UGT2B7

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Peter I. Mackenzie - One of the best experts on this subject based on the ideXlab platform.

  • hetero oligomer formation of mouse udp glucuronosyltransferase ugt 2b1 and 1a1 results in the gain of glucuronidation activity towards morphine an activity which is absent in homo oligomers of either ugt
    Biochemical and Biophysical Research Communications, 2020
    Co-Authors: Yuu Miyauchi, Shin Ichi Ikushiro, Peter I. Mackenzie, Ayumi Kurita, Ryohei Yamashita, Tomoyuki Takamatsu, Yoshitaka Tanaka, Yuji Ishii
    Abstract:

    UDP-Glucuronosyltransferase (UGT, Ugt) is a major drug metabolizing enzyme family involved in the glucuronidation and subsequent elimination of drugs and small lipophilic molecules. UGT forms homo- and hetero-oligomers that enhance or suppress UGT activity. In our previous study, we characterized mouse Ugt1a1 and all the Ugt isoform belonging to the Ugt2b subfamily and revealed that mouse Ugt2b1 and Ugt1a1 cannot metabolize morphine. Mouse Ugt2b1 had been believed to function similarly to rat UGT2B1, which plays a major role in morphine glucuronidation in rat liver. Thus, in this study, we hypothesized that hetero-oligomerization with another Ugt isoform may affect Ugt2b1 catalytic ability. We co-expressed Ugt1a1 and Ugt2b1 in a baculovirus-insect cell system, and confirmed hetero-oligomer formation by co-immunoprecipitation. As reported previously, microsomes singly expressing Ugt1a1 or Ugt2b1 were inactive towards the glucuronidation of morphine. Interestingly, in contrast, morphine-3-glucuronide, a major metabolite of morphine was formed, when Ugt2b1 and Ugt1a1 were co-expressed. This effect of hetero-oligomerization of Ugt1a1 and Ugt2b1 was also observed for 17β-estradiol glucuronidation. This is the first report demonstrating that UGT acquires a novel catalytic ability by forming oligomers. Protein-protein interaction of Ugts may contribute to robust detoxification of xenobiotics by altering the substrate diversity of the enzymes.

  • regulation of udp glucuronosyltransferases ugt2b4 and UGT2B7 by micrornas in liver cancer cells
    Journal of Pharmacology and Experimental Therapeutics, 2017
    Co-Authors: Dhilushi D Wijayakumara, Dong Gui Hu, Ross A Mckinnon, Peter I. Mackenzie, Robyn Meech
    Abstract:

    The transcriptional regulation of UGT2B4 and UGT2B7 has been well studied using liver cancer cell lines and recently post-transcriptional regulation of these two UGTs by miR-216b-5p was reported. The present study describes novel miRNA-mediated regulation of UGT2B4 and UGT2B7 in liver cancer cells. Bioinformatic analyses identified a putative miR-3664-3p binding site in the UGT2B7 39-UTR, and binding sites for both miR-135a-5p and miR-410-3p in the UGT2B4 39-UTR. These sites were functionally characterized using miRNA mimics and reporter constructs. A miR-3664-3p mimic induced repression of a luciferase reporter carrying the UGT2B7 39-UTR in liver cancer cell lines; mutation of the miR-3664-3p site abrogated the response of the reporter to the mimic. Similarly, mutation of the miR-135a-5p site or miR-410-3p site in a luciferase reporter bearing UGT2B4 39-UTR abrogated the ability of miR-135a-5p or miR-410-3p mimics to reduce reporter activity. Transfection of miR-3664-3p mimics in HepG2 liver cancer cells significantly reduced mRNA and protein levels of UGT2B7, and this led to reduced enzymatic activity. Transfection of miR-135a-5p or miR-410-3p mimics significantly decreased UGT2B4 mRNA levels in Huh7 liver cancer cells. The expression levels of miR-410-3p were inversely correlated with UGT2B4 mRNA levels in the TCGA cohort of Liver Hepatocellular Carcinoma (370 specimens) and a panel of 9 normal human tissues. Similarly, there was an inverse correlation between miR-135a and UGT2B4 mRNA levels in a panel of 18 normal human liver tissues. Together these data suggest that miR-135a and miR-410 control UGT2B4 and that miR-3664 controls UGT2B7 expression in liver cancer and/or normal liver cells.

  • regulation of udp glucuronosyltransferases ugt2b4 and UGT2B7 by micrornas in liver cancer cells
    Journal of Pharmacology and Experimental Therapeutics, 2017
    Co-Authors: Dhilushi D Wijayakumara, Ross A Mckinnon, Peter I. Mackenzie, Robyn Meech
    Abstract:

    The transcriptional regulation of UDP-glucuronosyltransferases UGT2B4 and UGT2B7 has been well studied using liver cancer cell lines, and post-transcriptional regulation of these two UGTs by microRNA (miRNA/miR) miR-216b-5p was recently reported. This study describes novel miRNA-mediated regulation of UGT2B4 and UGT2B7 in liver cancer cells. Bioinformatic analyses identified a putative miR-3664-3p binding site in the UGT2B7 3'-untranslated region (UTR) and binding sites for both miR-135a-5p and miR-410-3p in the UGT2B4 3'-UTR. These sites were functionally characterized using miRNA mimics and reporter constructs. A miR-3664-3p mimic induced repression of a luciferase reporter carrying the UGT2B7 3'-UTR in liver cancer cell lines; mutation of the miR-3664-3p site abrogated the response of the reporter to the mimic. Similarly, mutation of the miR-135a-5p site or miR-410-3p site in a luciferase reporter bearing UGT2B4 3'-UTR abrogated the ability of miR-135a-5p or miR-410-3p mimics to reduce reporter activity. Transfection of miR-3664-3p mimics in HepG2 liver cancer cells significantly reduced mRNA and protein levels of UGT2B7, and this led to reduced enzymatic activity. Transfection of miR-135a-5p or miR-410-3p mimics significantly decreased UGT2B4 mRNA levels in Huh7 liver cancer cells. The expression levels of miR-410-3p were inversely correlated with UGT2B4 mRNA levels in The Cancer Genome Atlas cohort of liver hepatocellular carcinoma (371 specimens) and a panel of ten normal human tissues. Similarly, there was an inverse correlation between miR-135a and UGT2B4 mRNA levels in a panel of 18 normal human liver tissues. Together, these data suggest that miR-135a and miR-410 control UGT2B4 and that miR-3664 controls UGT2B7 expression in liver cancer and/or normal liver cells.

  • induction of human udp glucuronosyltransferase 2b7 gene expression by cytotoxic anticancer drugs in liver cancer hepg2 cells
    Drug Metabolism and Disposition, 2015
    Co-Authors: Peter I. Mackenzie, Robyn Meech, Ross A Mckinnon
    Abstract:

    We recently reported induction of UGT2B7 by its substrate epirubicin, a cytotoxic anthracycline anticancer drug, via activation of p53 and subsequent recruitment of p53 to the UGT2B7 promoter in hepatocellular carcinoma HepG2 cells. Using the same HepG2 model cell line, the present study assessed the possibility of a similar induction of UGT2B7 by several other cytotoxic drugs. We first demonstrated by reverse transcriptase quantitative real-time polymerase chain reaction that, as observed with epirubicin, nine cytotoxic drugs including three anthracyclines (doxorubicin, daunorubicin, and idarubicin) and six nonanthracyclines (mitomycin C, 5-fluorouracil, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, and etoposide) significantly increased UGT2B7 mRNA levels. To investigate a potential involvement of p53 in this induction, we conducted further experiments with four of the nine drugs (doxorubicin, daunorubicin, idarubicin, and mitomycin C). The cytotoxic drugs studied increased p53 and UGT2B7 protein levels. Knockdown of p53 expression by small interfering RNA reduced cytotoxic drug-induced UGT2B7 expression. Luciferase reporter assays showed activation of the UGT2B7 promoter by cytotoxic drugs via a previously reported p53 site. Finally, chromatin immunoprecipitation assays demonstrated p53 recruitment to the UGT2B7 p53 site upon exposure to mitomycin C, the most potent UGT2B7 inducer among the nine tested drugs. Taken together, these results provide further evidence supporting UGT2B7 as a p53 target gene. The cytotoxic drug-induced UGT2B7 activity in target liver cancer cells or possibly in normal liver cells may affect the therapeutic efficacy of co-administered cytotoxic drugs (e.g., epirubicin) and noncytotoxic drugs (e.g., morphine), which are UGT2B7 substrates.

  • homodimerization of udp glucuronosyltransferase 2b7 UGT2B7 and identification of a putative dimerization domain by protein homology modeling
    Biochemical Pharmacology, 2011
    Co-Authors: Benjamin C Lewis, Peter I. Mackenzie, John O Miners
    Abstract:

    Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S(50) values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a 'mixed' UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B'-C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic π-π stacking interactions, two S-aromatic (face) interactions, and the presence of 'proline brackets'. The homology model further provides important insights into structure-function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.

Alain Belanger - One of the best experts on this subject based on the ideXlab platform.

  • Glucuronidation activity of the UGT2B17 enzyme toward xenobiotics. Drug Metab Dispos 2003;31: 670 –6
    2020
    Co-Authors: David Turgeon, Jeansebastien Carrier, And Sarah Chouinard, Alain Belanger
    Abstract:

    This article is available online at http://dmd.aspetjournals.org ABSTRACT: UDP-glucuronosyltransferase (UGT) 2B17 is one of the most important conjugating enzymes in androgen metabolism and shares more than 95% homology with UGT2B15. Although UGT2B15 has been fully characterized for its ability to conjugate drugs, environmental pollutants, and dietary components, UGT2B17 received less attention for its capacity to glucuronidate xenobiotics. In the present study, more than 55 exogenous compounds belonging to several categories of compounds were analyzed as potential substrates for UGT2B17. Glucuronidation activity was observed with several coumarins, anthraquinones, and flavonoids. The higher glucuronidation activity was measured with alizarin (125 pmol ⅐ min ؊1 ⅐ mg protein ؊1 ), whereas UGT2B17 conjugated eugenol, scopoletin, and galangin with glucuronidation rates of 102.5, 102, and 58 pmol ⅐ min ؊1 ⅐ mg protein ؊1 , respectively. The characterization of UGT2B17 as a xenobiotics-conjugating enzyme demonstrates that its role is not limited to androgen metabolism and that its specificity for exogenous substrates is different from other UGT2B isoforms. Taken together, these data suggest a role of UGT2B17 for the hepatic detoxification

  • isoform specific regulation of uridine diphosphate glucuronosyltransferase 2b enzymes in the human prostate differential consequences for androgen and bioactive lipid inactivation
    Endocrinology, 2006
    Co-Authors: Sarah Chouinard, Alain Belanger, Georges Pelletier, Olivier Barbier
    Abstract:

    Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1alpha, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5alpha-androstane-3alpha,17beta-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1alpha and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1alpha induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1alpha, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.

  • cellular specific expression of the androgen conjugating enzymes ugt2b15 and ugt2b17 in the human prostate epithelium
    Endocrine Research, 2004
    Co-Authors: Sarah Chouinard, Alain Belanger, Georges Pelletier, Olivier Barbier
    Abstract:

    In humans, 3β‐hydroxysteroid dehydrogenase (3β‐HSD), 17β‐HSD, and 5α‐reductase enzymes convert dehydroepiandrosterone (DHEA), androstenedione, and testosterone into the most potent natural androgen dihydrotestosterone (DHT) in the prostate. This androgen is transformed mainly in situ to two Phase I metabolites, androsterone (ADT) and androstane‐3α,17β‐diol (3α‐DIOL), which can, however, be back‐converted to DHT. Here, we report recent findings on the characterization of specific anti‐UDP‐glucuronosyltransferases (UGT)2B15 and 2B17 antibodies and their use to identify UGT2B expressing‐cells in the human prostate epithelium. We found that UGT2B17 is expressed in basal cells where DHEA is converted into 3α‐DIOL and ADT. By contrast, the expression of UGT2B15 was observed only in luminal cells, where DHT is formed from testosterone. These results demonstrate that, in the human prostate, UGT2B15 and UGT2B17 genes have complementary roles, and are expressed in cells where their specific substrates are synthesiz...

  • inactivation of androgens by udp glucuronosyltransferase enzymes in humans
    Trends in Endocrinology and Metabolism, 2003
    Co-Authors: Alain Belanger, Olivier Barbier, Georges Pelletier, Fernand Labrie, Sarah Chouinard
    Abstract:

    Abstract In humans, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD and 5α-reductase activities in androgen target tissues, such as the prostate and skin, convert dehydroepiandrosterone, androstenedione and testosterone into the most potent natural androgen dihydrotestosterone (DHT). This androgen is converted mainly in situ into two phase I metabolites, androsterone (ADT) and androstane-3α,17β-diol (3α-DIOL), which might be back converted to DHT. Here, we discuss the recent findings regarding the characterization of specific UDP-glucuronosyltransferases (UGTs), UGT2B7, B15 and B17, responsible for the glucuronidation of these metabolites. The tissue distribution and cellular localization of the UGT2B transcripts and proteins in humans clearly indicate that these enzymes are synthesized in androgen-sensitive tissues. It is postulated that the conjugating activity of UGT enzymes is the main mechanism for modulating the action of steroids and protecting the androgen-sensitive tissues from deleteriously high concentrations of DHT, ADT and 3α-DIOL.

  • inactivation of androgens by udp glucuronosyltransferase enzymes in humans
    Trends in Endocrinology and Metabolism, 2003
    Co-Authors: Alain Belanger, Olivier Barbier, Georges Pelletier, Fernand Labrie, Sarah Chouinard
    Abstract:

    In humans, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-HSD and 5alpha-reductase activities in androgen target tissues, such as the prostate and skin, convert dehydroepiandrosterone, androstenedione and testosterone into the most potent natural androgen dihydrotestosterone (DHT). This androgen is converted mainly in situ into two phase I metabolites, androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-DIOL), which might be back converted to DHT. Here, we discuss the recent findings regarding the characterization of specific UDP-glucuronosyltransferases (UGTs), UGT2B7, B15 and B17, responsible for the glucuronidation of these metabolites. The tissue distribution and cellular localization of the UGT2B transcripts and proteins in humans clearly indicate that these enzymes are synthesized in androgen-sensitive tissues. It is postulated that the conjugating activity of UGT enzymes is the main mechanism for modulating the action of steroids and protecting the androgen-sensitive tissues from deleteriously high concentrations of DHT, ADT and 3alpha-DIOL.

John O Miners - One of the best experts on this subject based on the ideXlab platform.

  • homodimerization of udp glucuronosyltransferase 2b7 UGT2B7 and identification of a putative dimerization domain by protein homology modeling
    Biochemical Pharmacology, 2011
    Co-Authors: Benjamin C Lewis, Peter I. Mackenzie, John O Miners
    Abstract:

    Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S(50) values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a 'mixed' UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B'-C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic π-π stacking interactions, two S-aromatic (face) interactions, and the presence of 'proline brackets'. The homology model further provides important insights into structure-function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.

  • characterization of niflumic acid as a selective inhibitor of human liver microsomal udp glucuronosyltransferase 1a9 application to the reaction phenotyping of acetaminophen glucuronidation
    Drug Metabolism and Disposition, 2011
    Co-Authors: John O Miners, Kushari Bowalgaha, Pawel Baranczewski, David J. Elliot, Kathleen M. Knights
    Abstract:

    Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50–100 μM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with Ki values ranging from 0.10 to 0.40 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (Ki range 14–18 μM), whereas competitive inhibition (Ki 62 μM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 μM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean Vmax for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 μM NFA. Mean Km values decreased in parallel with Vmax, although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

  • in vitro in vivo extrapolation predicts drug drug interactions arising from inhibition of codeine glucuronidation by dextropropoxyphene fluconazole ketoconazole and methadone in humans
    Journal of Pharmacology and Experimental Therapeutics, 2010
    Co-Authors: Pritsana Raungrut, Verawan Uchaipichat, Benjamas Janchawee, Andrew A. Somogyi, David J. Elliot, John O Miners
    Abstract:

    Because codeine (COD) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro–in vivo extrapolation approaches were used to identify potential drug–drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD-6-glucuronide (C6G) formation by human liver microsomes (HLM) and demonstrated an 88% reduction in the Michaelis constant ( K m ) (0.29 versus 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UDP-glucuronosyltransferase (UGT) enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S 50 values (0.32 and 0.27 mM) generated in the presence of BSA were comparable with the mean K m observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone. Inhibitor constant values generated for dextropropoxyphene (3.5 μM), fluconazole (202 μM), ketoconazole (0.66 μM), and methadone (0.32 μM) predicted 1.60- to 3.66-fold increases in the area under the drug plasma concentration–time curve ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4- and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone potentially affect the intensity and duration of COD analgesia.

  • influence of n terminal domain histidine and proline residues on the substrate selectivities of human udp glucuronosyltransferase 1a1 1a6 1a9 2b7 and 2b10
    Drug Metabolism and Disposition, 2009
    Co-Authors: Oranun Kerdpin, Kushari Bowalgaha, Peter I. Mackenzie, Moshe Finel, John O Miners
    Abstract:

    An N-terminal domain histidine [corresponding to position 39 of UDP-glucuronosyltransferase (UGT) 1A1] is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobiotic-metabolizing enzymes, UGT1A4 and UGT2B10 lack the ability to glucuronidate 4-methylumbelliferone (4MU) and 1-naphthol (1NP), both planar phenols, and naproxen (a carboxylic acid). However, only UGT1A4 glucuronidates the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP). In this study, we sought to elucidate the influence of specific N-terminal histidine and proline residues on UGT enzyme substrate selectivity. The conserved N-terminal domain histidine of UGT1A1, UGT1A6, UGT1A9, and UGT2B7 was mutated to proline and leucine 34 of UGT2B10 was substituted with histidine, and the capacity of the wild-type and mutant proteins to glucuronidate 4MU, 1NP, LTG, TFP, and naproxen was characterized. Whereas UGT1A1(H39P), UGT1A6(H38P), and UGT1A9(H37P) lacked the ability to metabolize 4MU, 1NP, and naproxen, all glucuronidated LTG. K(m) values for UGT1A1(H39P) and UGT1A9(H37P) were 774 and 3812 microM, respectively, compared with 1579 microM for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a V(max)/K(m) value comparable to that of UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective K(m) values of 260 and 118 microM. UGT2B7(H35P) lacked activity toward all substrates. The data confirm a pivotal role for an N-terminal domain proline in the glucuronidation of the tertiary amines LTG and TFP by UGT1A subfamily proteins, whereas glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes.

  • The Configuration of the 17-Hydroxy Group Variably Influences the Glucuronidation of β-Estradiol and Epiestradiol by Human UDP-Glucuronosyltransferases
    Drug Metabolism and Disposition, 2008
    Co-Authors: Katrina Itaaho, Shin Ichi Ikushiro, Peter I. Mackenzie, John O Miners, Moshe Finel
    Abstract:

    The glucuronidation of 17β-estradiol (β-estradiol) and 17α-estradiol (epiestradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. Recombinant human UDP-glucuronosyltransferases (UGTs) UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A10 conjugated one or both diastereomers, mainly at the 3-OH. The activity of UGT1A4 was low and unique because it was directed merely toward the 17-OH of both aglycones. UGT1A10 exhibited particularly high estradiol glucuronidation activity, the rate and affinity of which were significantly higher in the case of β-estradiol than with epiestradiol. UGT1A9 did not catalyze estradiol glucuronidation, but UGT1A9-catalyzed scopoletin glucuronidation was competitively inhibited by β-estradiol. UGT2B4, UGT2B7, and UGT2B17 exclusively conjugated the estradiols at the 17-OH position in a highly stereoselective fashion. UGT2B4 was specific for epiestradiol; UGT2B7 glucuronidated both diastereomers, with high affinity for epiestradiol, whereas UGT2B17 only glucuronidated β-estradiol. UGT2B15 glucuronidated both estradiols at the 3-OH, with a strong preference for epiestradiol. Human UGT2A1 and UGT2A2 glucuronidated both diastereoisomers at both hydroxyl groups. Microsomal studies revealed that human liver mainly yielded epiestradiol 17- O -glucuronide, and human intestine primarily yielded β-estradiol 3- O -glucuronide, whereas rat liver preferentially formed β-estradiol 17- O -glucuronide. Of the three recombinant rat UGTs that were examined in this study, rUGT2B1 was specific for the 17-OH of β-estradiol, rUGT2B2 did not catalyze estradiol glucuronidation, whereas rUGT2B3 exhibited high activity toward the 17-OH in both diastereoisomers. The results show that although many UGTs can catalyze estradiol glucuronidation, there are marked differences in their kinetics, regioselectivity, and stereoselectivity.

Chantal Guillemette - One of the best experts on this subject based on the ideXlab platform.

  • a rare UGT2B7 variant creates a novel n glycosylation site at codon 121 with impaired enzyme activity
    Drug Metabolism and Disposition, 2016
    Co-Authors: Camille Girardbock, Sylvie Desjardins, Lyne Villeneuve, Marieodile Benoitbiancamano, Chantal Guillemette
    Abstract:

    The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism.

  • Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential.
    Drug Metabolism and Disposition, 2015
    Co-Authors: Guillaume Margaillan, John K. Fallon, Patrick Caron, Kathrin Klein, Lyne Villeneuve, Michele Rouleau, Philip C Smith, Ulrich M Zanger, Chantal Guillemette
    Abstract:

    Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30–34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%–184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential.

  • expression of UGT2B7 is driven by two mutually exclusive promoters and alternative splicing in human tissues changes from prenatal life to adulthood and in kidney cancer
    Pharmacogenetics and Genomics, 2013
    Co-Authors: Vincent Menard, Lena Ekstrom, Anders Rane, Eric Levesque, Olivier Eap, Sylvia Chen, Melanie S Joy, Chantal Guillemette
    Abstract:

    OBJECTIVE: UDP-glucuronosyltransferase 2B7 (UGT2B7) plays a major detoxification role in commonly prescribed drugs and endogenous lipophilic molecules. Additional exons and multiple alternative splicing events (ASEs) at the UGT2B7 locus were recently discovered. MATERIALS AND METHODS: Novel and classical ASEs were quantified in 27 human tissues, as well as in fetal and tumoral tissues. The activity of the alternative UGT2B7 promoters was studied in cell lines. RESULTS: UGT2B7 expression is driven by an alternate promoter 1a associated with transcripts containing exon 1b, which is located ∼44 kb upstream of the known promoter 1 associated with transcripts containing exon 1 required for enzyme activity. The exon 1 was expressed most abundantly in the liver and gastrointestinal tract, whereas exon 1b was expressed predominantly in other extrahepatic tissues. Experimental evidence indicated endogenous translation that yields alternative UGT2B7s derived from the use of exon 1b are enzymatically inactive. Alternate 5' ASE predominates in fetal tissues (kidney, lung) and kidney tumor samples compared with normal adult kidney. These changes further correlate with reduced glucuronidation in neoplastic kidneys. This differential expression pattern was further confirmed using four liver and kidney cell lines and was consistent with the differential usage of alternate promoters in hepatic (promoter 1) and kidney cells (1a). CONCLUSION: UGT2B7 is characterized by two mutually exclusive exons 1, both flanked by a unique 5' promoter region. Data also indicated a switch toward functional enzyme upon maturation in the kidney and reversal of this process in neoplastic cells, considerably modifying the glucuronidation potential across human tissues and cells.

  • modulation of the UGT2B7 enzyme activity by c terminally truncated proteins derived from alternative splicing
    Drug Metabolism and Disposition, 2013
    Co-Authors: Vincent Menard, Guillaume Margaillan, Pierre Collin, Chantal Guillemette
    Abstract:

    The enzyme UGT2B7 is one of the most active UDP-glucuronosyltransferases (UGTs) involved in drug metabolism and in maintaining homeostasis of endogenous compounds. We recently reported the existence of 22 UGT2B7 mRNAs, two with a classic 5′ region but alternative 3′ ends namely UGT2B7\_v5 (containing a novel terminal exon 6b) and \_v7 (exon 5 excluded) that encode enzymatically inactive isoforms 2 and 4 (i2 and i4), respectively. The v1 mRNA encoding the UGT2B7 enzyme (renamed isoform 1 or i1) is coexpressed with the splice variants v5 and v7 in human liver, kidney, and small intestine and the hepatic cell lines HepG2 and C3A. The presence of alternate v5 and v7 transcripts in isolated polysomes from these hepatic cells further supports endogenous protein translation. Cellular fractionation of clonal HEK293 cell lines overexpressing UGT2B7 isoforms demonstrates that i1, i2, and i4 proteins colocalize in the microsomal/Golgi fraction, whereas i2 and i4 can also be found in the cytosol; a finding sustained by immunofluorescence experiments using tagged proteins. By modifying splice variant abundance in overexpression in HEK293 and HepG2 cells as well as RNA interference experiments in HepG2 and C3A cells, we observe drug glucuronidation phenotypes compatible with variant-mediated repression of UGT2B7 activity without consequent alteration of the apparent enzyme affinity ( K m). Finally, coimmunoprecipitation experiments support a direct protein–protein interaction of i2 and i4 proteins with the functional UGT2B7 enzyme as a potential causative mechanism. These findings point toward a novel autoregulatory mechanism of the UGT2B7 glucuronidation pathway by naturally occurring alternative i2 and i4 proteins.

  • worldwide variation in human drug metabolism enzyme genes cyp2b6 and UGT2B7 implications for hiv aids treatment
    Pharmacogenomics, 2012
    Co-Authors: Vincent Menard, Rebekah L Benish, Richard J Jurevic, Mark Stoneking, Peter A Zimmerman, Chantal Guillemette, Rajeev K Mehlotra
    Abstract:

    Aim: Hepatic enzymes, CYP2B6 and UGT2B7 play a major role in the metabolism of the widely used antiretroviral drugs efavirenz, nevirapine and zidovudine. In the present study, we provide a view of UGT2B7 haplotype structure, and quantify the genetic diversity and differentiation at both CYP2B6 and UGT2B7 genes on a worldwide scale. Materials & methods: We genotyped one intronic and three promoter SNPs, and together with three nonsynonymous SNPs, inferred UGT2B7 alleles in north American (n = 326), west African (n = 133) and Papua New Guinean (n = 142) populations. We also included genotype data for five CYP2B6 and six UGT2B7 SNPs from an additional 12 worldwide populations (n = 629) analyzed in the 1000 Genomes Project. Results: We observed significant differences in certain SNP and allele frequencies of CYP2B6 and UGT2B7 among worldwide populations. Diversity values were higher for UGT2B7 than for CYP2B6, although there was more diversity between populations for CYP2B6. For both genes, most of the geneti...

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  • inhibition of udp glucuronosyltransferase enzymes by major cannabinoids and their metabolites
    Drug Metabolism and Disposition, 2021
    Co-Authors: Shamema Nasrin, Christy J W Watson, Gang Chen, Keti Bardhi, Gabriela Fort, Philip Lazarus
    Abstract:

    The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the U.S. and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells over-expressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4,1A6 and 2B7, with binding- corrected IC50,u values of 0.12 {plus minus} 0.020 µM, 0.22 {plus minus} 0.045 µM, 0.40 {plus minus} 0.10 µM and 0.82 {plus minus} 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with IC50,u values of 0.45 {plus minus} 0.12 µM and 0.51 {plus minus} 0.063 µM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. Significance Statement Major cannabinoids found in the plasma of cannabis users inhibit several UGT enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all major kidney UGTs and the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinoids and therapeutics that are cleared renally.

  • potential regulation of ugt2b10 and UGT2B7 by mir 485 5p in human liver
    Molecular Pharmacology, 2019
    Co-Authors: Aimee K Sutliff, Jian Shi, Christy J W Watson, Martina S Hunt, Gang Chen, Hao Jie Zhu, Philip Lazarus
    Abstract:

    The UDP-glucuronosyltransferase (UGT) family of enzymes is important in the metabolic elimination of a variety of endogenous compounds such as bile acids, steroids, and fat-soluble vitamins, as well as exogenous compounds including many pharmaceuticals. The UGT2B subfamily is a major family of UGT enzymes expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contributes to interindividual variability in UGT2B expression and is a crucial component in predicting patient drug response. In the present study, a high-resolution liquid chromatography-tandem mass spectrometry method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples (n = 62). Concurrent in silico analysis identified eight candidate miRNAs as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant inverse correlation between UGT2B10 and UGT2B15 and one of these candidate miRNAs, miR-485-5p. A near-significant correlation was also observed between UGT2B7 and miR-485-5p expression. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the UGT2B10 3'-untranslated region (UTR), and significant reduction in luciferase activity was also observed for a luciferase vector containing the UGT2B7 3'-UTR; however, none was observed for the UBT2B15 3'-UTR. UGT2B10 and UGT2B7 activities were probed using nicotine and 3'-azido-3'-deoxythymidine, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells upon overexpression of miR-485-5p mimic. This is the first study demonstrating a regulatory role of miR-485-5p for multiple UGT2B enzymes. SIGNIFICANCE STATEMENT: The purpose of this study was to identify novel epigenetic miRNA regulators of the UGT2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that miRNA 485-5p is a novel regulator of UGT2B7 and UGT2B10, which play an important role in the metabolism of many commonly prescribed medications, carcinogens, and endogenous compounds. This study identified potential miRNA-UGT2B mRNA interactions using a novel proteomic approach, with in vitro experiments undertaken to validate these interactions.

  • glucuronidation of trans 3 hydroxycotinine by ugt2b17 and ugt2b10
    Pharmacogenetics and Genomics, 2012
    Co-Authors: Nino E. Giambrone, Philip Lazarus
    Abstract:

    trans-3′-Hydroxycotinine (3HC) and its glucuronide (3HC-Gluc) are major nicotine metabolites excreted in the urine of smokers and other tobacco users. While several members of the UDP-glucuronosyltransferase (UGT) family of enzymes were previously shown to be active in catalyzing the formation of 3HC-Gluc, a comprehensive screening of all known human UGT1A and 2B enzymes for glucuronidation activity against 3HC was not previously performed. In the present study, 8 UGT1A and 6 UGT2B enzymes were screened for activity against 3HC. UGT2B17 exhibited the highest O-glucuronidation activity, exhibiting a 4-fold lower (p<0.005) KM (8.3 mM) than that observed for UGTs 1A9 (35 mM) or 2B7 (31 mM) and a KM smaller than that observed for human liver microsomes (HLM; 26 mM). The KM for 3HC-O-Gluc formation was 3.1-fold lower (p<0.0005) in HLM from male subjects exhibiting the wild-type genotype UGT2B17 (*1/*1) than that in HLM from subjects homozygous for the UGT2B17 deletion genotype [UGT2B17 (*2/*2)]. Both UGTs 2B10 and 1A4 exhibited 3HC-N-Gluc formation activity, with UGT2B10 exhibiting a 4-fold lower (p<0.05) KM (13 mM) than that observed for UGT1A4 (57 mM) and which was similar to the KM observed in HLM (14 mM). There was a 91% (p<0.0001) and 39% (p<0.001) decrease in 3HC-N-Gluc formation activities in HLM from subjects with the UGT2B10 (*2/*2) and UGT2B10 (*1/*2) genotypes, respectively, compared to that of HLM from subjects with the wild-type UGT2B10 (*1/*1) genotype. These results suggest that UGT2B17 and UGT2B10 play key roles in the glucuronidation of 3HC in the human liver and that functional polymorphisms in UGT2B17 and UGT2B10 are associated with significantly reduced glucuronidation activities against 3HC.

  • genotype phenotype correlation between the polymorphic ugt2b17 gene deletion and nnal glucuronidation activities in human liver microsomes
    Pharmacogenetics and Genomics, 2005
    Co-Authors: Philip Lazarus, Joshua E. Muscat, Yan Zheng, Aaron E Runkle, Doris Wiener
    Abstract:

    The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1 -(3-pyridyl)-1-butanol (NNAL), is an important mechanism for NNK detoxification. In cigarette smokers and tobacco chewers, there is a wide variation in the urinary levels of the ratio of NNAL to NNAL glucuronide (NNAL-Gluc). To determine whether genetic variation plays a potential role in this inter-individual variability, NNAL-glucuronidating activities were analysed in a series of human liver microsomal specimens and compared with UGT2B17 deletion genotypes in the same subjects. Assays performed in vitro demonstrated that over-expressed UGT2B17 exhibits high O-glucuronidating activity against NNAL. When stratifying subjects by UGT2B17 genotype, a significant or near-significant decrease in NNAL-O-Gluc formation was observed in liver microsomes from individuals who were either heterozygous [(+/0), P=0.07] or homozygous [(0/0), P=0.016] for the UGT2B17 deletion compared to liver microsomes from individuals with intact UGT2B17 alleles [(+/+)]. There was a significant (P<0.01) association between the level of liver microsomal NNAL-O-glucuronide formation and increasing numbers of the UGT2B17 null alleles in the liver microsomal specimens examined in this study, and a significant decrease in NNAL-O-Gluc formation was observed when comparing liver microsomes from individuals who had at least one UGT2B17 allele deleted [(+/0)+(0/ 0)] versus microsomes from UGT2B17 (+/+) subjects (P=0.004). When stratifying by the median value of NNAL-O-Gluc formation activity, a significantly (P=0.015) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B17 null genotype compared to subjects with liver microsomes exhibiting high NNAL-O-Gluc formation activity. When stratifying by UGT2B7/UGT2B17 haplotypes, the association between the level of liver microsomal NNAL-O-glucuronide formation and increasing numbers of the UGT2B17 null allele was at the level of statistical significance for subjects with the UGT2B7 (*1/*2) (P=0.05) or UGT2B7 (*2/*2) (P<0.02) genotypes. These data suggest that the UGT2B17 deletion polymorphism is associated with a reduced rate of NNAL detoxification in vivo and may increase individual susceptibility to tobacco-related cancers.

  • O-Glucuronidation of the Lung Carcinogen 4-(methylnitrosamino)-1- (3-Pyridyl)-1-Butanol (nnal) by Human Udp-Glucuronosyltransferases 2b7 and 1a9
    Drug Metabolism and Disposition, 2000
    Co-Authors: Sharon E Murphy, Zhong Zheng, Philip Lazarus
    Abstract:

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UGT-mediated O-glucuronidation of NNAL is an important detoxification pathway for these carcinogens. To better characterize this pathway in humans, we screened a series of UGT-overexpressing cell lines and baculosome preparations for their ability to O-glucuronidate NNAL and examined multiple human liver and lung specimens for NNAL-glucuronidating activity and their levels of expression of NNAL-glucuronidating UGTs. Human liver microsomal fractions exhibited significant levels of NNAL-glucuronidating activity, with the NNAL-Gluc II diastereomer formed at a rate 3.4 times that observed for NNAL-Gluc I. As with liver microsomal fractions, NNAL-Gluc II was the major diastereomer formed by homogenates from UGT2B7-overexpressing HK293 cells or UGT2B7-overexpressing baculosomes; the major diastereomer formed by homogenates from UGT1A9-overexpressing V79 cells was NNAL-Gluc I. No significant O-glucuronidating activity of NNAL was detected in UGT1A1-, UGT1A4-, UGT1A6-, UGT2B4-, or UGT2B15-overexpressing HK293 or V79 cell homogenates, or in UGT1A1-, UGT1A3-, UGT1A7-, or UGT1A10-overexpressing baculosomes. Significant levels of UGT2B7 mRNA were detected by reverse transcriptase-polymerase chain reaction in human liver and at low levels in human lung specimens. UGT1A9 mRNA was detected in liver but not in lung. These results suggest that although both UGT2B7 and UGT1A9 play an important role in the overall glucuronidation of NNAL in humans, UGT2B7 potentially plays an important role in the detoxification of NNAL in the lung.