hydrophilic end of CNX, and chimera 2 exhibited a suppressive effect on CYP3A4 activity. These results suggest that the hydrophobic residues in the transmembrane helix of UGT2B7 but not CNX are important. These observations convincingly support a view that the C-terminal hydrophobic region of UGT2B7 plays an important role in the negative regulation of CYP3A4 activity.

In addition to the hydrophobic area near the C terminus of UGT2B7, our data demonstrate the important role of the internal region spanning the 183–200th residues for interaction with CYP3A4. In UGT1A6, the corresponding region is assumed to serve as a part of the internal membrane-anchoring region (Ouzzine et al., 1999). Furthermore, it has been suggested that this region is the domain needed for homodimerization of UGT2B7 (Lewis et al., 2011). Chimera 3 was a UGT2B7-based mutant in the 183–200th region that was replaced with the luminal amphiphilic helix of CNX (Table 4). This chimera failed to reduce CYP3A4 activity, although it retained the wild-type UGT2B7 hydrophobic region in the transmembrane helix (Fig. 9B; Supplemental Table 9). Therefore, it is likely that a part of the internal membrane-anchoring domain is also necessary for interaction with CYP3A4. However, because chimera 3 was catalytically inactive (unpublished data), it is conceivable that its inability to suppress CYP3A4 activity may result from inappropriate protein folding. From these lines of evidence, UGT2B7 would need at least two domains to exhibit its suppressive effect on CYP3A4: 1) the hydrophobic region in the transmembrane helix, and 2) part of the internal membrane-anchoring domain. It is suggested that these domains work to negatively regulate CYP3A4 function (Fig. 10).

Fig. 10.
Fig. 10.

Cooperative suppression of CYP3A4 function by different domains of UGT2B7. (A) Two UGT2B7 domains that were suggested to contribute to the interaction with CYP3A4 are schematically shown together with the signal peptide. (B) The cooperative suppression of CYP3A4 by the two domains is illustrated. The C-terminal hydrophobic domain serves as a transmembrane helix. The helix in this model consists of 35 residues, and it was longer than the previous model (Mackenzie, 1986) by being extended at both ends. The internal membrane anchoring segment and a possible dimerization domain are predicted on the basis of previous work (Ouzzine et al., 1999; Lewis et al., 2011).

This study suggests that the functional interaction of CYP3A4 and UGT2B7 is one of the reasons underlying the interindividual differences in CYP3A4 activity. From a physiologic viewpoint, it may not be beneficial that CYP3A4, a major defensive mechanism protecting against hazardous xenobiotics, is inhibited by UGT. However, interaction of CYP3A4 and UGT2B7 is expected to reduce the CYP3A4-dependent production of cytotoxic hydrogen peroxide by suppression of the uncoupling that occurs under physiologic conditions. In other words, it is reasonable to suppose that the P450-UGT interaction prevents cells from being exposed to undesirable stress. This hypothesis is supported by the effect of UGT2B7 on the catalytic cycle of CYP3A4 in testosterone oxidation (Fig. 4B): that is, this UGT suppressed both the H2O2 production and the uncoupling reaction.

In conclusion, this is the first report describing the modulation of P450 activity by UGT. However, there are likely to be a number of P450-UGT interactions involving a variety of P450 and UGT isoforms (Thummel and Wilkinson, 1998; Ishii et al., 2010, 2014), and their effects on P450s have not yet been elucidated. Further studies are necessary to understand the details and physiologic significance of these P450-UGT interactions.

Acknowledgments

The authors thank Ayumi Furukawa and Dr. Yoshio Nishimura for constructing the C-terminal truncated mutants of UGT2B7 in pTargeT and expression plasmid for pFastBac1-CNX, respectively. The authors also thank the Research Support Center, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, for technical support.

Authorship Contributions

Participated in research design: Miyauchi, Nagata, Yamazoe, Mackenzie, Yamada, Ishii.

Conducted experiments: Miyauchi, Ishii.

Contributed new reagents or analytic tools: Miyauchi, Nagata.

Performed data analysis: Miyauchi, Yamada, Ishii.

Wrote or contributed to the writing of the manuscript: Miyauchi, Mackenzie, Yamada, Ishii.

Footnotes

    • Received February 23, 2015.
    • Accepted July 31, 2015.

  • This study was supported in part by the Japan Research Foundation for Clinical Pharmacology; a Grant-in-Aid for Scientific Research (B) [Grant 25293039] from the Japanese Society of Promotion of Science; and a Grant-in-Aid for Scientific Research (C) [Grant 19590147] from the Ministry of Science, Education, Sports and Technology to Y.I.

  • This work was presented in part at the 132nd Annual Meeting of the Pharmaceutical Society of Japan, Sapporo, Japan, March 2012 (Miyauchi Y, Ishii Y, Oizaki T, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, Suppression of cytochrome P450 3A4 function by UDP-glucuronosyltransferase 2B7: Role of C-terminal cytosolic region of UGT2B7); the 19th Microsomes and Drug Oxidation Meeting/12th European Regional Meeting of the International Society for the Study of Xenobiotics, Noordwijk ann Zee, The Netherlands, June 2012 (Miyauchi Y, Ishii Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, UDP-Glucuronosyltransferase (UGT) 2B7 and 1A9 suppress cytochrome P450 3A4 function: evidence for the involvement of the cytosolic tail of UGT in the suppression); the 28th Annual Meeting of the Japanese Society of the Study of Xenobiotics, Tokyo, Japan, October 2013 (Miyauchi Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, Ishii Y, Suppression of cytochrome P450 3A4 activity by UDP-glucuronosyltransferase 2B7: Crucial role of the length of UGT2B7 cytosolic tail in the suppression); the 134th Annual Meeting of the Pharmaceutical Society of Japan, Kumamoto, Japan, March 2014 (Miyauchi Miyauchi Y, Ishii Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, Alteration of cytochrome P450 3A4 activity through a protein-protein interaction with UDP-glucuronosyltransferase 2B7: Cooperation of the luminal domain and cytosolic tail of UGT2B7 is required in the suppression of CYP3A4 function); 19th North American Regional Meeting of the International Society for the Study of Xenobiotics/29th Annual Meeting of the Japanese Society of the Study of Xenobiotics, San Francisco, October, 2014 (Miyauchi Y, Ishii Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, Suppression of cytochrome P450 3A4 activity by UDP-glucuronosyltransferase (UGT) 2B7: the role of charged residue(s) in the cytosolic tail of UGT2B7); and the 20th Microsomes and Drug Oxidation Meeting, Stuttgart, Germany, May, 2014 (Ishii Y, Miyauchi Y, Yamada H, Functional interactions between cytochrome P450 and UDP-glucuronosyltransferase: a new insight into the inter-individual variation of drug metabolism).

  • dx.doi.org/10.1124/mol.115.098582.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

b5
cytochrome b5
CNX
calnexin
CPR
NADPH-cytochrome P450 reductase
ER
endoplasmic reticulum
HLM
human liver microsomes
HRP
horseradish peroxidase
P450
cytochrome P450
PCR
polymerase chain reaction
UGT
UDP-glucuronosyltransferase

References

  1. ↵
    1. Ahn T,
    2. Guengerich FP, and
    3. Yun CH
    (1998) Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids. Biochemistry 37:12860–12866.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Backes WL and
    2. Kelley RW
    (2003) Organization of multiple cytochrome P450s with NADPH-cytochrome P450 reductase in membranes. Pharmacol Ther 98:221–233.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Barbier O,
    2. Girard C,
    3. Breton R,
    4. Bélanger A, and
    5. Hum DW
    (2000) N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes. Biochemistry 39:11540–11552.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Bergeron JJ,
    2. Brenner MB,
    3. Thomas DY, and
    4. Williams DB
    (1994) Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci 19:124–128.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Bridges A,
    2. Gruenke L,
    3. Chang YT,
    4. Vakser IA,
    5. Loew G, and
    6. Waskell L
    (1998) Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase. J Biol Chem 273:17036–17049.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chefson A,
    2. Zhao J, and
    3. Auclair K
    (2006) Replacement of natural cofactors by selected hydrogen peroxide donors or organic peroxides results in improved activity for CYP3A4 and CYP2D6. ChemBioChem 7:916–919.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Cole C,
    2. Barber JD, and
    3. Barton GJ
    (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36:W197–W201.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Crespi CL and
    2. Miller VP
    (1999) The use of heterologously expressed drug metabolizing enzymes—state of the art and prospects for the future. Pharmacol Ther 84:121–131.
    OpenUrlCrossRefPubMed
  9. ↵
    1. David V,
    2. Hochstenbach F,
    3. Rajagopalan S, and
    4. Brenner MB
    (1993) Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J Biol Chem 268:9585–9592.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Davydov DR
    (2011) Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions. Expert Opin Drug Metab Toxicol 7:543–558.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Fujiwara R,
    2. Nakajima M,
    3. Yamanaka H,
    4. Katoh M, and
    5. Yokoi T
    (2007) Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab Dispos 35:1781–1787.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Guengerich FP
    (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39:1–17.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hildebrandt A and
    2. Estabrook RW
    (1971) Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions. Arch Biochem Biophys 143:66–79.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Hirota T,
    2. Ieiri I,
    3. Takane H,
    4. Maegawa S,
    5. Hosokawa M,
    6. Kobayashi K,
    7. Chiba K,
    8. Nanba E,
    9. Oshimura M,
    10. Sato T,
    11. et al.
    (2004) Allelic expression imbalance of the human CYP3A4 gene and individual phenotypic status. Hum Mol Genet 13:2959–2969.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Imai Y
    (1976) The use of 8-aminooctyl sepharose for the separation of some components of the hepatic microsomal electron transfer system. J Biochem 80:267–276.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Ishii Y,
    2. Iwanaga M,
    3. Nishimura Y,
    4. Takeda S,
    5. Ikushiro S,
    6. Nagata K,
    7. Yamazoe Y,
    8. Mackenzie PI, and
    9. Yamada H
    (2007) Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex. Drug Metab Pharmacokinet 22:367–376.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Ishii Y,
    2. Koba H,
    3. Kinoshita K,
    4. Oizaki T,
    5. Iwamoto Y,
    6. Takeda S,
    7. Miyauchi Y,
    8. Nishimura Y,
    9. Egoshi N,
    10. Taura F,
    11. et al.
    (2014) Alteration of the function of the UDP-glucuronosyltransferase 1A subfamily by cytochrome P450 3A4: different susceptibility for UGT isoforms and UGT1A1/7 variants. Drug Metab Dispos 42:229–238.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Ishii Y,
    2. Miyoshi A,
    3. Watanabe R,
    4. Tsuruda K,
    5. Tsuda M,
    6. Yamaguchi-Nagamatsu Y,
    7. Yoshisue K,
    8. Tanaka M,
    9. Maji D,
    10. Ohgiya S,
    11. et al.
    (2001) Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltransferase 2B21-catalyzed morphine-6-glucuronide formation. Mol Pharmacol 60:1040–1048.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Ishii Y,
    2. Takeda S, and
    3. Yamada H
    (2010) Modulation of UDP-glucuronosyltransferase activity by protein-protein association. Drug Metab Rev 42:145–158.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Ishii Y,
    2. Takeda S,
    3. Yamada H, and
    4. Oguri K
    (2005) Functional protein-protein interaction of drug metabolizing enzymes. Front Biosci 10:887–895.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Isin EM and
    2. Guengerich FP
    (2008) Substrate binding to cytochromes P450. Anal Bioanal Chem 392:1019–1030.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Jin CJ,
    2. Miners JO,
    3. Burchell B, and
    4. Mackenzie PI
    (1993) The glucuronidation of hydroxylated metabolites of benzo[α]pyrene and 2-acetylaminofluorene by cDNA-expressed human UDP-glucuronosyltransferases. Carcinogenesis 14:2637–2639.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Kaminsky LS,
    2. Guengerich FP,
    3. Dannan GA, and
    4. Aust SD
    (1983) Comparisons of warfarin metabolism by liver microsomes of rats treated with a series of polybrominated biphenyl congeners and by the component-purified cytochrome P-450 isozymes. Arch Biochem Biophys 225:398–404.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kim KH,
    2. Ahn T, and
    3. Yun CH
    (2003) Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4. Biochemistry 42:15377–15387.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Lakhman SS,
    2. Ma Q, and
    3. Morse GD
    (2009) Pharmacogenomics of CYP3A: considerations for HIV treatment. Pharmacogenomics 10:1323–1339.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Lamba JK,
    2. Lin YS,
    3. Schuetz EG, and
    4. Thummel KE
    (2002) Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev 54:1271–1294.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Laursen T,
    2. Jensen K, and
    3. Møller BL
    (2011) Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450. Biochim Biophys Acta 1814:132–138.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Lewis BC,
    2. Mackenzie PI, and
    3. Miners JO
    (2011) Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling. Biochem Pharmacol 82:2016–2023.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Locuson CW,
    2. Wienkers LC,
    3. Jones JP, and
    4. Tracy TS
    (2007) CYP2C9 protein interactions with cytochrome b5: effects on the coupling of catalysis. Drug Metab Dispos 35:1174–1181.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Lowry OH,
    2. Rosebrough NJ,
    3. Farr AL, and
    4. Randall RJ
    (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
    OpenUrlFREE Full Text
  31. ↵
    1. Lu AY,
    2. Junk KW, and
    3. Coon MJ
    (1969) Resolution of the cytochrome P-450-containing ω-hydroxylation system of liver microsomes into three components. J Biol Chem 244:3714–3721.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Mackenzie PI
    (1986) Rat liver UDP-glucuronosyltransferase. cDNA sequence and expression of a form glucuronidating 3-hydroxyandrogens. J Biol Chem 261:14112–14117.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Mackenzie PI
    (1990) The effect of N-linked glycosylation on the substrate preferences of UDP glucuronosyltransferases. Biochem Biophys Res Commun 166:1293–1299.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Mackenzie PI,
    2. Hjelmeland LM, and
    3. Owens IS
    (1984) Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver. Arch Biochem Biophys 231:487–497.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Meech R and
    2. Mackenzie PI
    (1998) Determinants of UDP glucuronosyltransferase membrane association and residency in the endoplasmic reticulum. Arch Biochem Biophys 356:77–85.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Miwa GT and
    2. Lu AY
    (1984) The association of cytochrome P-450 and NADPH-cytochrome P-450 reductase in phospholipid membranes. Arch Biochem Biophys 234:161–166.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Möller S,
    2. Croning MD, and
    3. Apweiler R
    (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Nakajima M,
    2. Koga T,
    3. Sakai H,
    4. Yamanaka H,
    5. Fujiwara R, and
    6. Yokoi T
    (2010) N-Glycosylation plays a role in protein folding of human UGT1A9. Biochem Pharmacol 79:1165–1172.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Omura T
    (2011) Recollection of the early years of the research on cytochrome P450. Proc Jpn Acad, Ser B, Phys Biol Sci 87:617–640.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Omura T and
    2. Sato R
    (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378.
    OpenUrlFREE Full Text
  41. ↵
    1. Ouzzine M,
    2. Magdalou J,
    3. Burchell B, and
    4. Fournel-Gigleux S
    (1999) An internal signal sequence mediates the targeting and retention of the human UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum. J Biol Chem 274:31401–31409.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Pan YZ,
    2. Gao W, and
    3. Yu AM
    (2009) MicroRNAs regulate CYP3A4 expression via direct and indirect targeting. Drug Metab Dispos 37:2112–2117.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Radominska-Pandya A,
    2. Czernik PJ,
    3. Little JM,
    4. Battaglia E, and
    5. Mackenzie PI
    (1999) Structural and functional studies of UDP-glucuronosyltransferases. Drug Metab Rev 31:817–899.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Reed JR and
    2. Backes WL
    (2012) Formation of P450∣P450 complexes and their effect on P450 function. Pharmacol Ther 133:299–310.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Schenkman JB and
    2. Jansson I
    (2006) Spectral analyses of cytochromes P450. Methods Mol Biol 320:11–18.
    OpenUrlPubMed
  46. ↵
    1. Schenkman JB,
    2. Remmer H, and
    3. Estabrook RW
    (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol Pharmacol 3:113–123.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y, and
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Singh D,
    2. Kashyap A,
    3. Pandey RV, and
    4. Saini KS
    (2011) Novel advances in cytochrome P450 research. Drug Discov Today 16:793–799.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Sonderfan AJ,
    2. Arlotto MP,
    3. Dutton DR,
    4. McMillen SK, and
    5. Parkinson A
    (1987) Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch Biochem Biophys 255:27–41.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Takagi S,
    2. Nakajima M,
    3. Mohri T, and
    4. Yokoi T
    (2008) Post-transcriptional regulation of human pregnane X receptor by micro-RNA affects the expression of cytochrome P450 3A4. J Biol Chem 283:9674–9680.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Takeda S,
    2. Ishii Y,
    3. Iwanaga M,
    4. Mackenzie PI,
    5. Nagata K,
    6. Yamazoe Y,
    7. Oguri K, and
    8. Yamada H
    (2005) Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4. Mol Pharmacol 67:665–672.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Takeda S,
    2. Ishii Y,
    3. Iwanaga M,
    4. Nurrochmad A,
    5. Ito Y,
    6. Mackenzie PI,
    7. Nagata K,
    8. Yamazoe Y,
    9. Oguri K, and
    10. Yamada H
    (2009) Interaction of cytochrome P450 3A4 and UDP-glucuronosyltransferase 2B7: evidence for protein-protein association and possible involvement of CYP3A4 J-helix in the interaction. Mol Pharmacol 75:956–964.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Taura K,
    2. Naito E,
    3. Ishii Y,
    4. Mori MA,
    5. Oguri K, and
    6. Yamada H
    (2004) Cytochrome P450 1A1 (CYP1A1) inhibitor α-naphthoflavone interferes with UDP-glucuronosyltransferase (UGT) activity in intact but not in permeabilized hepatic microsomes from 3-methylcholanthrene-treated rats: possible involvement of UGT-P450 interactions. Biol Pharm Bull 27:56–60.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Taura K,
    2. Yamada H,
    3. Hagino Y,
    4. Ishii Y,
    5. Mori MA, and
    6. Oguri K
    (2000) Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase. Biochem Biophys Res Commun 273:1048–1052.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Thummel KE and
    2. Wilkinson GR
    (1998) In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol 38:389–430.
    OpenUrlCrossRefPubMed
  56. ↵
    1. West SB and
    2. Lu AY
    (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. V. Competition between cytochromes P-450 and P-448 for reductase in 3,4-benzpyrene hydroxylation. Arch Biochem Biophys 153:298–303.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Wood AW,
    2. Ryan DE,
    3. Thomas PE, and
    4. Levin W
    (1983) Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes. J Biol Chem 258:8839–8847.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Yasukochi Y and
    2. Masters BS
    (1976) Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J Biol Chem 251:5337–5344.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Zhang H,
    2. Patana AS,
    3. Mackenzie PI,
    4. Ikushiro S,
    5. Goldman A, and
    6. Finel M
    (2012) Human UDP-glucuronosyltransferase expression in insect cells: ratio of active to inactive recombinant proteins and the effects of a C-terminal his-tag on glucuronidation kinetics. Drug Metab Dispos 40:1935–1944.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

UGT2B7 Suppresses CYP3A4 Function

Yuu Miyauchi, Kiyoshi Nagata, Yasushi Yamazoe, Peter I. Mackenzie, Hideyuki Yamada and Yuji Ishii
Molecular Pharmacology October 1, 2015, 88 (4) 800-812; DOI: https://doi.org/10.1124/mol.115.098582
Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement