Uridine 5'-diphospho-glucronosyltrasferase: Its role in pharmacogenomics and human disease.
ABSTRACT: Biotransformation is an enzyme-catalyzed process in which the body converts endogenous compounds, xenobiotics and toxic substances into harmless or easily excreted metabolites. The biotransformation reactions are classified as phase I and II reactions. Uridine 5'-diphospho (UDP)-glucuronosyltransferases (UGTs) are a superfamily of phase II enzymes which have roles in the conjugation of xenobiotics or endogenous compounds, including drugs and bilirubin, with glucuronic acid to make them easier to excrete. The method the human body uses to achieve glucuronidation may be affected by a large interindividual variation due to changes in the sequences of the genes encoding these enzymes. In the last five years, the study of the genetic variants of the UGTs at a molecular level has become important due to its association with several diseases and the ability to predict adverse events due to drug metabolism. In the present review, the structure and the prominent genetic variants of the UGT1A subfamily and their metabolic and clinical implications are described.
Project description:A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.
Project description:Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro-in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2-4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction's K(m), increasing its V(max), or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions' K(m) are concerned. In the cases of V(max) values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V(max) increases. Additionally, the BSA effects may be UGT subfamily dependent since K(m) decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V(max) increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.
Project description:BACKGROUND AND PURPOSE: NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA. EXPERIMENTAL APPROACH: We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity. KEY RESULTS: Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect. CONCLUSIONS AND IMPLICATIONS: UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs' substrates.
Project description:UDP-glucuronosyltransferases (UGTs) play an important role in the phase II metabolism of exogenous and endogenous compounds. As colorectal cancer (CRC) etiology is thought to involve the biotransformation of dietary factors, UGT polymorphisms may affect CRC risk by altering levels of exposure. Genotyping of over 1800 Caucasian subjects was completed to identify the role of genetic variation in nine UGT1A and five UGT2B genes on CRC risk. Unconditional logistic regression and haplotype analyses were conducted to identify associations with CRC risk and potential gene-environment interactions. UGT1A haplotype analysis found that the T-G haplotype in UGT1A10 exon 1 (block 2: rs17864678, rs10929251) decreased cancer risk for the colon [proximal (OR?=?0.28, 95% CI?=?0.11–0.69) and for the distal colon (OR?=?0.32, 95% CI?=?0.12–0.91)], and that the C-T-G haplotype in the 3? region flanking the UGT1A shared exons (block 11: rs7578153, rs10203853, rs6728940) increased CRC risk in males (OR?=?2.56, 95% CI?=?1.10–5.95). A haplotype in UGT2B15 containing a functional variant (rs4148269, K523T) and an intronic SNP (rs6837575) was found to affect rectal cancer risk overall (OR?=?2.57, 95% CI?=?1.21–5.04) and in females (OR?=?3.08, 95% CI?=?1.08–8.74). An interaction was found between high NSAID use and the A-G-T haplotype (block 10: rs6717546, rs1500482, rs7586006) in the UGT1A shared exons that decreased CRC risk. This suggests that UGT genetic variation alters CRC risk differently by anatomical sub-site and gender and that polymorphisms in the UGT1A shared exons may have a regulatory effect on gene expression that allows for the protective effect of NSAIDs on CRC risk.
Project description:UNLABELLED:?-lapachone (?-lap), an NAD(P)H:quinone oxidoreductase 1 (NQO1) targeting antitumor drug candidate in phase II clinical trials, is metabolically eliminated via NQO1 mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation. This study intends to explore the inner link between the cellular glucuronidation and pharmacokinetics of ?-lap and its apoptotic effect in human colon cancer cells. HT29 cells S9 fractions exhibited high glucuronidation activity towards ?-lap, which can be inhibited by UGT1A9 competitive inhibitor propofol. UGT1A siRNA treated HT29 cells S9 fractions displayed an apparent low glucuronidation activity. Intracellular accumulation of ?-lap in HCT116 cells was much higher than that in HT29 cells, correlated with the absence of UGT1A in HCT116 cells. The cytotoxic and apoptotic effect of ?-lap in HT29 cells were much lower than that in HCT116 cells; moreover, ?-lap triggered activation of SIRT1-FOXO1 apoptotic pathway was observed in HCT116 cells but not in HT29 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol significantly decreased ?-lap's cytotoxic and apoptotic effects, due to the repression of glucuronidation and the resultant intracellular accumulation. In conclusion, UGT1A is an important determinant, via switching NQO1-triggered redox cycle to metabolic elimination, in the intracellular accumulation of ?-lap and thereafter its cytotoxicity in human colon cancer cells. Together with our previous works, we propose that UGTs determined cellular pharmacokinetics is an important determinant in the apoptotic effects of NQO1 targeting substrates serving as chemotherapeutic drugs.
Project description:UDP-glucuronosyltransferases (UGTs) catalyze the addition of UDP-glucuronic acid to endo- and xenobiotics, enhancing their water solubility and elimination. Many exogenous compounds, such as microsomal enzyme inducers (MEIs), alter gene expression through xenobiotic-responsive transcription factors, namely, the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor erythroid 2-related factor 2 (Nrf2). These transcription factors regulate xenobiotic-inducible expression of hepatic and intestinal biotransformation enzymes and transporters. The purpose of this study was to determine hepatic and intestinal inducibility of mouse Ugt mRNA by MEIs. Male C57BL/6 mice were treated for four consecutive days with activators of AhR [2,3,7,8-tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126, and beta-naphthoflavone], CAR [1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenobarbital, and diallyl sulfide], PXR [pregnenolone-16alpha-carbonitrile (PCN), spironolactone, and dexamethasone], PPARalpha (clofibrate, ciprofibrate, and diethylhexylphthalate), and Nrf2 (oltipraz, ethoxyquin, and butylated hydroxyanisole), respectively. Ugt1a1 mRNA expression in liver was induced by activators of all five transcription factor pathways, Ugt1a5 by Nrf2 activators, Ugt1a6 by all the pathways except CAR, and Ugt1a9 by all the pathways except Nrf2. Ugt2b35 mRNA in liver was induced by AhR activators and Ugt2b36 by CAR and PPARalpha activators. Throughout the small and large intestine, the AhR ligand TCDD increased Ugt1a6 and Ugt1a7 mRNA. In small intestine, the PXR activator PCN increased Ugt1a1, Ugt1a6, Ugt1a7, Ugt2b34, and Ugt2b35 mRNA in the duodenum. In conclusion, chemical activation of AhR, CAR, PXR, PPARalpha, and Nrf2 in mouse results in induction of distinct Ugt gene sets in liver and intestine, predominantly the Ugt1a isoforms.
Project description:Increased levels of bile acids (BAs) due to the various hepatic diseases could interfere with the metabolism of xenobiotics, such as drugs, and endobiotics including steroid hormones. UDP-glucuronosyltransferases (UGTs) are involved in the conjugation and elimination of many xenobiotics and endogenous compounds. The present study sought to investigate the potential for inhibition of UGT enzymes by BAs. The results showed that taurolithocholic acid (TLCA) exhibited the strongest inhibition toward UGTs, followed by lithocholic acid. Structure-UGT inhibition relationships of BAs were examined and in vitro-in vivo extrapolation performed by using in vitro inhibition kinetic parameters (Ki) in combination with calculated in vivo levels of TLCA. Substitution of a hydrogen with a hydroxyl group in the R1, R3, R4, R5 sites of BAs significantly weakens their inhibition ability toward most UGTs. The in vivo inhibition by TLCA toward UGT forms was determined with following orders of potency: UGT1A4 > UGT2B7 > UGT1A3 > UGT1A1 ? UGT1A7 ? UGT1A10 ? UGT2B15. In conclusion, these studies suggest that disrupted homeostasis of BAs, notably taurolithocholic acid, found in various diseases such as cholestasis, could lead to altered metabolism of xenobiotics and endobiotics through inhibition of UGT enzymes.
Project description:Mammalian UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from UDP-glucuronic acid to various xenobiotics and endobiotics. Since UGTs comprise rate-limiting enzymes for metabolism of various compounds, co-administration of UGT-inhibiting drugs and genetic deficiency of UGT genes can cause an increased blood concentration of these compounds. During the last few decades, extensive efforts have been made to advance the understanding of gene structure, function, substrate specificity, and inhibition/induction properties of UGTs. However, molecular mechanisms and physiological importance of the oligomerization and protein-protein interactions of UGTs are still largely unknown. While three-dimensional structures of human UGTs can be useful to reveal the details of oligomerization and protein-protein interactions of UGTs, little is known about the protein structures of human UGTs due to the difficulty in solving crystal structures of membrane-bound proteins. Meanwhile, soluble forms of plant and bacterial UGTs as well as a partial domain of human UGT2B7 have been crystallized and enabled us to predict three-dimensional structures of human UGTs using a homology-modeling technique. The homology-modeled structures of human UGTs do not only provide the detailed information about substrate binding or substrate specificity in human UGTs, but also contribute with unique knowledge on oligomerization and protein-protein interactions of UGTs. Furthermore, various in vitro approaches indicate that UGT-mediated glucuronidation is involved in cell death, apoptosis, and oxidative stress as well. In the present review article, recent understandings of UGT protein structures as well as physiological importance of the oligomerization and protein-protein interactions of human UGTs are discussed.
Project description:The 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 ?-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.
Project description:The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P < 0.05) associated with mRNA expression and/or activities of UGT1A1, UGT1A3 and UGT2B17. We found novel SNPs in the UGT2B17 CNV region accounting for variability in UGT2B17 gene transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.