RESUMEN
This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity.
Asunto(s)
Benzofenonas , Bromobencenos , Enfermedad Hepática Inducida por Sustancias y Drogas , Uridina Difosfato Ácido Glucurónico , Humanos , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato Ácido Glucurónico/farmacología , Microsomas Hepáticos/metabolismo , Glucuronosiltransferasa/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Lactamas/metabolismo , Lactamas/farmacología , Glucurónidos/metabolismoRESUMEN
UXS1-mediated clearance of the sugar nucleotide UDPGA is a specific vulnerability of cancer cells.
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Neoplasias , Uridina Difosfato Ácido Glucurónico , Humanos , Uridina Difosfato Ácido Glucurónico/metabolismo , Nucleótidos , Neoplasias/genéticaRESUMEN
Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.
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Neoplasias , Uridina Difosfato Ácido Glucurónico , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Uridina Difosfato Ácido Glucurónico/biosíntesis , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato Xilosa/biosíntesis , Uridina Difosfato Xilosa/metabolismo , Adenocarcinoma del Pulmón , Neoplasias PulmonaresRESUMEN
Uridine diphosphate glucuronic acid (UDPGA) is an essential substrate in the glucuronidation of exogenous and endogenous lipophilic compounds via the liver glucuronic acid pathway, and its synthesis depends on glucose and energy in the body. Bisphenol S (BPS), as a lipophilic environmental pollutant, has been widely utilized in the manufacturing of daily necessities. The biological effect of BPS in interference with liver energy metabolism might affect UDPGA synthesis and the excretion of lipophilic compounds, but this was not clearly revealed. Here, female zebrafish that were exposed to BPS for 35 days exhibited a significant decrease in UDPGA in the liver with significant accumulation of exogenous BPS and endogenous bilirubin in the body. One vital reason may be that the exposure to BPS for 35 days promoted the lipid formation through PPARg signaling and reduced energy levels in the liver, resulting in the decreased raw materials for UDPGA production in glucuronic acid pathway. Meanwhile, transcriptome analysis showed that BPS inhibited the mRNA expression levels of genes related to the glucuronic acid pathway. The accumulation of endogenous and exogenous lipophilic compounds can trigger a variety of toxicological effect. Thus, weakened liver detoxification might be the primary cause of the toxicological effects of lipophilic pollutants.
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Uridina Difosfato Ácido Glucurónico , Pez Cebra , Animales , Femenino , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato Ácido Glucurónico/farmacología , Ácido Glucurónico/farmacología , Pez Cebra/metabolismo , Hígado/metabolismoRESUMEN
BACKGROUND: Saccharolactone is used as a ß-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors. METHODS: Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM. RESULTS: The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC50 values of less than 4 mM. CONCLUSION: The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations.
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Citocromo P-450 CYP1A2 , Sistema Enzimático del Citocromo P-450 , Humanos , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromo P-450 CYP3A/metabolismo , Glucurónidos/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Quinidina/farmacología , Xenobióticos/farmacología , NADP/metabolismo , Fenacetina/metabolismo , Microsomas Hepáticos , Isoformas de Proteínas/metabolismo , Peptaiboles/metabolismoRESUMEN
Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of Salmonella enterica serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD+ binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.
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NAD , Polimixinas , Humanos , Polimixinas/farmacología , Polimixinas/química , Ligandos , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismo , OxidorreductasasRESUMEN
Uridine diphosphate glucose (UDP-Glc) is able to accelerate the decay of snail family transcriptional repressor 1 (SNAI1) mRNA by inhibiting Hu antigen R (HuR, an RNA-binding protein), thereby preventing cancer invasiveness and drug resistance. Nevertheless, the phosphorylation of tyrosine 473 (Y473) of UDP-glucose dehydrogenase (UGDH is capable of converting UDP-Glc to uridine diphosphate glucuronic acid (UDP-GlcUA)) weakens the inhibition of UDP-Glc to HuR, thus initiating the epithelial-mesenchymal transformation of tumor cells and promoting tumor cell migration and metastasis. To address the mechanism, we performed molecular dynamics simulations combined with molecular mechanics generalized Born surface area (MM/GBSA) analysis on wild-type and Y473 phosphorylated UGDH and HuR, UDP-Glc, UDP-GlcUA complexes. We demonstrated that Y473 phosphorylation was able to enhance the binding between UGDH and the HuR/UDP-Glc complex. Compared with HuR, UGDH has a stronger binding ability with UDP-Glc; therefore, UDP-Glc was inclined to bind to UGDH and then was catalyzed to UDP-GlcUA by UGDH, which relieved the inhibition of UDP-Glc to HuR. In addition, the binding ability of HuR for UDP-GlcUA was lower than its affinity for UDP-Glc, significantly reducing the inhibition of HuR. Hence, HuR bound to SNAI1 mRNA more easily to increase the stability of mRNA. Our results revealed the micromolecular mechanism of Y473 phosphorylation of UGDH regulating the interaction between UGDH and HuR as well as relieving the inhibition of UDP-Glc on HuR, which contributed to understanding the role of UGDH and HuR in tumor metastasis and developing small molecule drugs targeting the interaction between UGDH and HuR.
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Uridina Difosfato Glucosa , Uridina Difosfato Ácido Glucurónico , Uridina Difosfato Glucosa/metabolismo , Fosforilación , Uridina Difosfato Ácido Glucurónico/metabolismo , Glucosa , ARN MensajeroRESUMEN
UDP-glucuronosyltransferase 2B15 (UGT2B15) is a crucial phase II drug-metabolizing enzyme, which glucuronidates various compounds, including clinical drugs and hormones. Mutants might affect glucuronidation, leading to a disruption of drug metabolism in vivo and decrease of therapeutic effect. Here, we mainly analyzed two representative mutants, H401P and L446S, on UGT2B15 activity using glucuronidation assays, molecular dynamic (MD) simulation and X-ray diffraction methods. The enzyme activity of L446S obviously increased six-fold than the wild type, although the enzyme activities of P191L, T374A, and H401P were lost apparently. Furthermore, we used MD simulations to calculate the energy change in the catalytic process of H401P and L446S, and the results indicated the free binding energies of H401P mutant to oxazepam and UDPGA were -30.98 ± 1.00 kcal/mol and -36.42 ± 1.04 kcal/mol, respectively, increased obviously compared to wild type, suggesting the mutation on position 401 had a crucial effect on the catalysis. Moreover, the three-dimensional structure of UGT2B15 C-terminal domain L446S was determined through protein crystallography and X-ray diffraction technology and the results suggested that one more hydrogen bonding between S446 and K410 was formed in the S446 crystal structure, compared to the wild type. Isothermal titration calorimetry assay further revealed the Kd values of C-terminal domain of UGT2B15 harbored L446S towards the cofactor UDPGA was similar to the value of wild type. Above all, our results pointed out that H401P and L446S affected the enzyme activity by different mechanism. Our work provided a helpful mechanism for variance explained in the UGTs catalyzation process.
Asunto(s)
Glucuronosiltransferasa , Uridina Difosfato Ácido Glucurónico , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/química , Glucuronosiltransferasa/metabolismo , Difracción de Rayos X , CinéticaRESUMEN
UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.
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Racemasas y Epimerasas , Uridina Difosfato Ácido Glucurónico , Uridina Difosfato Ácido Glucurónico/metabolismo , Descarboxilación , Rotación , Ácido Glucurónico , Racemasas y Epimerasas/metabolismo , Cetosas , NAD/químicaRESUMEN
Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.
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Tortugas , Uridina Difosfato Ácido Glucurónico , Animales , Uridina Difosfato Ácido Glucurónico/metabolismo , Tortugas/metabolismo , Magnesio , Uridina Difosfato , Glucuronosiltransferasa/metabolismo , Microsomas Hepáticos/metabolismo , Alameticina/farmacologíaRESUMEN
Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.
Asunto(s)
Glucuronosiltransferasa/química , Glucuronosiltransferasa/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Vías de Eliminación de Fármacos , Interacciones Farmacológicas , Humanos , Cinética , Fenotipo , Especificidad por SustratoRESUMEN
The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.
Asunto(s)
Cryptococcus/efectos de los fármacos , Farmacorresistencia Fúngica , Flucitosina/farmacología , Cromosomas Fúngicos/genética , Células Clonales , Cryptococcus/genética , Cryptococcus/crecimiento & desarrollo , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Duplicación de Gen , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Supresores , Variación Genética , Genoma Fúngico , Espacio Intracelular/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación/genética , Reproducibilidad de los Resultados , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O-GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.
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Vías Biosintéticas , Metabolismo Energético , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/metabolismo , Animales , Humanos , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.
Asunto(s)
Biocatálisis , Glucosiltransferasas/metabolismo , Ácido Glucurónico/metabolismo , Saponinas/biosíntesis , Vías Biosintéticas , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicosilación , Glycyrrhiza uralensis/genética , Ácido Glicirrínico/metabolismo , Funciones de Verosimilitud , Lotus/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Saponinas/química , Glycine max/genética , Especificidad por Sustrato , Triterpenos/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.
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Arginina/genética , Glucuronosiltransferasa/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Sitios de Unión/genética , Coenzimas/metabolismo , Cristalografía por Rayos X , Glucosiltransferasas/genética , Glucosiltransferasas/ultraestructura , Glucurónidos/metabolismo , Glucuronosiltransferasa/genética , Glicósidos/metabolismo , Células HEK293 , Humanos , Himecromona/metabolismo , Medicago truncatula , Simulación de Dinámica Molecular , Morfina/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestructura , Homología de Secuencia de Aminoácido , Especificidad por Sustrato/genética , Zidovudina/metabolismoRESUMEN
Dysregulated metabolism is a hallmark of cancer that supports tumor growth and metastasis. One understudied aspect of cancer metabolism is altered nucleotide sugar biosynthesis, which drives aberrant cell surface glycosylation known to support various aspects of cancer cell behavior including migration and signaling. We examined clinical association of nucleotide sugar pathway gene expression and found that UGDH, encoding UDP-glucose 6-dehydrogenase which catalyzes production of UDP-glucuronate, is associated with worse breast cancer patient survival. Knocking out the mouse homolog Ugdh in highly-metastatic 6DT1 breast cancer cells impaired migration ability without affecting in vitro proliferation. Further, Ugdh-KO resulted in significantly decreased metastatic capacity in vivo when the cells were orthotopically injected in syngeneic mice. Our experiments show that UDP-glucuronate biosynthesis is critical for metastasis in a mouse model of breast cancer.
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Neoplasias de la Mama/patología , Neoplasias Pulmonares/secundario , Uridina Difosfato Glucosa Deshidrogenasa/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Uridina Difosfato Ácido Glucurónico/biosíntesisRESUMEN
The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER.
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Retículo Endoplásmico/metabolismo , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Proteínas de Transporte de Monosacáridos/deficiencia , Uridina Difosfato Ácido Glucurónico/metabolismo , Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen/métodos , Glucurónidos/genética , Glucuronosiltransferasa/genética , Células HEK293 , Hepatocitos/metabolismo , Humanos , Proteínas de Transporte de Monosacáridos/genética , Uridina Difosfato Ácido Glucurónico/genéticaRESUMEN
Rosmarinic acid (RA) is a phenolic acid originally isolated from the herb medicine Rosmarinus officinalis. The purpose of this study was to identify the metabolites of RA. RA was incubated with human liver microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate tetrasodium salt and/or uridine diphosphate glucuronic acid using glutathione (GSH) as a trapping agent. After 60-min incubation, the samples were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Under the current conditions, 14 metabolites were detected and identified. Our data revealed that RA was metabolized through the following pathways: the first pathway is the oxidation of catechol to form ortho-quinone intermediates, which react with GSH to form mono-GSH adducts (M1, M2, and M3) and bis-GSH adducts (M4 and M5); the second pathway is conjugation with glucuronide to yield acylglucuronide (M7), which further reacts with GSH to form RA-S-acyl-GSH adduct (M9); the third pathway is hydroxylation to form M10, M11, and M12, which further react with GSH to form mono-GSH adducts (M13 and M14); the fourth pathway is conjugation with GSH through Michael addition (M6); the fifth pathway is conjugation with glucuronidation, forming M8, which is the major metabolic pathway of RA.
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Cromatografía Liquida/métodos , Cinamatos/metabolismo , Depsidos/metabolismo , Microsomas Hepáticos/metabolismo , Espectrometría de Masas en Tándem/métodos , Glutatión , Humanos , Modelos Moleculares , NADP , Espectrometría de Masa por Ionización de Electrospray , Uridina Difosfato Ácido Glucurónico , Ácido RosmarínicoRESUMEN
A highly efficient di-C-glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra. GgCGT catalyzes a two-step di-C-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-C-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-C-glycosylation. GgCGT is the first di-C-glycosyltransferase with a crystal structure, and the first C-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C-glycosides with medicinal potential.
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Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Glycyrrhiza/enzimología , Clonación Molecular , Cristalografía por Rayos X , Glicosilación , Glicosiltransferasas/genética , Glycyrrhiza/genética , Ligandos , Modelos Moleculares , Floretina/química , Floretina/metabolismo , Especificidad por Sustrato , Transcriptoma , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato Xilosa/química , Uridina Difosfato Xilosa/metabolismoRESUMEN
Liver X receptors (LXRs), LXRα and LXRß, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 (hUGT1) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrß -/- , and hUGT1/Lxrαß -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXRα, with the induction pattern paralleling induction of LXRα-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαß -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXRα, showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXRα is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXRα, and not LXRß, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXRα is critical for the developmental expression of UGT1A1.