Personne : Rouleau, Mélanie
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Rouleau
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Mélanie
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CHU de Québec - Université Laval
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ncf11896158
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- PublicationAccès libreÉtude fonctionnelle des variants d'épissage des UGT1A humains(2014) Rouleau, Mélanie; Guillemette, ChantalLa réaction de glucuronidation prise en charge par les enzymes UDP-glucuronosyltransférases est une voie majeure du système de détoxification cellulaire qui influence la biodisponibilité de molécules endogènes et exogènes. Notre laboratoire a récemment découvert l’existence de nouvelles protéines UGT nommées i2 dérivées de l’épissage alternatif du gène UGT1A. Ces protéines sont dépourvues d’activité transférase. Nous avons démontré par immunohistochimie que les enzymes i1 et les protéines alternatives i2 sont coexprimées dans plusieurs tissus du tractus gastro-intestinal, ainsi que dans les mêmes types cellulaires de ces tissus. Les i2 sont localisées dans la membrane du réticulum endoplasmique (RE) avec les i1, mais sont également présentes dans le cytosol. Étant donné la proximité physique des i1 et i2 au RE, nous avons généré des modèles cellulaires surexprimant différentes combinaisons d’enzymes i1 et de protéines i2. Nos données démontrent que la coexpression de ces deux types de protéines diminue l’activité de glucuronidation de 20 à 80 % dépendamment du substrat et de l’enzyme testés. Par co-immunoprécipitation, nous avons démontré que cette répression survient via l’interaction physique des i1 avec les i2. À l’inverse, l’augmentation de l’activité de glucuronidation suite à la répression des formes i2 endogènes a permis de confirmer ce rôle de modulateur négatif des i2. Il semble aussi que les i2 soient en mesure de moduler significativement l’activité UGT même lorsque leur niveau d’expression est inférieur à celui des i1, tel que retrouvé dans plusieurs tissus humains. Nos données supportent également l’influence de ces protéines alternatives sur la réponse pharmacologique. En effet, la répression de l’expression des i2 endogènes dans une lignée de cancer de côlon entraîne un avantage de survie sous traitement chimiothérapeutique. Enfin, l’identification de l’interactome tissulaire des isoformes des UGT1A démontre qu’elles ont le potentiel d’interagir avec des enzymes impliquées dans le métabolisme énergétique et la migration cellulaire. Nos données supportent que les i2 ont même la capacité de modifier le potentiel migratoire de cellules cancéreuses. Nous avons également démontré que les i2 ont la capacité de moduler le stress oxydatif cellulaire, entre autres via l’interaction avec des protéines antioxydantes, telle la catalase. En conclusion, nos résultats démontrent que les protéines alternatives i2 auraient le potentiel de moduler le système de défense cellulaire à plusieurs niveaux, en plus d’influencer la réponse aux médicaments.
- 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 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 libreCrosstalk between alternatively spliced UGT1A isoforms and colon cancer cell metabolism(American Society for Pharmacology and Experimental Therapeutics, 2017-01-03) Rouleau, Michèle; Picard, Frédéric; Têtu, Bernard; Roberge, Joannie; Audet-Delage, Yannick; Guillemette, Chantal; Rouleau, Mélanie; Miard, StéphanieAlternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function into a complex protein network connecting other metabolic pathways with influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues, namely the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by co-immunoprecipitation in the HT115 colon cancer cells and was supported by a partial co-localization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect with higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites including many intermediates derived from the glycolysis and TCA cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2s mRNA in colon tumors compared to normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity.
- PublicationAccès libreEndogenous protein interactome of human UDP-glucuronosyltransferases exposed by untargeted proteomics(Frontiers Research Foundation, 2017-02-03) Rouleau, Michèle; Desjardins, Sylvie; Audet-Delage, Yannick; Guillemette, Chantal; Rouleau, Mélanie; Girard-Bock, CamilleThe conjugative metabolism mediated by UDP-glucuronosyltransferase enzymes (UGTs) significantly influences the bioavailability and biological responses of endogenous molecule substrates and xenobiotics including drugs. UGTs participate in the regulation of cellular homeostasis by limiting stress induced by toxic molecules, and by controlling hormonal signaling networks. Glucuronidation is highly regulated at genomic, transcriptional, post-transcriptional and post-translational levels. However, the UGT protein interaction network, which is likely to influence glucuronidation, has received little attention. We investigated the endogenous protein interactome of human UGT1A enzymes in main drug metabolizing non-malignant tissues, where UGT expression is most prevalent, using an unbiased proteomics approach. Mass spectrometry analysis of affinity-purified UGT1A enzymes and associated protein complexes in liver, kidney and intestine tissues revealed an intricate interactome linking UGT1A enzymes to multiple metabolic pathways. Several proteins of pharmacological importance such as transferases (including UGT2 enzymes), transporters and dehydrogenases were identified, upholding a potential coordinated cellular response to small lipophilic molecules and drugs. Furthermore, a significant cluster of functionally related enzymes involved in fatty acid ß-oxidation, as well as in the glycolysis and glycogenolysis pathways were enriched in UGT1A enzymes complexes. Several partnerships were confirmed by co-immunoprecipitations and co-localization by confocal microscopy. An enhanced accumulation of lipid droplets in a kidney cell model overexpressing the UGT1A9 enzyme supported the presence of a functional interplay. Our work provides unprecedented evidence for a functional interaction between glucuronidation and bioenergetic metabolism.