Interacting selectively and non-covalently with a drug, any naturally occurring or synthetic substance, other than a nutrient, that, when administered or applied to an organism, affects the structure or functioning of the organism; in particular, any such substance used in the diagnosis, prevention, or treatment of disease.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37 degrees C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5- O-glucuronide) and metabolite 2 (prunetin-4'- O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8, and UGT1A9 were mainly responsible for the formation of metabolite 1, whereas UGT1A1, UGT1A8, and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform-specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that the thermostability of the microsomes was isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat-stable (37 degrees C) than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermostable than human UGT1A10 and UGT1A8. The organ-specific thermostability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differs significantly from rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ-, and UGT-isoform-dependent, all of which may be impacted by the thermostability of specific UGT isoforms involved in the metabolism.
13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.
Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
Finding rapid, reversible down-regulation of human UDP-glucuronosyltransferases (UGTs) in LS180 cells following curcumin treatment led to the discovery that UGTs require phosphorylation. UGTs, distributed primarily in liver, kidney, and gastrointestinal tract, inactivate aromatic-like metabolites and a vast number of dietary and environmental chemicals, which reduces the risk of toxicities, mutagenesis, and carcinogenesis. Our aim here is to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGT cDNA-transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [(33)P]orthophosphate in UGTs and protein kinase Cepsilon (PKCepsilon), following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C, suggested UGT phosphorylation is supported by active PKC(s). Immunofluorescent and co-immunoprecipitation studies with UGT-transfected cells showed co-localization of UGT1A7His and PKCepsilon and of UGT1A10His and PKCalpha or PKCdelta. Inhibition of UGT activity by PKCepsilon-specific antagonist peptide or by PKCepsilon-targeted destruction with PKCepsilon-specific small interference RNA and activation of curcumin-down-regulated UGTs with typical PKC agonists verified a central PKC role in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCepsilon confirms it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide-activated-UGTs demonstrated that reactive oxygen species-related oxidants act as second messengers in maintaining constitutive PKC-dependent signaling evidently sustaining UGT phosphorylation and activity. Because cells use signal transduction collectively to detect and respond appropriately to environmental changes, this report, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggests regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs to function efficiently.
13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
The chemical reactions and pathways involving a drug, a substance used in the diagnosis, treatment or prevention of a disease; as used here antibiotic substances (see antibiotic metabolism) are considered to be drugs, even if not used in medical or veterinary practice.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
The elimination by an organism of the waste products that arise as a result of metabolic activity. These products include water, carbon dioxide (CO2), and nitrogenous compounds.
Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.
The chemical reactions and pathways involving flavones, a class of pigmented plant compounds based on 2-phenyl-4H-1-benzopyran-4-one (2-phenylchromone).
We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37 degrees C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5- O-glucuronide) and metabolite 2 (prunetin-4'- O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8, and UGT1A9 were mainly responsible for the formation of metabolite 1, whereas UGT1A1, UGT1A8, and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform-specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that the thermostability of the microsomes was isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat-stable (37 degrees C) than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermostable than human UGT1A10 and UGT1A8. The organ-specific thermostability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differs significantly from rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ-, and UGT-isoform-dependent, all of which may be impacted by the thermostability of specific UGT isoforms involved in the metabolism.
The modification of a flavonoid by the conjugation of glucuronic acid. The resultant flavonoid glucuronosides are often much more water-soluble than the precursor.
Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.
The modification of a xenobiotic substance by the conjugation of glucuronic acid. The resultant glucuronosides are often much more water-soluble than the xenobiotic precursor, enabling efficient excretion.
Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.
Enzymes that catalyze the transfer of glycosyl (sugar) residues to an acceptor, both during degradation (cosubstrates= water or inorganic phosphate) and during biosynthesis of polysaccharides, glycoproteins and glycolipids. In biosynthetic glycosyl transfers, the common activated monomeric sugar intermediate is a nucleoside diphosphate sugar.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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