Personne : Guillemette, Chantal
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Université Laval. Faculté de pharmacie
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- PublicationAccès libreUnravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing(Nature, 2015-04-14) Lévesque, Éric; Margaillan, Guillaume; Rouleau, Michèle; Gilbert, Isabelle; Droit, Arnaud; Tourancheau, Alan; Guillemette, Chantal; Villeneuve, LyneA comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.
- PublicationAccès libreThe relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro(American Society for Pharmacology and Experimental Therapeutics, etc., 2013-04-01) Roberge, Joannie; Guillemette, Chantal; Falardeau, Sarah-Ann; Rouleau, Mélanie; Villeneuve, LyneAlternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38–263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 = i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 = i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.
- PublicationAccès librePharmacogenomics of human uridine diphospho-glucuronosyltransferases (UGTs) and clinical implications(Wiley, 2014-06-12) Lévesque, Éric; Rouleau, Michèle; Guillemette, ChantalGlucuronidation, mediated by UDP-glucuronosyltransferase enzymes (UGTs), is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanism responsible for the inactivation of therapeutic drugs, other xenobiotics and numerous endogenous molecules. Individual variability in UGT enzymatic pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes are underlying this variability and are discussed in the context of drug metabolism and diseases such as cancer.
- PublicationAccès libreDual roles for splice variants of the glucuronidation pathway as regulators of cellular metabolism(American Society for Pharmacology and Experimental Therapeutics, etc., 2014-01-01) Bellemare, Judith.; Roberge, Joannie; Guillemette, Chantal; Rouleau, MélanieTranscripts of the UGT1A gene, encoding half of human UDP-glucuronosyltransferase (UGT) enzymes, undergo alternative splicing, resulting in active enzymes named isoforms 1 (i1s) and novel truncated isoforms 2 (i2s). Here, we investigated the effects of depleting endogenous i2 on drug response and attempted to unveil any additional biologic role(s) for the truncated novel UGT proteins. We used an integrated systems biology approach that combines RNA interference with unbiased global genomic and proteomic screens, and used HT115 colorectal cancer cells as a model. Consistent with previous evidence suggesting that i2s negatively regulate i1s through protein-protein interactions, i2-depleted cells were less sensitive to drug-induced cell death (IC50 of 0.45 ± 0.05 µM versus 0.22 ± 0.03 µM; P = 0.006), demonstrating that modulation of i2 levels meaningfully impacts drug bioavailability and cellular response. We also observed reduced production of reactive oxygen species by 30% (P < 0.05), and an enhanced expression (>1.2-fold; P < 0.05) of several proteins, such as hemoglobin α genes and superoxide dismutase 1, that have network functions associated with antioxidant properties. Interaction proteomics analysis of endogenous proteins from the cellular model, mainly in human intestine but also in kidney tissues, further uncovered interactions between i2s (but not i1s) and the antioxidant enzymes catalase and peroxiredoxin 1, which may influence antioxidant potential through sequestration of these novel partners. Our findings demonstrate for the first time dual roles for i2s in the cellular defense system as endogenous regulators of drug response as well as in oxidative stress.
- PublicationAccès libreImmunohistochemical expression of conjugating UGT1A-derived splice proteins in normal and tumoral drug-metabolising tissues in humans(Wiley, 2010-10-29) Bellemare, Judith.; Pelletier, Georges; Popa, Ion; Têtu, Bernard; Harvey, Mario.; Guillemette, Chantal; Rouleau, MélanieGlucuronidation by UDP-glucuronyltransferase (UGT) enzymes is the prevailing conjugative pathway for the metabolism of both xenobiotics and endogenous compounds. Alterations in this pathway, such as those generated by common genetic polymorphisms, have been shown to significantly impact on the health of individuals, influencing cancer susceptibility, responsiveness to drugs and drug-induced toxicity. Alternative usage of terminal exons leads to UGT1A-derived splice variants, namely the classical and enzymatically active isoforms 1 (i1) and the novel enzymatically inactive isoforms 2 (i2). In vitro functional data from heterologous expression and RNA interference experiments indicate that these i2 isoforms act as negative modulators of glucuronidation, likely by forming inactive complexes with active isoform 1. We used specific antibodies against either active i1 or inactive i2 proteins to examine their distribution in major drug-metabolizing tissues. Data revealed that UGT1A_i1 and inactive UGT1A_i2 are co-produced in the same tissue structures, including liver, kidney, stomach, intestine and colon. Examination of the cellular distribution and semi-quantitative level of expression of UGT1As revealed heterogeneous expression of i1 and i2 proteins, with increased expression of i2 in liver tumours and decreased levels of i1 and i2 in colon cancer specimens, compared to normal tissues. These differences in expression may be relevant to human colon and liver cancer tumorigenesis. Our data clearly demonstrate the similar immunolocalization of active and inactive UGT1A isoforms in most UGT1A-expressing cell types of major tissues involved in drug metabolism. These expression patterns are consistent with a dominant-negative function for the i2 encoded by the UGT1A gene.
- PublicationAccès libreA pharmacogenetics study of the human glucuronosyltransferase UGT1A4(Lippincott Williams & Wilkins, 2009-12-01) Benoît-Biancamano, Marie-Odile; Leblanc, Marie-Hélène; Bernard, Olivier; Court, Michael H.; Guillemette, Chantal; Caron, Patrick; Adam, Jean-PhilippeUGT1A4 is primarily expressed in the liver and exhibits catalytic activities for various drugs. Amongst the few UGT1A4 polymorphisms evaluated, studies support the alteration of UGT1A4-mediated glucuronidation by a few variations including the Pro24Thr and Leu48Val variants (referred to as UGT1A4*2 and *3). We therefore investigated genetic mechanisms that might contribute to interindividual variation in UGT1A4 expression and activity. The UGT1A4 gene was sequenced from -4963 bp relative to the ATG to 2000 bp after the first exon in 184 unrelated Caucasians and African-Americans. We identified a large number of genetic variations, including 13 intronic, 39 promoter, as well as 14 exonic polymorphisms, with 10 that lead to aminoacid changes. Of the nucleotide variations found in the -5kb promoter region, 5 are located in the proximal region (first 500 bp), and positioned in putative HNF-1 and OCT-1 binding sites. Four of these variants, placed at -163, -219, -419 and -463, are in complete linkage disequilibrium with the Leu48Val coding region variant and with several variants in the upstream region of the promoter. Transient transfections of reference and variant promoter constructs (from position -500 to +1) in different cell lines with or without co-expression of HNF-1 and/or OCT-1, demonstrated limited effect of these variations. However, several coding variants significantly modified the enzyme kinetics for tamoxifen and Z-4-hydroxytamoxifen (Val48, Asp50, Gln56, Phe176, Asn250, Leu276). Our results reveal that, despite a large number of polymorphisms located in the promoter region, the exonic variants are those expected to have a potential in vivo effect.
- PublicationAccès libreUGT genomic diversity : beyond gene duplication(Informa Healthcare, 2009-10-26) Lévesque, Éric; Bellemare, Judith.; Harvey, Mario.; Ménard, Vincent; Guillemette, ChantalThe human uridine diphospho (UDP)-glucuronosyltransferase (UGT) superfamily comprises enzymes responsible for a major biotransformation phase II pathway: the glucuronidation process. The UGT enzymes are located in the endoplasmic reticulum of almost all tissues, where they catalyze the inactivation of several endogenous and exogenous molecules, including bilirubin, sex steroids, numerous prescribed drugs, and environmental toxins. This metabolic pathway is particularly variable. The influence of inheritable polymorphisms in human UGT-encoding genes has been extensively documented and was shown to be responsible for a fraction of the observed phenotypic variability. Other key genomic processes are likely underlying this diversity; these include copy-number variations, epigenetic factors, and newly discovered splicing mechanisms. This review will discuss novel molecular aspects that may be determinant to UGT phenotypes.
- PublicationAccès libreOverexpression of uridine diphospho glucuronosyltransferase 2B17 in high risk chronic lymphocytic leukemia(American Society of Hematology, 2012-11-20) Gruber, Michaela; Lévesque, Éric; Bellemare, Judith.; Hörmann, Gregor; Guillemette, Chantal; Gleiss, Andreas; Porpaczy, Edith; Bilban, Martin; Le, Trang; Zehetmayer, Sabine; Mannhalter, Christine; Gaiger, Alexander; Shehata, Medhat; Fleiss, Karin; Skrabs, Cathrin; Vanura, Katrina; Jäger, UlrichUridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications.
- PublicationAccès libreA rare UGT2B7 variant creates a novel N-glycosylation site at codon 121 with impaired enzyme activity(American Society for Pharmacology and Experimental Therapeutics, 2016-09-12) Benoît-Biancamano, Marie-Odile; Desjardins, Sylvie; Guillemette, Chantal; Girard-Bock, Camille; Villeneuve, LyneUDP-glucuronosyltransferase (UGT) superfamily are glycoproteins resident of the endoplasmic reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and are only examining a small fraction of the genetic diversity. However, rare variants (frequency <1%) might have significant effect as they are predicted to greatly outnumber common variants in the human genome. Here, 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 (Asn) instead of an aspartic acid (Asp) (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 from HEK293 cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and 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 due to changes in PTM such as N-linked glycosylation with consequences on drug metabolism.
- PublicationRestreintFactors affecting interindividual variability of hepatic UGT2B17 protein expression examined using a novel specific monoclonal antibody(American Society for Pharmacology and Experimental Therapeutics, etc., 2019-02-28) Lévesque, Éric; Rouleau, Michèle; Labriet, Adrien; Emond, Jean-Philippe; Hovington, Hélène; Périgny, Martine; Desjardins, Sylvie; Têtu, Bernard; Lacombe, Louis; Brisson, Hervé.; Guillemette, Chantal; Caron, Patrick; Fallon, John K.; Villeneuve, Lyne; Klein, Kathrin; Simonyan, David; Smith, Philip; Zanger, Ulrich M.The accurate quantification of the metabolic enzyme UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers reveals a strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93) and three major expression patterns (absent, low or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, which is located in the binding site for the transcription factor Forkhead Box A1 (FOXA1) of the UGT2B17 promoter. The highest expression was observed for individuals carrying at least one rs59678213 A allele. A multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213 and reached 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize UGT2B17 level in various disease states and establish more precisely the UGT2B17 enzyme contribution to drug and hormone metabolism