Dataset Information

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

ABSTRACT: 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.

SUBMITTER: Osborne MJ

PROVIDER: S-EPMC6309593 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

Osborne Michael J MJ Coutinho de Oliveira Luciana L Volpon Laurent L Borden Katherine L B KLB

Biomolecular NMR assignments 20180622 2

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 biolog ...[more]

PMID: 29934866

Similar Datasets

Project description:Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.

Project description:Background and purposeNAD(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 approachWe 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 resultsMultiple 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 implicationsUGT1A 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:HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.

Dataset Information

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

Publications

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure