UGT) 2B7 in Human Liver Microsomes

Drug Metab. Pharmacokinet. 29 (3): 259–265 (2014). Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Extensive Protein-Protein Interactions Involving UDP- glucuronosyltransferase (UGT) 2B7 in Human Liver Microsomes

Ryoichi FUJIWARA* and Tomoo ITOH School of Pharmacy, Kitasato University, Tokyo, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: UDP-glucuronosyltransferase (UGT) 2B7 is a membrane protein that catalyzes glucuronidation of endogenous and exogenous substrates. Because UGTs are expressed in the endoplasmic reticulum (ER), their substrates and metabolites need to be transported through the ER membrane. However, insight into the mechanism underlying the transport of substrates/metabolites of UGTs through the ER membrane has not been elucidated. Metabolosome is a functional unit of metabolism consisting of multiple metabolism- related proteins. UGTs might form a metabolosome to facilitate the transport of their substrates and/or metabolites through the ER membrane. In the present study, therefore, extensive protein-protein interactions involving UGT2B7 were determined by a shotgun analysis of immunoprecipitate. Our shotgun analysis revealed that 92 proteins were immunoprecipitated with anti-UGT2B7 antibody in human liver microsomes. We further determined that 42 proteins out of the 92 proteins were speciﬁcally immunoprecipitated with the anti-UGT2B7 antibody. In addition to UGT2B7, other microsomal enzymes such as UGT1A, CYP3A4, CYP1A2, and a monoamine oxidase, were included in the list of proteins immunoprecipitated with the anti-UGT2B7 antibody, suggesting that these proteins might form a metabolosome to regulate their functions in the liver. Further analyses are needed to elucidate the roles of those proteins in the enzymatic activity of human UGTs.

Keywords: UDP-glucuronosyltransferase; protein-protein interactions; metabolosome; UGT2B7; CYP3A4

UGT1A4 is a unique UGT isoform that is responsible for N- Introduction glucuronidation of primary, secondary, and tertiary amine-contain- UDP-glucuronosyltransferases (UGTs; EC 2.4.1.17) are impor- ing xenobiotics such as imipramine and trifluoperazine.6,7) tant membrane-bound enzymes that metabolize drugs by trans- The active site of UGT proteins is located in the luminal side of ferring the glucuronic acid moiety of UDP-glucuronic acid to the the endoplasmic reticulum (ER).8) Therefore, substrates and a co- substrates.1) Human UGTs are divided into two distinct families, substrate of UGTs need to be transported into the ER lumen from UGT1 and UGT2, on the basis of evolutionary divergence and cytoplasm to be metabolized by UGTs. Metabolites also need to homology.2) The UGT1 gene is located on chromosome 2q37 be transported to cytoplasm to be further excreted from the body. and produces nine functional enzymes (UGT1A1, UGT1A3- Because glucuronides are extremely hydrophilic, they are not UGT1A10) by exon sharing.3) The UGT2A and UGT2B genes easily translocated across the ER membrane by passive diffusion. are located on chromosome 4q13, encoding three and seven func- The presence of multiple glucuronide transporters mediating the tional proteins, respectively.2) UGT2A1 and UGT2A2 are formed transport of glucuronides through the ER membrane has been by differential splicing of variable first exons and common exons 2 demonstrated, although those transporters have not been identi- to 6, likely on the UGT1A gene. Meanwhile, UGT2A3 and each fied.9) In contrast, a nucleotide sugar transporter, which is involved UGT2B isoform are encoded by individual genes.2) Each UGT in the transport of UDP-glucuronic acid from cytosol to the luminal enzyme expresses in a tissue-specific manner and exhibits sub- side of ER, has been identified.10) strate specificity.4) Among 19 functional UGT enzymes, UGT2B7 Cytochrome P450s (CYPs) are phase I drug-metabolizing is the most responsible isoform for glucuronidation of clinically enzymes. Similar to UGTs, CYP is a superfamily of enzymes used drugs.5) While most UGT isoforms catalyze the transfer of involved in the metabolism of a wide variety of endogenous and glucuronic acid to hydroxyl- and carboxyl-group of substrates, exogenous compounds. Among all CYP enzymes, CYP3A4 is the

Received September 10, 2013; Accepted December 14, 2013 J-STAGE Advance Published Date: December 24, 2013, doi:10.2133/dmpk.DMPK-13-RG-096 *To whom correspondence should be addressed: Ryoichi FUJIWARA, Ph.D., School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel. ©81-3-5791-6249, E-mail: [email protected] This work was supported by the Naito Foundation (RF).

259 260 Ryoichi FUJIWARA and Tomoo ITOH most abundant in both the liver and intestinal tract and is involved Immunoprecipitation: Dynabeads were resuspended in a vial in the metabolism of more than 50% of clinically used drugs.5) for 5 min. Fifty µL (1.5 mg) of Dynabeads was transferred to a tube Although the active site of CYPs is located in the cytoplasmic side and the tube was placed on a magnet to remove the supernatant. of ER, the function of UGTs and CYPs is similar, as they share Two hundred µL of antibodies was added and the tube was rotated substrates such as codeine and acetaminophen. There is also a for 10 min at room temperature. The tube was again placed on functional relationship between UGTs and CYPs in that CYPs a magnet to remove the supernatant, and the Dynabead-antibody catalyze hydroxylation of substrates so that UGTs can transfer complex was washed using 200 µL PBS containing 0.1% Tween glucuronic acid to the hydroxyl group of the substrates. For 20. After the supernatant was removed by placing the tube on a example, UGTs introduce glucuronic acid to the hydroxyl groups magnet, 400 µL of human liver microsomes, which were sus- of trans-3A-hydroxycotinine and trans-4-hydroxyl tamoxifen, which pended in 0.25 M sucrose, was added to the tube. The tube was are oxidative metabolites of nicotine and tamoxifen by CYPs.11,12) then rotated for 20 min at room temperature. The tube was placed This fact led to the hypothesis that UGTs and CYPs interact with on the magnet to remove the supernatant, and the Dynabead- each other to transport substrates and metabolites across the ER antibody-antigen complex was washed three times using 200 µL membrane. To support this hypothesis, protein-protein interactions washing buffer. The supernatant was removed and the beads were between UGTs and CYPs have been demonstrated previously.13,14) resuspended in 100 µL washing buffer. After the bead solution was Furthermore, functional interactions between UGTs and CYPs transferred to a new tube and the supernatant was removed, 30 µL have also been demonstrated by investigating a sequential metabo- of SDS-PAGE buffer was added to elute the immune complex. lism of 7-ethoxycoumarin as a model substrate.15) It has also been Digestion of the immunoprecipitate: Ten µL of reduc- suggested that homo- and hetero-oligomerization of UGT proteins tion buffer (1.5 mg of dithiothreitol dissolved in 1 mL of 100 mM might be involved in the transport of substrates and glucuronides ammonium bicarbonate) was added to the eluted sample from the across the ER membrane.16) Homo- and hetero-oligomers of UGTs Dynabead-antibody-antigen complex. After a 30-min incubation at have been demonstrated by gel permeation chromatography, radia- room temperature, 10 µL of alkylating buffer (10 mg of iodoacet- tion inactivation analyses, cross-linking studies, double-UGT amide dissolved in 1 mL 100 mM ammonium bicarbonate) was isoforms-expression systems, native-PAGE analyses, and fluo- added. After a 30-min incubation at room temperature, 2 µL of rescence resonance energy transfer (FRET) analyses.16–19) How- trypsin solution (10 µg/mL) and 10 µL of 50 mM ammonium ever, insight into the mechanism underlying the transport of bicarbonate were added to the reaction tube. The tube was substrates of UGTs and their metabolites across the ER membrane incubated overnight at 30°C and the solution was evaporated to has not been clarified yet. dryness. Seven µL of 1% formic acid was added to the tube to Metabolosome and transportsome are functional units of dissolve the peptides. The peptide solution was then subjected to metabolism and membrane transport. Metabolosome and trans- nano-LC-MS/MS analysis. portsome are a multi-molecular assembly composed of metaboliz- Nano-LC-MS/MS analysis: Nano-LC-MS/MS analysis was ing enzymes, transport-related proteins (transporters, channels or carried out in a CapLC system (Waters, Milford, MA) with Q-tof pumps), regulatory proteins, scaffold proteins, and other functional microtechnology (Micromass, Manchester, UK). Peptides were cellular components, which are assembled by means of multiple injected into a Symmetry C18 5 µm, 180 µm © 20 mm precolumn protein-protein interactions and/or protein-lipid interactions.20) and subsequently separated using a 1.7 µm BEH 130 C18 100 Formation of metabolosome and/or transportsome assembled by µm © 25 mm separation column maintained at 30°C. Mobile extensive protein-protein interactions involving UGTs might be phases A and B were water with 0.1% formic acid and 0.1% associated with the transport of compounds across the ER mem- formic acid in acetonitrile, respectively. Peptide separation was brane. In the present study, large-scale analysis of protein-protein obtained by a gradient of 1–50% mobile phase B over 120 min at a interaction involving UGTs was carried out by shotgun liquid flow rate of 300 nL/mim, followed by a gradient of 50–85% mobile chromatography–mass spectrometry (LC-MS/MS) proteomic anal- phase B over 1 min and a 25 min rinse with 85% mobile phase B. ysis of immunoprecipitated proteins to identify proteins interacting Data acquisition and processing of the MS/MS analysis were with human UGT2B7 in human liver microsomes. carried out using MassLynx software (Micromass). A cone voltage of 45 V and a capillary voltage of 2.3 kV were used. The collision Methods energy was 25–35 eV and the collision gas was argon for the MS/ Chemicals and reagents: Human liver microsomes, UGT MS operation. The resulting files were submitted to a MASCOT Supersomes, and rabbit anti-human UGT2B7 antibody were (Matrix Science, London, UK) query search and searched against obtained from BD Gentest (Woburn, MA). Rabbit anti-human the Swiss-Prot database. The sensitivity, specificity and repro- UGT2B antibody (H-300) was purchased from Santa Cruz ducibility of the nano-LC-MS/MS analysis used in this study have Biotechnology (Santa Cruz, CA). Control antibodies (IgG from been described elsewhere.23) rabbit serum) were purchased from Sigma-Aldrich (St. Louis, Results MO) and Abcam (Cambridge, MA). Immunoprecipitation Kit Dynabeads ProteinG was purchased from Invitrogen (Carlsbad, Confirmation of the specificity of anti-UGT2B7 antibody: CA). All other chemicals and solvents were of analytical grade or Prior to the immunoprecipitation assays, the specificity of the the highest grade commercially available. antibodies was determined using UGT expression systems, such SDS/PAGE and immunoblotting: Ten µg of human liver as UGT2B4, UGT2B7, UGT2B10, UGT2B15, UGT2B17, and microsomes and 2 µg of each UGT expression system (BD UGT1A1. The anti-UGT2B7 antibody specifically reacted with Gentest) were separated on 4–12% NuPAGE Bis-Tris polyacryl- UGT2B7, as a strong band was only observed against the UGT2B7 amide gels (Invitrogen). Immunoblotting with anti-UGT2B7 and expression system (Fig. 1, top). In contrast, the anti-UGT2B anti-UGT2B antibodies was performed as previously outlined.21,22) antibody reacted with multiple UGT2B isoforms such as UGT2B4,

that the anti-UGT2B7 antibody used in the co-immunoprecipitation assay reacted non-specifically with some proteins in human liver microsomes. To obtain more reliable data, immunoprecipitation assays were further conducted using different antibodies, followed by shotgun analyses. Identification of proteins immuno-precipitated with control antibodies: To further exclude the possibility that antibodies used in the immunoprecipitation assays non-specifically reacted to proteins in human liver microsomes, we carried out immunoprecipitation assays with control rabbit IgG, followed by shotgun analysis, because anti-UGT2B7 was rabbit antibodies. The shotgun analysis with rabbit IgG antibody revealed that 101 proteins were precipitated with the antibody. Peptide sequences of UGT2B7 were not detected with this control antibody, demonstrating that peptide Fig. 1. Immunoblot analysis of human liver microsomes and UGT sequences of UGT2B7 obtained in the assay with anti-UGT2B7 expression systems antibody could be attributed to specific reaction with this antibody. Ten µg of HLM and 2 µg of each UGT expression system were subjected to As we expected, peptide sequences of most PDIs were detected an immunoblot analysis with anti-UGT2B7 antibody (top) and anti-UGT2B antibody (bottom). Arrows indicate the presence of bands. M, marker. in the shotgun analysis, suggesting that the peptide sequences of PDIs obtained in the assay with anti-UGT2B7 antibody could be attributed to non-specific reaction with rabbit IgG. UGT2B7, and UGT2B17 (Fig. 1, bottom). The purpose of our Rabbit IgG antibody from another vendor was also subjected study was to identify the proteins specifically interacting with to the immunoprecipitation assay, followed by shotgun analysis. UGT2B7; therefore, in the present study, the specific anti-UGT2B7 In this assay, peptide sequences of 155 proteins were detected. antibody was used for the immunoprecipitation assay. Peptide sequences of UGT2B7 were not detected with this control Identification of proteins immunoprecipitated with human antibody, demonstrating that peptide sequences of UGT2B7 UGT2B7 antibody: UGT2B7 is the primary UGT isoform in- obtained in the assay with anti-UGT2B7 antibody could be attrib- volved in the metabolism of a wide variety of drugs in the human uted to specific reaction with these antibodies. Although peptide liver. To identify proteins that are interacting with UGT2B7, sequences of calnexin were not detected in the former analysis with human liver microsomes were subjected to an immunoprecipitation the control antibody, they were detected in this analysis, suggesting assay with rabbit anti-human UGT2B7 antibody, followed by a that the peptide sequences of calnexin obtained in the assay with shotgun analysis of the immunoprecipitate by an LC-MC/MC anti-UGT2B7 antibody could be attributed to non-specific reaction analysis. Our shotgun analysis revealed that 92 proteins including with rabbit IgG. human UGT2B7 were co-immunoprecipitated with anti-human Out of the 92 proteins that were detected with anti-UGT2B7 UGT2B7 antibody (Fig. 2). Human UGTs have been reported antibody, these control antibodies detected 50 proteins, including to form homo- and hetero-oligomers. Protein-protein interactions carboxylesterase, glutathione S-transferases, epoxide hydrolase, between UGT2B7 and other UGT isoforms such as UGT1A1, and alcohol dehydrogenases. In contrast to these findings, peptide UGT1A4, UGT1A6, and UGT1A9 were demonstrated previ- sequences of CYP3A4 and CYP1A2 were not detected with the ously.17) In the present study, the peptide sequences of human control antibodies, indicating that these proteins were specifically UGT1A have been identified by the shotgun analysis (Fig. 3), co-immunoprecipitated with UGT2B7 antibody. showing the reproducibility of the previous finding. Protein-protein Summary of the results: Proteins that are specifically co- interaction between UGT2B7 and CYP3A4 was also demonstrated immunoprecipitated by UGT2B7 antibody were summarized in previously.24,25) To support this finding, a peptide sequence of Table 1. Because these proteins were not co-immunoprecipitated human CYP3A4 was obtained in our immunoprecipitate followed by control rabbit IgG, it is considered that they were interacting by the shotgun analysis (Fig. 4). Our shotgun analysis revealed with UGT2B7 protein in human liver microsomes, possibly by peptide sequences of epoxide hydrolase 1, which is an enzyme that forming a metabolosome. CYPs have been reported to interact with to alter their functions,26) Discussion suggesting that not only CYPs, but also UGTs might interact with epoxide hydrolases. Several peptide sequences of other drug- An immunoprecipitation assay, followed by immunoblotting, metabolizing enzymes such as calboxylestetase 1, alcohol de- is a classic method of identifying proteins that are interacting with hydrogenases, and glutathione S-transferases were also obtained a targeted protein. While this method has been widely applied in the shotgun analysis. This finding indicates that UGT2B7 might in numerous scientific papers due to its convenience, it also has interact with multiple drug metabolism-associated proteins to form disadvantages. For example, in addition to a primary antibody to a metabolosome. immunoprecipitate a targeted protein, it requires a primary anti- Protein disulfide-isomerases (PDIs) and calnexine are non-drug body to detect candidate proteins, which are interacting with the metabolizing enzymes that are expressed in ER. These proteins targeted protein, in the immunoblotting analysis. Therefore, this were reported not to interact with CYP1A1 by affinity chromatog- method cannot be used to identify truly new proteins interacting raphy using CYP1A1-conjugated Sepharose 4B columns.14) In the with target proteins. Furthermore, the detection limit of immuno- present study, however, peptide sequences of PDIs and calnexin precipitation, followed by immunoblotting, is not sufficient to were obtained in the immunoprecipitation assay with anti-UGT2B7 detect proteins poorly interacting with the targeted protein. To antibodies followed by shotgun analysis, suggesting a possibility overcome this disadvantage, overexpression systems need to be

Fig. 2. Peptide sequence analysis of human UGT2B7 Three peptide sequences of human UGT2B7 were obtained with the shotgun analysis of the immunoprecipitate with anti-UGT2B7 antibody (A). The middle panel indicates the MS/MS spectra of the peptide (B), whereas the bottom panel indicates ion tables (C). Fig. 3. Peptide sequence analysis of human UGT1A4 Five peptide sequences of human UGT1A4 were obtained with the shotgun analysis of the immunoprecipitate with anti-UGT2B7 antibody (A). The middle panel indicates the MS/MS spectra of the peptide (B), whereas the bottom panel indicates ion tables (C).

established. Cross-linking methods are alternative ways to identify proteins that are interacting with a targeted protein; however, those methods face the same issues described above. Although a mass spectrometry analysis of bands, which can be obtained with silver- staining of a gel used in SDS-PAGE of immunoprecipitate, does not require primary antibodies to detect interacting proteins and can be applied to identify new interacting proteins, this method, too, cannot be used to determine extensive protein-protein interactions, because each band on the gel needs to be excised and applied for the MS analysis. Recently, a new technique, shotgun analysis, has been developed to carry out large-scale analysis of protein-protein interactions. To identify new proteins interacting with human UGT2B7 in liver microsomes, therefore, shotgun LC-MS/MS proteomic analysis of immunoprecipitated proteins was carried out in the present study. As a preliminary study, we have performed an immunoprecipitation assay using a buffer with or without 0.05% Tween-20. When both immunoprecipitates were subjected to the shotgun analysis, we found that the number of detected proteins in both methods was very similar. To identify more new proteins interacting with UGT2B7 in the microsomes, Fig. 4. Peptide sequence analysis of human CYP3A4 therefore, we employed the immunoprecipitation without the Two peptide sequences of human CYP3A4 were obtained with the shotgun detergent. analysis of the immunoprecipitate with anti-UGT2B7 antibody (A). The middle panel indicates the MS/MS spectra of the peptide (B), whereas the bottom panel As mentioned in Introduction, physical and functional inter- indicates ion tables (C). actions between UGT-UGT and UGT-CYP have been widely

Table 1. Proteins that were co-immunoprecipitated with anti-UGT2B7 antibody

No. Accession Score Mass Sequences emPAI Description 1 UD17_HUMAN 303 60,578 6 0.43 UDP-glucuronosyltransferase 1–7 2 UD14_HUMAN 276 60,613 6 0.43 UDP-glucuronosyltransferase 1–4 3 H2B1C_HUMAN 249 13,898 2 1.11 Histone H2B type 1-C/E/F/G/I 4 VDAC1_HUMAN 81 30,868 2 0.26 Voltage-dependent anion-selective channel protein 1 5 UD2B7_HUMAN 75 61,168 2 0.13 UDP-glucuronosyltransferase 2B7 6 OST48_HUMAN 74 50,940 2 0.15 Dolichyl-diphosphooligosaccharide–protein glycosyltransferase 48 kDa subunit 7 PRDX4_HUMAN 72 30,749 2 0.26 Peroxiredoxin-4 8 MYH11_HUMAN 63 228,054 1 0.02 Myosin-11 9 NNTM_HUMAN 63 114,564 2 0.07 NAD(P) transhydrogenase, mitochondrial 10 MOGS_HUMAN 62 92,032 1 0.04 Mannosyl-oligosaccharide glucosidase 11 ACTN4_HUMAN 58 105,245 1 0.04 Alpha-actinin-4 12 PHB_HUMAN 57 29,843 2 0.27 Prohibitin 13 CP4F2_HUMAN 55 60,442 1 0.06 Leukotriene-B(4) omega-hydroxylase 1 14 ERP29_HUMAN 54 29,032 1 0.13 Endoplasmic reticulum resident protein 29 15 BAP31_HUMAN 53 28,031 1 0.13 B-cell receptor-associated protein 31 16 THIM_HUMAN 52 42,354 1 0.09 3-Ketoacyl-CoA thiolase, mitochondrial 17 TMEDA_HUMAN 51 25,131 1 0.15 Transmembrane emp24 domain-containing protein 10 18 PDIA4_HUMAN 46 73,229 1 0.05 Protein disulfide-isomerase A4 19 S27A5_HUMAN 45 76,420 1 0.05 Bile acyl-CoA synthetase 20 APMAP_HUMAN 44 46,622 1 0.08 Adipocyte plasma membrane-associated protein 21 CP3A4_HUMAN 44 57,705 1 0.06 Cytochrome P450 3A4 22 NB5R3_HUMAN 41 34,441 1 0.11 NADH-cytochrome b5 reductase 3 23 MTP_HUMAN 39 99,916 1 0.04 Microsomal triglyceride transfer protein large subunit 24 CP1A2_HUMAN 39 58,656 1 0.06 Cytochrome P450 1A2 25 PXMP2_HUMAN 39 22,238 1 0.17 Peroxisomal membrane protein 2 26 LRC59_HUMAN 38 35,308 1 0.11 Leucine-rich repeat-containing protein 59 27 M2OM_HUMAN 37 34,211 1 0.11 Mitochondrial 2-oxoglutarate/malate carrier protein 28 ATP5L_HUMAN 36 11,421 1 0.35 ATP synthase subunit g, mitochondrial 29 MPC2_HUMAN 34 14,327 1 0.27 Mitochondrial pyruvate carrier 2 30 TBB1_HUMAN 33 50,865 1 0.07 Tubulin beta-1 chain 31 RL7_HUMAN 33 29,264 1 0.13 60S ribosomal protein L7 32 PLAG1_HUMAN 31 56,786 1 0.07 Zinc finger protein PLAG1 33 NDUV1_HUMAN 28 51,469 1 0.07 NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial 34 ATPD_HUMAN 27 17,479 1 0.22 ATP synthase subunit delta, mitochondrial 35 NDUA9_HUMAN 26 42,654 1 0.09 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial 36 UDB15_HUMAN 24 61,510 1 0.06 UDP-glucuronosyltransferase 2B15 37 THIK_HUMAN 24 44,834 1 0.08 3-Ketoacyl-CoA thiolase, peroxisomal 38 RET3_HUMAN 23 135,734 1 0.03 Retinol-binding protein 3 39 CEPT1_HUMAN 22 47,435 1 0.08 Choline/ethanolaminephosphotransferase 1 40 GLT10_HUMAN 21 69,689 1 0.05 Polypeptide N-acetylgalactosaminyltransferase 10 41 AOFB_HUMAN 19 59,238 1 0.06 Amine oxidase [flavin-containing] B 42 B3A2_HUMAN 14 137,493 1 0.03 Anion exchange protein 2 Proteins specifically immunoprecipitated with the anti-UGT2B7 are shown. investigated in UGT-overexpressing cells. However, these inter- protein and pyruvate carrier 2, were also identified as proteins actions had not been determined under physiological conditions interacting with human UGT2B7 in liver microsomes. The func- until recently, when a functional interaction between human UGTs tional relationship between UGT2B7 and newly identified micro- in human hepatocytes was demonstrated.27) Nevertheless, physical somal proteins is still unknown. However, as 5-hydroxytryptamine interactions between UGTs under physiological conditions, such as (5-HT, serotonin)—a monoamine neurotransmitter—is metabo- in human hepatocytes and liver microsomes, have not been proved lized by both UGT and MAO, there might be a functional coopera- yet. In our large-scale analysis of protein-protein interactions by tion between these enzymes to enhance their catalytic properties. shotgun analysis, the presence of various UGT-UGT and UGT- The role of other proteins identified in the present study in a CYP interactions in human liver microsomes was revealed. Taking glucuronidation reaction catalyzed by UGTs is unclear. However, these and previous findings together, it can be speculated that by forming a metabolosome, they might indirectly regulate the drug-metabolizing enzymes interact with each other to alter their function of UGTs. For example, PSD-95/Discs-large/ZO-1 (PDZ) function under physiological conditions. proteins interact with drug transporters to form a transportsome. Our study further identified multiple proteins interacting with Although PDZ protein itself does not function as a transporter, human UGT2B7 in liver microsomes (Table 1). Most of these are it has been suggested that PDZ proteins regulate the function of microsomal enzymes such as protein glycosyltransferase, man- transporters by stabilizing the transporters at the cell surface.28) nosyl-oligosaccharide glucosidase, 3-ketoacyl-CoA thiolase, bile Therefore, like the transportsome involving transporters and other acyl-CoA synthetase, triglyceride transfer protein, ATP synthase, proteins such as PDZs, formation of a metabolosome might affect NADH dehydrogenase, and monoamine oxidase (MAO). Channel the stability of UGT proteins, causing an alteration of UGT func- proteins, such as voltage-dependent anion-selective channel pro- tions. Further analyses are needed to elucidate the roles of the tein 1, and carrier proteins, such as 2-oxoglutarate/malate carrier proteins listed in Table 1 in the enzymatic activity of human

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