DOCSLIB.ORG

Molecular Drivers of Specificity in Human Ribonucleotide

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Molecular Drivers of Specificity in Human Ribonucleotide MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE by ANDREW JOHN KNAPPENBERGER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation advisor: Dr. Michael E. Harris Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Andrew John Knappenberger Candidate for the degree of Doctor of Philosophy*. (signed) Committee Chair Hung-Ying Kao Committee Member Chris Dealwis Committee Member Michael Harris Committee Member Arne Rietsch Committee Member Martin Snider (date) March 10, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2017 by Andrew Knappenberger All rights reserved Table of Contents Acknowledgements ............................................................................................................ ix Chapter 1 : Introduction .................................................................................................... 13 Molecular recognition by enzymes is central to life ..................................................... 14 Recognition of multiple substrates and regulation by allostery are common features of enzymes .................................................................................................................... 16 Ribonucleotide reductase is an essential enzyme for all known cellular organisms .... 18 Loop 2 is central to specificity regulation in ribonucleotide reductase ........................ 21 Measuring multiple substrate kinetics through internal competition permits comprehensive description of enzymatic specificity .................................................... 22 Chapter 2 : Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity ......................................................................................................................... 28 Abstract ...................................................................................................................... 29 Introduction ................................................................................................................... 30 Results and discussion .................................................................................................. 42 Conclusions ................................................................................................................... 70 Materials and methods .................................................................................................. 72 Chapter 3 : Phylogenetic Comparative Sequence Analysis and Functional Studies of Mutant Enzymes Reveal Compensatory Amino Acid Substitutions in Loop 2 of Human Ribonucleotide Reductase .................................................................................... 77 i Abstract ......................................................................................................................... 78 Introduction ................................................................................................................... 79 Results and discussion .................................................................................................. 85 Conclusions ................................................................................................................. 119 Materials and methods ................................................................................................ 122 Chapter 4 : Summary and Future Directions .................................................................. 126 Summary ..................................................................................................................... 126 Future directions ......................................................................................................... 131 Bibliography ................................................................................................................... 137 ii List of Tables Table 3-1. Numerical values from activity assays in Figure 3-6. .............................. 107 Table 3-2. Numerical values from activity assays in Figure 3-9. .............................. 116 iii List of Figures Figure 1-1. Structure of eukaryotic R1. ....................................................................... 25 Figure 1-2. Conformational changes in loop 2. ............................................................ 26 Figure 2-1. Structure of human ribonucleotide reductase and location of the S-site and C-site. ................................................................................................... 35 Figure 2-2. Comparison of RR loop 2 across species. ................................................. 37 Figure 2-3. Measurements of RR specificity from this and other studies. ................. 39 Figure 2-4. Comparison of eukaryotic and bacterial RRs with dGTP or dTTP bound in the S-site. .......................................................................................................... 40 Figure 2-5. Application of internal competition to measurement of hRR specificity. ............................................................................................................... 56 Figure 2-6. Measurement of native hRR and ScRR specificities. ............................... 58 Figure 2-7. The specificities directed by a series of pyrimidine effector analogs. ..... 60 Figure 2-8. Structures of eukaryotic RR bound to S-site ligands. .............................. 62 Figure 2-9. The specificities directed by a series of purine effector analogs. ............ 64 Figure 2-10. Competition between purine effector analogs and the natural S-site effector dTTP. ................................................................................................................. 66 Figure 2-11. Effects of the D287A substitution on hRR substrate recognition. ........ 68 Figure 3-1. Three-dimensional structure of hRR. ........................................................ 83 Figure 3-2. Sequence conservation in eukaryotic ribonucleotide reductase and the extent of variation in loop 2. .................................................................................. 100 Figure 3-3. Loop 2 sequences from Figure 3-2C mapped onto a eukaryotic tree of life. .............................................................................................................................. 102 iv Figure 3-4. Crystal structure of hR1 bound to dTTP and GDP. .............................. 103 Figure 3-5. Circular dichroism (CD) of hRR variants. ............................................. 104 Figure 3-6. Activity and specificity of the hRR variants. .......................................... 105 Figure 3-7. Size exclusion chromatography (SEC) of hRR variants in the presence of dGTP, dTTP or ATP. ............................................................................... 109 Figure 3-8. SEC profiles of wild-type and P294K hRR large subunit in the presence of 3 mM ATP. ................................................................................................ 111 Figure 3-9. Activity and specificity of N291G mutant hR1 in the presence of 2- aminopurine-drTP, dITP, and dZeb. .......................................................................... 112 Figure 3-10. Activity and specificity of the hRR variants in the presence of both ATP and dGTP/dTTP................................................................................................... 114 Figure 3-11. Correspondence between the present in vitro experiments and “natural experiments” of evolution. .................................................................... 118 v List of Abbreviations • RR – ribonucleotide reductase. • DNA – deoxyribonucleic acid. • dNTP – deoxyribonucleotide diphosphate. • ATP – adenosine triphosphate. • dATP – deoxyadenosine triphosphate. • dGTP – deoxyguanosine triphosphate. • dTTP – deoxythymidine triphosphate. • tRNA – transfer ribonucleic acid. • ATC – aspartate transcarbamylase. • CTP – cytidine triphosphate. • SAMHD1 – SAM domain and HD domain-containing protein 1. • HIV – human immunodeficiency virus. • ADP – adenosine diphosphate. • CDP – cytidine diphosphate. • GDP – guanosine diphosphate. • UDP – uridine diphosphate. • R1 – ribonucleotide reductase large subunit. • R2 – ribonucleotide reductase small subunit. • C-site – catalytic site, the active site of ribonucleotide reductase. • S-site – specificity site. • A-site – activity site. vi • dNDP – deoxynucleotide diphosphate. • hRR – human ribonucleotide reductase. • ScRR – S. cerevisiae ribonucleotide reductase. • AMPPNP – adenylylimidodiphosphate. • PDB – Protein Data Bank. • HPLC – high-performance liquid chromatography. • TmRR – Thermotoga maritima RR. • EcRR – E. coli RR. • aa – amino acid. • hR1 – human ribonucleotide reductase large subunit. • 5FdUTP – 5-fulorodeoxyuridine triphosphate. • dUTP – deoxyuridine triphosphate. • dZeb – deoxyzebularine triphosphate. • 2-aminopurine-drTP – 2-aminopurine-deoxyribose triphosphate. • N2dATP – 2-amino-deoxyadenosine triphosphate. • dITP – deoxyinosine triphosphate. • hRRM1 – human ribonucleotide reductase large subunit. • hRRM2 – human ribonucleotide reductase small subunit. • ScRR1 – S. cerevisiae ribonucleotide reductase large subunit. • ScR2R4 – S. cerevisiae ribonucleotide reductase small subunit. • DTT – dithiothreitol.
Load more

Recommended publications
  • The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (Cad) by Phosphorylation and Protein-Protein Interactions
    THE REGULATION OF CARBAMOYL PHOSPHATE SYNTHETASE-ASPARTATE TRANSCARBAMOYLASE-DIHYDROOROTASE (CAD) BY PHOSPHORYLATION AND PROTEIN-PROTEIN INTERACTIONS Eric M. Wauson A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2007 Approved by: Lee M. Graves, Ph.D. T. Kendall Harden, Ph.D. Gary L. Johnson, Ph.D. Aziz Sancar M.D., Ph.D. Beverly S. Mitchell, M.D. 2007 Eric M. Wauson ALL RIGHTS RESERVED ii ABSTRACT Eric M. Wauson: The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (CAD) by Phosphorylation and Protein-Protein Interactions (Under the direction of Lee M. Graves, Ph.D.) Pyrimidines have many important roles in cellular physiology, as they are used in the formation of DNA, RNA, phospholipids, and pyrimidine sugars. The first rate- limiting step in the de novo pyrimidine synthesis pathway is catalyzed by the carbamoyl phosphate synthetase II (CPSase II) part of the multienzymatic complex Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, Dihydroorotase (CAD). CAD gene induction is highly correlated to cell proliferation. Additionally, CAD is allosterically inhibited or activated by uridine triphosphate (UTP) or phosphoribosyl pyrophosphate (PRPP), respectively. The phosphorylation of CAD by PKA and ERK has been reported to modulate the response of CAD to allosteric modulators. While there has been much speculation on the identity of CAD phosphorylation sites, no definitive identification of in vivo CAD phosphorylation sites has been performed. Therefore, we sought to determine the specific CAD residues phosphorylated by ERK and PKA in intact cells.
    [Show full text]
  • Difluorodeoxyguanosine in Chinese Hamster Ovary Cells'
    [CANCER RESEARCH55, 1517-1524, April 1, 19951 Cytotoxicity, Metabolism, and Mechanisms of Action of 2',2'- Difluorodeoxyguanosine in Chinese Hamster Ovary Cells' Varsha Gandhi,2 Shin Mineishi, Peng Huang, Amy J. Chapman, Yandan Yang, Feng Chen, Biffie Nowak, Shern Chubb, Larry W. Hertel, and William Plunkett Department of Clinical Investigation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 (V. G., S. M., P. H.. A. J. C.. Y. 1'., F. C., B. N., S. C., w. p.]. andLillyResearchLaboratories,Indianapolis,indiana46285(L W.H.) ABSTRACT treatment of hematological malignancies (4—6).Arabinosyladenine, although not successful in cancer therapy, has proven antiviral activity The emerging clinical success of gemcItabine (2',2'-difluorodeoxycytl dine) Stimulated interest in the synthesis and evaluation of purine conge (7). The importance of the 2'-pentose moiety for the design of chemo ners. The cytotoxicity, metabolism, and mechanisms of action of the lead therapeutic agents and the fact that fluorine has a van der Waals radius candidate, 2',2'-difluorodeoxyguanoslne (dFdGuo), were StUdied In ChI nese hamster ovary cells Unlike the natural nucleoside deoxyguanosine similar to that of hydrogen led to the synthesis and evaluation of (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase 2'-fluorinated analogues. 2'-Deoxy-2'-fluorocytidine and 2'-deoxy Wild-type Chinese hamster ovary cells and a mutant line deficient in 2'-fluoroguanosine possess inhibitory activities against several deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% lymphoid cell lines and influenza viruses, respectively (8, 9). Inter inhibitory concentration, 7.5 and 6.5 pM, respectively), suggesting that estingly, substitution of the 2'-hydrogen of dCyd3 with a fluorine unlike gemcitabine, dCyd kinase was not responsible for activation of atom in the arabinose configuration produced 10-fold greater cyto dFdGuo This was further confirmed by separation of nucleoside kinases toxicity than when placed in the ribose position (10).
    [Show full text]
  • DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents
    molecules Review DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents Maria Valeria Raimondi 1,*,† , Ornella Randazzo 1,†, Mery La Franca 1 , Giampaolo Barone 1 , Elisa Vignoni 2, Daniela Rossi 2 and Simona Collina 2,* 1 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, via Archirafi 32, 90123 Palermo, Italy; [email protected] (O.R.); [email protected] (M.L.F.); [email protected] (G.B.) 2 Drug Sciences Department, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, via Taramelli 12, 27100 Pavia, Italy; [email protected] (E.V.); [email protected] (D.R.) * Correspondence: [email protected] (M.V.R.); [email protected] (S.C.); Tel.: +390-912-389-1915 (M.V.R.); +390-382-987-379 (S.C.) † These Authors contributed equally to this work. Academic Editors: Simona Collina and Mariarosaria Miloso Received: 25 February 2019; Accepted: 20 March 2019; Published: 22 March 2019 Abstract: Dihydrofolate reductase inhibitors are an important class of drugs, as evidenced by their use as antibacterial, antimalarial, antifungal, and anticancer agents. Progress in understanding the biochemical basis of mechanisms responsible for enzyme selectivity and antiproliferative effects has renewed the interest in antifolates for cancer chemotherapy and prompted the medicinal chemistry community to develop novel and selective human DHFR inhibitors, thus leading to a new generation of DHFR inhibitors. This work summarizes the mechanism of action, chemical, and anticancer profile of the DHFR inhibitors discovered in the last six years. New strategies in DHFR drug discovery are also provided, in order to thoroughly delineate the current landscape for medicinal chemists interested in furthering this study in the anticancer field.
    [Show full text]
  • 1611 REGULATION of PYRIMIDINE METABOLISM in PLANTS Chris
    [Frontiers in Bioscience 9, 1611-1625, May 1, 2004] REGULATION OF PYRIMIDINE METABOLISM IN PLANTS 1, 2 1, 3 1, 4 1, 5 1, 6 1, 7 Chris Kafer , Lan Zhou , Djoko Santoso , Adel Guirgis , Brock Weers , Sanggyu Park and Robert Thornburg 1 1 Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, 2 BASF Plant Science LLC, 2901 South Loop Drive, Ste 3800, Ames, Iowa 50014, 3 Lan Zhou, Pioneer Hi-Bred International, Inc. 7300 NW 62nd Avenue, PO Box 1004, Johnston, Iowa 50131-1004, 4 Indonesian Biotechnology Research Institute for Estate Crops, Jl, Taman Kencana No 1, Bogor 16151 Indonesia, 5 Institute of Genetic Engineering and Biotechnology, Menofiya University, PO Box 79/22857, Sadat City, Egypt, 6 Department of Biochemistry, University of Iowa, 4/511 Bowen Science Building, Iowa City, Iowa 52242-1109, 7 Division of Life and Environment, College of Natural Resources, Daegu University, Gyongsan City, Gyongbuk, Korea 712-714 TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Pyrimidine metabolic pathways 3.1. De novo pyrimidine biosynthesis 3.1.1. CPSase 3.1.2. ATCase 3.1.3. DHOase 3.1.4. DHODH 3.1.5. UMPS 3.1.6. Intracellular Organization of the de novo Pathway 3.2. Pyrimidine Salvage and Recycling 3.2.1. Cytosine deaminase 3.2.2. Cytidine deaminase 3.2.3. UPRTase 3.3. Pyrimidine Modification 3.3.1. UMP/CMP kinase 3.3.2. NDP kinase 3.3.3. CTP synthase, NDP reductase, dUTPase 3.3.4. Thymidylate synthase/Dihydrofolate reductase 3.4. Pyrimidine Catabolism 4. Regulation of pyrimidine metabolism 4.1.
    [Show full text]
  • TITLE Adenylate Kinase 2 Deficiency Causes NAD+ Depletion and Impaired Purine Metabolism During Myelopoiesis
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.05.450633; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. TITLE Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis AUTHORS Wenqing Wang1, Andew DeVilbiss2, Martin Arreola1, Thomas Mathews2, Misty Martin-Sandoval2, Zhiyu Zhao2, Avni Awani1, Daniel Dever1, Waleed Al-Herz3, Luigi Noratangelo4, Matthew H. Porteus1, Sean J. Morrison2, Katja G. Weinacht1, * 1. Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305 USA 2. Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390 USA 3. Department of Pediatrics, Faculty of Medicine, Kuwait University, Safat, 13110 Kuwait 4. Laboratory of Clinical Immunology and Microbiology, National Institute of Health, BETHESDA MD 20814 USA * Corresponding author ABSTRACT Reticular Dysgenesis is a particularly grave from of severe combined immunodeficiency (SCID) that presents with severe congenital neutropenia and a maturation arrest of most cells of the lymphoid lineage. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis in the mitochondria. Accordingly, it has long been hypothesized that a decline in OXPHOS metabolism is the driver of the disease. The mechanistic basis for Reticular Dysgenesis, however, remained incompletely understood, largely due to lack of appropriate model systems to phenocopy the human disease.
    [Show full text]
  • NADPH Homeostasis in Cancer: Functions, Mechanisms and Therapeutic Implications
    Signal Transduction and Targeted Therapy www.nature.com/sigtrans REVIEW ARTICLE OPEN NADPH homeostasis in cancer: functions, mechanisms and therapeutic implications Huai-Qiang Ju 1,2, Jin-Fei Lin1, Tian Tian1, Dan Xie 1 and Rui-Hua Xu 1,2 Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms, and provides the reducing power for anabolic reactions and redox balance. NADPH homeostasis is regulated by varied signaling pathways and several metabolic enzymes that undergo adaptive alteration in cancer cells. The metabolic reprogramming of NADPH renders cancer cells both highly dependent on this metabolic network for antioxidant capacity and more susceptible to oxidative stress. Modulating the unique NADPH homeostasis of cancer cells might be an effective strategy to eliminate these cells. In this review, we summarize the current existing literatures on NADPH homeostasis, including its biological functions, regulatory mechanisms and the corresponding therapeutic interventions in human cancers, providing insights into therapeutic implications of targeting NADPH metabolism and the associated mechanism for cancer therapy. Signal Transduction and Targeted Therapy (2020) 5:231; https://doi.org/10.1038/s41392-020-00326-0 1234567890();,: BACKGROUND for biosynthetic reactions to sustain their rapid growth.5,11 This In cancer cells, the appropriate levels of intracellular reactive realization has prompted molecular studies of NADPH metabolism oxygen species (ROS) are essential for signal transduction and and its exploitation for the development of anticancer agents. cellular processes.1,2 However, the overproduction of ROS can Recent advances have revealed that therapeutic modulation induce cytotoxicity and lead to DNA damage and cell apoptosis.3 based on NADPH metabolism has been widely viewed as a novel To prevent excessive oxidative stress and maintain favorable and effective anticancer strategy.
    [Show full text]
  • TRACE: Tennessee Research and Creative Exchange
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2009 Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae James Wesley Fairman University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Fairman, James Wesley, "Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae. " PhD diss., University of Tennessee, 2009. https://trace.tennessee.edu/utk_graddiss/49 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by James Wesley Fairman entitled "Structure- Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Biochemistry and Cellular and Molecular Biology. Chris G. Dealwis,
    [Show full text]
  • Erythrocyte Metabolism in Purine Nucleoside Phosphorylase Deficiency After Enzyme Replacement Therapy by Infusion of Erythrocytes
    Erythrocyte metabolism in purine nucleoside phosphorylase deficiency after enzyme replacement therapy by infusion of erythrocytes. G E Staal, … , S K Wadman, D W Martin J Clin Invest. 1980;65(1):103-108. https://doi.org/10.1172/JCI109639. Research Article Purine nucleoside phosphorylase deficiency is associated with a severely defective T-cell immunity. A patient with purine nucleoside phosphorylase deficiency was treated with transfusions of irradiated erythrocytes and plasma. This resulted in a remarkable correction of the metabolic disturbances in the patient. The urinary excretion of inosine, deoxyinosine, guanosine, and deoxyguanosine decreased, whereas uric acid excretion as well as serum uric acid concentration increased. It could be shown that the enzyme activity of the circulating erythrocytes correlated inversely with the urinary excretion of nucleosides and directly with the excretion of uric acid. As a consequence of the therapy, several glycolytic intermediates of the erythrocytes were increased, especially 2,3-diphosphoglycerate. The high 2,3-diphosphoglycerate level caused a shift to the right of the oxygen dissociation curve (P50 = 32.9 mm Hg). The immunological status of the patient showed definite improvement after the enzyme replacement therapy. Find the latest version: https://jci.me/109639/pdf Erythrocyte Metabolism in Purine Nucleoside Phosphorylase Deficiency after Enzyme Replacement Therapy by Infusion of Erythrocytes GERARD E. J. STAAL, JAN W. STOOP, BEN J. M. ZEGERS, Louis H. SIEGENBEEK VAN HEUKELOM, MARGREET
    [Show full text]
  • Transient State Analysis of Porcine Dihydropyrimidine Dehydrogenase Reveals Reductive Activation by NADPH
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
    [Show full text]
  • ANABOLISM III: Biosynthesis Amino Acids & Nucleotides
    BI/CH 422/622 ANABOLISM OUTLINE: Photosynthesis Carbohydrate Biosynthesis in Animals Biosynthesis of Fatty Acids and Lipids Biosynthesis of Amino Acids and Nucleotides Nitrogen fixation nitrogenase Nitrogen assimilation Glutamine synthetase Glutamate synthase Amino-acid Biosynthesis non-essential essential Nucleotide Biosynthesis RNA precursors purines pyrimidines DNA precursors deoxy-nucleotides Biosynthesis of secondary products of amino acids ANABOLISM III: Biosynthesis Amino Acids & Nucleotides Dr. Kornberg: Lecture 04.26.17 (0:00-5:06) 5 min 1 Biosynthesis Amino Acids & Nucleotides How are Ribonucleic Acid Precursors So far: converted to Deoxyribonucleic Acid GMPàGDPàGTP Precursors? ….....and how is dTTP made? AMPàADPàATP 2’C-OH bond is directly reduced to 2’-H UMPàUDPàUTPà bond …without activating the carbon for CDPßCTP dehydration, etc.! catalyzed by ribonucleotide reductase Specific kinases, Non-specific kinase, e.g., UMP kinase, nucleoside GMP kinase, diphosphate kinase Very unique enzyme in all of biochemistry – use of free Adenylate kinase (works on both oxy- and radicals etc. deoxy-ribose GDPàdGDP nucleosides) Mechanism: Two H atoms are donated ADPàdADP by NADPH and carried by thioredoxin or glutaredoxin to the active site. UDPàdUDP –Substrates are the NDPs and the products CDPàdCDP are dNDP. Biosynthesis Amino Acids & Nucleotides Source of Reducing Structure of Ribonucleotide Reductase a2 are regulatory Electrons for and half the Ribonucleotide catalytic site; need to be reduced. Reductase b 2 are the other half (a b ) of the active site, 2 2 and the free- radical generators • NADPH serves as the electron donor. • Funneled through glutathione or JoAnne Stubbe thioredoxin pathways (1946– ) 2 •Most forms of enzyme have two catalytic/ regulatory subunits and two radical- generating subunits.
    [Show full text]
  • Genetic Factors Influencing Pyrimidine- Antagonist Chemotherapy
    The Pharmacogenomics Journal (2005) 5, 226–243 & 2005 Nature Publishing Group All rights reserved 1470-269X/05 $30.00 www.nature.com/tpj REVIEW Genetic factors influencing Pyrimidine- antagonist chemotherapy JG Maring1 ABSTRACT 2 Pyrimidine antagonists, for example, 5-fluorouracil (5-FU), cytarabine (ara-C) HJM Groen and gemcitabine (dFdC), are widely used in chemotherapy regimes for 2 FM Wachters colorectal, breast, head and neck, non-small-cell lung cancer, pancreatic DRA Uges3 cancer and leukaemias. Extensive metabolism is a prerequisite for conversion EGE de Vries4 of these pyrimidine prodrugs into active compounds. Interindividual variation in the activity of metabolising enzymes can affect the extent of 1Department of Pharmacy, Diaconessen Hospital prodrug activation and, as a result, act on the efficacy of chemotherapy Meppel & Bethesda Hospital Hoogeveen, Meppel, treatment. Genetic factors at least partly explain interindividual variation in 2 The Netherlands; Department of Pulmonary antitumour efficacy and toxicity of pyrimidine antagonists. In this review, Diseases, University of Groningen & University Medical Center Groningen, Groningen, The proteins relevant for the efficacy and toxicity of pyrimidine antagonists will Netherlands; 3Department of Pharmacy, be summarised. In addition, the role of germline polymorphisms, tumour- University of Groningen & University Medical specific somatic mutations and protein expression levels in the metabolic Center Groningen, Groningen, The Netherlands; pathways and clinical pharmacology
    [Show full text]
  • Genome-Wide Transcriptional Analysis of Carboplatin Response in Chemosensitive and Chemoresistant Ovarian Cancer Cells
    1605 Genome-wide transcriptional analysis of carboplatin response in chemosensitive and chemoresistant ovarian cancer cells David Peters,1,2 John Freund,2 and Robert L. Ochs2 variety of disease processes, including cancer. Patterns of global gene expression can reveal the molecular pathways 1 Department of Pharmacology and Therapeutics, University of relevant to the disease process and identify potential new Liverpool, United Kingdom and 2Precision Therapeutics, Inc., Pittsburgh, Pennsylvania therapeutic targets. The use of this technology for the molecular classification of cancer was recently shown with the identification of an expression profile that was Abstract predictive of patient outcome for B-cell lymphoma (1). We have recently described an ex vivo chemoresponse In addition, this study showed that histologically similar assay for determining chemosensitivity in primary cultures tumors can be differentiated based on their gene expression of human tumors. In this study, we have extended these profiles. Ultimately, these unique patterns of gene expres- experiments in an effort to correlate chemoresponse data sion may be used as guidelines to direct different modes of with gene expression patterns at the level of transcription. therapy. Primary cultures of cells derived from ovarian carcinomas Although it is widely recognized that patients with the of individual patients (n = 6) were characterized using same histologic stage and grade of cancer respond to the ChemoFx assay and classified as either carboplatin therapies differently, few clinical tests can predict indi- sensitive (n = 3) or resistant (n = 3). Three representa- vidual patient responses. The next great challenge will be tive cultures of cells from each individual tumor were then to use the power of post-genomic technology, including subjected to Affymetrix gene chip analysis (n = 18) using microarray analyses, to correlate gene expression patterns U95A human gene chip arrays.
    [Show full text]