Differential Control of Dntp Biosynthesis and Genome Integrity
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Origin Sites of Calcium Release and Calcium Oscillations in Frog Sympathetic Neurons
The Journal of Neuroscience, December, 15, 2000, 20(24):9059–9070 Origin Sites of Calcium Release and Calcium Oscillations in Frog Sympathetic Neurons Stefan I. McDonough, Zolta´ n Cseresnye´ s, and Martin F. Schneider Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201 In many neurons, Ca 2ϩ signaling depends on efflux of Ca 2ϩ from levels within the cell body could increase or decrease indepen- intracellular stores into the cytoplasm via caffeine-sensitive ryan- dently of neighboring regions, suggesting independent action of odine receptors (RyRs) of the endoplasmic reticulum. We have spatially separate Ca 2ϩ stores. Confocal imaging of fluorescent used high-speed confocal microscopy to image depolarization- analogs of ryanodine and thapsigargin, and of MitoTracker, and caffeine-evoked increases in cytoplasmic Ca 2ϩ levels in showed potential structural correlates to the patterns of Ca 2ϩ individual cultured frog sympathetic neurons. Although caffeine- release and propagation. High densities of RyRs were found in a evoked Ca 2ϩ wave fronts propagated throughout the cell, in ring around the cell periphery, mitochondria in a broader ring just most cells the initial Ca 2ϩ release was from one or more discrete inside the RyRs, and sarco-endoplasmic reticulum Ca 2ϩ ATPase sites that were several micrometers wide and located at the cell pumps in hot spots at the cell edge. Discrete sites at the cell edge, even in Ca 2ϩ-free external solution. During cell-wide cy- edge primed to release Ca 2ϩ from intracellular stores might toplasmic [Ca 2ϩ] oscillations triggered by continual caffeine ap- preferentially convert Ca 2ϩ influx through a local area of plasma plication, the initial Ca 2ϩ release that began each Ca 2ϩ peak membrane into a cell-wide Ca 2ϩ increase. -
DUT (NM 001948) Human Tagged ORF Clone Product Data
OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC221635 DUT (NM_001948) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: DUT (NM_001948) Human Tagged ORF Clone Tag: Myc-DDK Symbol: DUT Synonyms: dUTPase Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RC221635 representing NM_001948 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGCCCTGCTCTGAAGAGACACCCGCCATTTCACCCAGTAAGCGGGCCCGGCCTGCGGAGGTGGGCGGCA TGCAGCTCCGCTTTGCCCGGCTCTCCGAGCACGCCACGGCCCCCACCCGGGGCTCCGCGCGCGCCGCGGG CTACGACCTGTACAGTGCCTATGATTACACAATACCACCTATGGAGAAAGCTGTTGTGAAAACGGACATT CAGATAGCGCTCCCTTCTGGGTGTTATGGAAGAGTGGCTCCACGGTCAGGCTTGGCTGCAAAACACTTTA TTGATGTAGGAGCTGGTGTCATAGATGAAGATTATAGAGGAAATGTTGGTGTTGTACTGTTTAATTTTGG CAAAGAAAAGTTTGAAGTCAAAAAAGGTGATCGAATTGCACAGCTCATTTGCGAACGGATTTTTTATCCA GAAATAGAAGAAGTTCAAGCCTTGGATGACACCGAAAGGGGTTCAGGAGGTTTTGGTTCCACTGGAAAGA AT ACGCGTACGCGGCCGCTCGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATT ACAAGGATGACGACGATAAGGTTTAA Protein Sequence: >RC221635 representing NM_001948 Red=Cloning site Green=Tags(s) MPCSEETPAISPSKRARPAEVGGMQLRFARLSEHATAPTRGSARAAGYDLYSAYDYTIPPMEKAVVKTDI QIALPSGCYGRVAPRSGLAAKHFIDVGAGVIDEDYRGNVGVVLFNFGKEKFEVKKGDRIAQLICERIFYP EIEEVQALDDTERGSGGFGSTGKN TRTRPLEQKLISEEDLAANDILDYKDDDDKV Chromatograms: -
Crucial Roles of Thymidine Kinase 1 and Deoxyutpase in Incorporating the Antineoplastic Nucleosides Trifluridine and 2'-Deoxy-5-Fluorouridine Into DNA
INTERNATIONAL JOURNAL OF ONCOLOGY 46: 2327-2334, 2015 Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA KAzUKI SAKAMoTo, Tatsushi YoKogAwA, HIRoYUKI UENo, KEI ogUcHI, HIRoMI KAzUNo, KEIJI ISHIDA, NozoMU TANAKA, AKIKo oSADA, YUKARI YAMADA, HIRoYUKI oKABE and KENIcHI Matsuo Drug Discovery and Development I, Discovery and Preclinical Research Division, Taiho Pharmaceutical co., Ltd., Tsukuba, Ibaraki 300-2611, Japan Received March 6, 2015; Accepted April 9, 2015 DoI: 10.3892/ijo.2015.2974 Abstract. Trifluridine (FTD) and 2'-deoxy-5-fluorouridine transported into cells by ENT1 and ENT2 and were phosphor- (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor ylated by thymidine kinase 1, which showed a higher catalytic agents that inhibit thymidylate synthase activity and their activity for FTD than for FdUrd. deoxyUTPase (DUT) did not nucleotides are incorporated into DNA. However, it is evident recognize dTTP and FTD-triphosphate (F3dTTP), whereas that several differences occur in the underlying antitumor deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate mechanisms associated with these nucleoside analogues. (FdUTP) were efficiently degraded by DUT. DNA poly- Recently, TAS-102 (composed of FTD and tipiracil hydrochlo- merase α incorporated both F3dTTP and FdUTP into DNA at ride, TPI) was shown to prolong the survival of patients with sites aligned with adenine on the opposite strand. FTD-treated colorectal cancer who received a median of 2 prior therapies, cells showed differing nuclear morphologies compared to including 5-FU. TAS-102 was recently approved for clinical FdUrd-treated cells. These findings indicate that FTD and use in Japan. -
Electronic Reprint Phosphorylation Adjacent to the Nuclear Localization
electronic reprint Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights Gergely Rona,´ Mary Marfori, Mat´ e´ Borsos, Ildiko´ Scheer, EnikoTak˝ acs,´ Judit Toth,´ Fruzsina Babos, Anna Magyar, Anna Erdei, Zoltan´ Bozoky,´ Laszl´ o´ Buday, Bostjan Kobe and Beata´ G. Vertessy´ Acta Cryst. (2013). D69, 2495–2505 Copyright c International Union of Crystallography Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the struc- tures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure–function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallo- graphic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crys- tallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. -
Regulation of Human Dutpase Gene Expression and P53-Mediated Transcriptional Repression in Response to Oxaliplatin-Induced DNA Damage Peter M
78–95 Nucleic Acids Research, 2009, Vol. 37, No. 1 Published online 16 November 2008 doi:10.1093/nar/gkn910 Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage Peter M. Wilson1, William Fazzone1, Melissa J. LaBonte1, Heinz-Josef Lenz2 and Robert D. Ladner1,* 1Department of Pathology and 2Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received May 19, 2008; Revised October 28, 2008; Accepted October 29, 2008 ABSTRACT INTRODUCTION Deoxyuridine triphosphate nucleotidohydrolase Deoxyuridine triphosphate nucleotidohydrolase (dUT (dUTPase) catalyzes the hydrolysis of dUTP to Pase) is the sole enzyme responsible for the hydrolysis of dUMP and PPi. Although dUTP is a normal intermedi- dUTP to dUMP and pyrophosphate simultaneously pro- ate in DNA synthesis, its accumulation and misincor- viding substrate for thymidylate synthase (TS) and elim- poration into DNA is lethal. Importantly, uracil inating dUTP from the DNA biosynthetic pathway. Although dUTP is a normal intermediate in DNA synth- misincorporation is a mechanism of cytotoxicity esis, its extensive accumulation and misincorporation induced by fluoropyrimidine chemotherapeutic into DNA is lethal in both prokaryotic and eukaryotic agents including 5-fluorouracil (5-FU) and elevated organisms as evidenced from knockout models (1,2). expression of dUTPase is negatively correlated Importantly, uracil misincorporation also represents with clinical response to 5-FU-therapy. In this study a major mechanism of cytotoxicity induced by the we performed the first functional characterization of TS-inhibitor class of chemotherapeutic agents including the dUTPase promoter and demonstrate a role for the fluoropyrimidines 5-fluorouracil (5-FU), fluorodeox- E2F-1 and Sp1 in driving dUTPase expression. -
Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
International Journal of Molecular Sciences Article Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases Kate McGraphery and Wilfried Schwab * Biotechnology of Natural Products, Technische Universität München, 85354 Freising, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-8161-712-912; Fax: +49-8161-712-950 Received: 27 January 2020; Accepted: 21 March 2020; Published: 23 March 2020 Abstract: The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP2-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP2-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible KM, vmax, and kcat values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications. -
Caffeine and Caffeic Acid Inhibit Growth and Modify Estrogen Receptor and Insulin-Like Growth Factor I Receptor Levels in Human Breast Cancer Ann H
Published OnlineFirst February 17, 2015; DOI: 10.1158/1078-0432.CCR-14-1748 Cancer Therapy: Clinical Clinical Cancer Research Caffeine and Caffeic Acid Inhibit Growth and Modify Estrogen Receptor and Insulin-like Growth Factor I Receptor Levels in Human Breast Cancer Ann H. Rosendahl1, Claire M. Perks2, Li Zeng2, Andrea Markkula1, Maria Simonsson1, Carsten Rose3, Christian Ingvar4, Jeff M.P. Holly2, and Helena Jernstrom€ 1 Abstract Purpose: Epidemiologic studies indicate that dietary factors, 0.018), compared with patients with low consumption (1 cup/ such as coffee, may influence breast cancer and modulate hor- day). Moderate to high consumption was associated with lower þ mone receptor status. The purpose of this translational study was risk for breast cancer events in tamoxifen-treated patients with ER to investigate how coffee may affect breast cancer growth in tumors (adjusted HR, 0.51; 95% confidence interval, 0.26–0.97). þ relation to estrogen receptor-a (ER) status. Caffeine and caffeic acid suppressed the growth of ER (P 0.01) À Experimental Design: The influence of coffee consumption on and ER (P 0.03) cells. Caffeine significantly reduced ER and þ patient and tumor characteristics and disease-free survival was cyclin D1 abundance in ER cells. Caffeine also reduced the assessed in a population-based cohort of 1,090 patients with insulin-like growth factor-I receptor (IGFIR) and pAkt levels in þ À invasive primary breast cancer in Sweden. Cellular and molecular both ER and ER cells. Together, these effects resulted in impaired effects by the coffee constituents caffeine and caffeic acid were cell-cycle progression and enhanced cell death. -
Targeting Nucleotide Metabolism Enhances the Efficacy of Anthracyclines and Anti-Metabolites in Triple-Negative Breast Cancer
www.nature.com/npjbcancer ARTICLE OPEN Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer Craig Davison1, Roisin Morelli1, Catherine Knowlson1, Melanie McKechnie1, Robbie Carson1, Xanthi Stachtea1, Kylie A. McLaughlin2, Vivien E. Prise2, Kienan Savage 1, Richard H. Wilson3, Karl A. Mulligan2, Peter M. Wilson2, Robert D. Ladner1,5 and ✉ Melissa J. LaBonte 1,4,5 Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines 1234567890():,; to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy. -
The Role of a Key Amino Acid Position in Species- Specific Proteinaceous Dutpase Inhibition
Article The Role of a Key Amino Acid Position in Species- Specific Proteinaceous dUTPase Inhibition András Benedek 1,2,*, Fanni Temesváry-Kis 1, Tamjidmaa Khatanbaatar 1, Ibolya Leveles 1,2, Éva Viola Surányi 1,2, Judit Eszter Szabó 1,2, Lívius Wunderlich 1 and Beáta G. Vértessy 1,2,* 1 Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, H -1111 Budapest, Szent Gellért tér 4, Hungary; [email protected] (F.T-K.); [email protected] (T.K.); [email protected] (L.W.) 2 Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Magyar tudósok körútja 2, Hungary; [email protected] (I.L.); [email protected] (É.V.S.); [email protected] (J.E.S.) * Correspondence: [email protected] (A.B.); [email protected] (B.G.V.) Received: 14 May 2019; Accepted: 27 May 2019; Published: 6 June 2019 Abstract: Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. -
PURINE SALVAGE in HELICOBACTER PYLORI by ERICA FRANCESCA MILLER (Under the Direction of Robert J. Maier) ABSTRACT Purines Are Es
PURINE SALVAGE IN HELICOBACTER PYLORI by ERICA FRANCESCA MILLER (Under the Direction of Robert J. Maier) ABSTRACT Purines are essential for all living cells. This fact is reflected in the high degree of pathway conservation for purine metabolism across all domains of life. The availability of purines within a mammalian host is thought to be a limiting factor for infection, as demonstrated by the importance of purine synthesis and salvage genes among many bacterial pathogens. Helicobacter pylori, a primary causative agent of peptic ulcers and gastric cancers, colonizes a niche that is otherwise uninhabited by bacteria: the surface of the human gastric epithelium. Despite many studies over the past 30 years that have addressed virulence mechanisms such as acid resistance, little knowledge exists regarding this organism’s purine metabolism. To fill this gap in knowledge, we asked whether H. pylori can carry out de novo purine biosynthesis, and whether its purine salvage network is complete. Based on genomic data from the fully sequenced H. pylori genomes, we combined mutant analysis with physiological studies to determine that H. pylori, by necessity, must acquire purines from its human host. Furthermore, we found the purine salvage network to be complete, allowing this organism to use any single purine nucleobase or nucleoside for growth. In the process of elucidating these pathways, we discovered a nucleoside transporter in H. pylori that, in contrast to the biochemically- characterized homolog NupC, aids in uptake of purine rather than pyrimidine nucleosides into the cell. Lastly, we investigated an apparent pathway gap in the genome annotation—that of adenine degradation—and in doing so uncovered a new family of adenosine deaminase that lacks sequence homology with all other adenosine deaminases studied to date. -
CRISPR/Cas9-Mediated Knock-Out of Dutpase in Mice Leads to Early Embryonic Lethality
bioRxiv preprint doi: https://doi.org/10.1101/335422; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. CRISPR/Cas9-mediated knock-out of dUTPase in mice leads to early embryonic lethality Hajnalka Laura Pálinkás1,2*, Gergely Rácz 1,3, Zoltán Gál4, Orsolya Hoffmann4, Gergely Tihanyi1,3, Elen Gócza4*, László Hiripi4*, Beáta G. Vértessy1,3* *Corresponding authors 1Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest, Hungary 2Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, Hungary 3Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary 4Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, Gödöllő, Hungary Correspondence and requests for materials should be addressed to Beáta G. Vértessy (email: [email protected]). Correspondence may also be addressed to Hajnalka Laura Pálinkás ([email protected]), Elen Gócza ([email protected]), and László Hiripi ([email protected]). 1 bioRxiv preprint doi: https://doi.org/10.1101/335422; this version posted May 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Sanitization of nucleotide pools is essential for genome maintenance. Among the enzymes significant in this mechanism, deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) performs cleavage of dUTP into dUMP and inorganic pyrophosphate. By this reaction the enzyme efficiently prevents uracil incorporation into DNA and provides dUMP, the substrate for de novo thymidylate biosynthesis. -
Androgen-Stimulated UDP-Glucose Dehydrogenase Expression Limits
Published OnlineFirst February 24, 2009; DOI: 10.1158/0008-5472.CAN-08-3083 Research Article Androgen-Stimulated UDP-Glucose Dehydrogenase Expression Limits Prostate Androgen Availability without Impacting Hyaluronan Levels Qin Wei,1 Robert Galbenus,1 Ashraf Raza,1 Ronald L. Cerny,2 and Melanie A. Simpson1 Departments of 1Biochemistry and 2Chemistry, University of Nebraska, Lincoln, Nebraska Abstract AR loss of expression or constitutive activation, or oncogenic UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to transformation through other growth control pathways (3). UDP-glucuronate, an essential precursor for production of Pathways involved in regulation of androgen availability have hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. been investigated as an obvious link to hormone-independent High levels of HA turnover in prostate cancer are correlated cancer progression. Typically, the focus of these studies has been the biosynthetic enzymes such as hydroxysteroid dehydrogenase with aggressive progression. UGDH expression is high in the a normal prostate, although HA accumulation is virtually and 5 -reductase that complete activation of testosterone undetectable. Thus, its normal role in the prostate may be precursors to their potent growth stimulatory forms (4–7). Some to provide precursors for glucuronosyltransferase enzymes, therapeutic success has been achieved by targeting these enzymes, which inactivate and solubilize androgens by glucuronidation. but excess hormones from other pathways can also be converted