DOCSLIB.ORG

Purine Nucleoside Phosphorylase, Or Adenosine Deaminase (Lesch-Nyhan Syndrome/Immunodeficiency) L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Purine Nucleoside Phosphorylase, Or Adenosine Deaminase (Lesch-Nyhan Syndrome/Immunodeficiency) L Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3722 -3726, August 1978 Biochemistry Purine metabolism in cultured human fibroblasts derived from patients deficient in hypoxanthine phosphoribosyltransferase, purine nucleoside phosphorylase, or adenosine deaminase (Lesch-Nyhan syndrome/immunodeficiency) L. F. THOMPSON*, R. C. WILLIS*, J. W. STOOPt, AND J. E. SEEGMILLER* * Department of Medicine, University of California, San Diego, La Jolla, California 92093; and t University Children's Hospital, Het Wilhelmina Kinderziekenhuis, Nieuwe Gracht 137, Utrecht, The Netherlands Contributed by J. Edwin Seegmiller, June 8, 1978 ABS14RACT Rates of purine synthesis de novo, as measured tive, high molecular weight form. Thus, either an increase in by the incorporation of [4C]formate into newly synthesized the concentration of PP-ribose-P or a decrease in the levels of purines, have been determined in cultured human fibroblasts derived from normal individuals and from patients deficient inhibitory nucleotides could potentially accelerate the rate of in adenosine deaminase, purine nucleoside phosporylase, or purine synthesis de novo and result in purine "overproduction." hypoxanthine phosphoribosyltransferase, three consecutive This communication concerns the study of purine metabolism enzymes of the purnne salvage pathway. All four types of cell in cultured human fibroblasts deficient in each of three en- lines are capable of incorporating [14C]formate into purines at zymes of the purine salvage pathway and attempts to define approximately the same rate when the assays are conducted in better a "normal" rate of purine synthesis de novo as compared purine-free medium. The purine overproduction that is char- to In particular, we have attempted acteristic of a deficiency in either the transferase or the phos- purine "overproduction." phorylase and that results from a block in purine reutilization to rectify the previously reported discrepancy in rates of purine can be demonstrated by the resistance of [14Clformate incor- synthesis de now in patients deficient in PNP and in their poration into purines to inhibition by hypoxanthine in the case cultured cells (1). of hypoxanthine phosphoribosyltransferase-deficient fibroblasts The three enzyme deficiencies studied are: HPRT, PNP, and and by resistance to inhibition by inosine in the case of purine adenosine deaminase (ADA). A lack of the enzyme HPRT (17) nucleoside phosphorylast-deficient fibroblasts. leads to the Lesch-Nyhan syndrome (2) with its characteristic Human cells in culture utilize purines derived from two met- neurological dysfunction and purine overproduction de novo abolic routes-either by synthesis de nowo beginning with 5- resulting in pronounced hyperuricemia. A lack of PNP (18) phosphoribosyl-1-pyrophosphate (PP-ribose-P) and glutamine results in an immunodeficiency disease with a specific defect or by "salvage" from PP-ribose-P and purines present in the in T-cell function as well as purine overproduction de novo; medium or arising from the intracellular degradation of nucleic however, hypouricemia rather than hyperuricemia is seen acids. Individuals who lack purine nucleoside phosphorylase because purine catabolism stops with the formation of purine (PNP) or hypoxanthine phosphoribosyltnferase (HPRT), two nucleosides. Large quantities of inosine and guanosine and consecutive enzymes of the purine salvage pathway, synthesize smaller amounts of the corresponding deoxynucleosides are purines de novo at an accelerated rate (1-3). Although several excreted in the urine (1, 3). ADA deficiency leads to a severe cell culture systems have been devised to demonstrate the pu- combined immunodeficiency with T-cell and usually B-cell rine overproduction characteristic of patients with a deficiency dysfunction (19). No purine overproduction has been found in of HPRT (4-8), none has been devised to show the comparable patients lacking ADA (20). overproduction in PNP deficiency. The mechanism by which the rate of purine synthesis de novo is controlled has been the MATERUILS AND METHODS subject of intensive study by numerous investigators. It is be- Source and Growth of Cells. Fibroblasts were grown from lieved to involve the regulation of PP-ribose-P synthetase or foreskins obtained from apparently normal male infants or from PP-ribose-P amidotransferase, the enzymes which catalyze the skin biopsies obtained from normal volunteers or from patients first two reactions of this pathway. In Ehrlich ascites tumor cells, deficient in HPRT, ADA (20), or PNP (3). Fibroblast cultures the rate of synthesis of PP-ribose-P has been shown to be in- were routinely maintained in Eagle's minimal essential medium versely related to the concentration of adenine and guanine supplemented with 10% fetal calf serum, 2 mM glutamine, nucleotides (9). Partially purified human PP-ribose-P synthetase penicillin (100 units/ml), and streptomycin (100 ,ug/ml). In has been shown to be inhibited by ADP and AMP (10). The some experiments, the medium was supplemented with fetal activity of PP-ribose-P amidotransferase and of the de novo calf serum that had been extensively dialyzed against 0.9% pathway as a whole may be controlled by the intracellular levels NaCl. Low serum medium contained AutoPow minimal es- of PP-ribose-P and of adenine and guanine nucleotides (4, sential medium (Flow Laboratories), supplemented with 0.1% 11-16). PP-ribose-P is normally present at concentrations well fetal calf serum, 2 mM glutamine, penicillin (100 units/ml), below the Km for the enzyme. It also converts the human en- streptomycin (100 ;g/ml), 4% bovine serum albumin, trans- zyme to its more active, low molecular weight form. Adenine ferrin (5'Mg/ml), and 4% (vol/vol) Coon's F-12 medium (minus and guanine nucleotides act as inhibitors of the partially puri- hypoxanthine, thymidine, and folic acid) (21). fied enzyme and also convert the human enzyme to its less ac- Rate of Purine Synthesis De Novo. Confluent fibroblasts The costs of publication of this article were defrayed in part by the Abbreviations: PP-ribose-P, 5-phosphoribosyl 1-pyrophosphate; HPRT, payment of page charges. This article must therefore be hereby marked hypoxanthine phosphoribosyltransferase; PNP, purine nucleoside "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate phosphorylase; ADA, adenosine deaminase; Hepes, 4-(2-hydroxy- this fact. ethyl)-l-piperazineethanesulfonic acid. 3722 Downloaded by guest on September 27, 2021 Biochemistry: Thompson et al. Proc. Nat!. Acad. Sci. USA 75(1978) 3723 were trypsinized, plated at a density of approximately 3 X 105 cells per 35-mm dish in minimal essential medium containing 30,OOC en 0% dialyzed fetal calf serum, and allowed to grow in a3if Z incubator maintained at 5% CO2 and 95% air for 15-20 hr. Two C) 0 3 or hr prior to the initiation of the experiment, the cells were le20,00C washed three times with medium supplemented with 40 mM x x' sodium 4- (2- hydroxyethyl) - 1- piperazineethanesulfonate (Hepes), pH 7.4, and any appropriate additions, overlaid with m l0,000 -- - 0.6 ml of the same medium, and allowed to equilibrate in 5% E=Is m CO2 and 95% air. The assay was begun by the addition of 5 c) ACi - of [14C]formate (60AGCi/Mmol) per dish and terminated by as- l piration of the medium and subsequent addition of 1 ml of 0.4 11,800 18,800 4,700 11,200 13,500 M HC104 to extract the intracellular purines. In some experi- ±5,600 ±7,700 ±2,000 ±2,000 ±5,500 ments the newly synthesized purines excreted into the medium A B C D E were also quantitated after precipitation of protein with 0.4 M FIG. 1. Rates of incorporation of ['4Clformate into acid-soluble HC104. In either case, the deproteinized solution was heated intracellular purines by normal, HPRT-, PNP-, and ADA- fibro- to 1000 for 1 hr, and the free purine bases were separated by blasts. The method is the standard assay as described in Materials chromatography on Dowex-50 and quantitated by liquid and Methods with medium supplemented with either 1096 dialyzed or fetal calf serum C). scintillation counting as described by Hershfield and Seegmiller (columns A, B, D, and E) undialyzed (column The results are a compilation of data obtained from multiple assays (22). Each time point required three dishes: two for duplicate using three normal fibroblast lines, four HPRT-deticient lines, one determination of the amount of ['14C]formate incorporated into PNP-deficient line, and two ADA-deficient lines. The mean +SD for total acid-soluble intracellular purines and one for the deter- each group of assays is indicated at the bottom of the appropriate column. Columns: A, normal; B, HPRT-; C, HPRT+; D, ADA-; E, mination- of cell numbers. PNP-. Cell Extracts and Enzyme Assays. ADA was assayed by a modification of the procedure of Snyder etal. (23) but with 100 mM Tris-HCl (pH 7.0) as buffer, which eliminated the further case, the rate of incorporation is 4-5 times greater for HPRT- conversion of the product inosine to hypoxanthine. PNP activity cells. PNP and ADA- fibroblasts showed. rates of ['4C]formate was measured by the same procedure, but with 100mM sodium incorporation that were diminished to the same extent as in phosphate (pH 7.4) as buffer and 300 AM f8-14C]inosine as control cells when the assays were conducted in medium sup- substrate. Protein concentrations were determined by the plemented with 10% undialyzed fetal calf serum (data not method of Lowry et al. (24). shown). In all four types of cell lines, the rate of incorporation Cell extracts were prepared from one 75-cm2 flask of con- of ['4C]formate was increased by an additional 25-60% when fluent fibroblasts. The cells were washed three times with a the amount of newly synthesized purines excreted into the buffer containing 0.04 M sodium Hepes (pH 7.4), 0.13 M NaCl, medium was also examined. The proportion of newly synthe- 0.4% bovine serum albumin, 100AM MgCl2, and 10 gM CaC12, sized purines excreted in the medium was approximately the and were lysed by freezing and thawing three times in liquid same in all four types of cell lines, but varied from experiment nitrogen.
Load more

Recommended publications
  • 35 Disorders of Purine and Pyrimidine Metabolism
    35 Disorders of Purine and Pyrimidine Metabolism Georges van den Berghe, M.- Françoise Vincent, Sandrine Marie 35.1 Inborn Errors of Purine Metabolism – 435 35.1.1 Phosphoribosyl Pyrophosphate Synthetase Superactivity – 435 35.1.2 Adenylosuccinase Deficiency – 436 35.1.3 AICA-Ribosiduria – 437 35.1.4 Muscle AMP Deaminase Deficiency – 437 35.1.5 Adenosine Deaminase Deficiency – 438 35.1.6 Adenosine Deaminase Superactivity – 439 35.1.7 Purine Nucleoside Phosphorylase Deficiency – 440 35.1.8 Xanthine Oxidase Deficiency – 440 35.1.9 Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency – 441 35.1.10 Adenine Phosphoribosyltransferase Deficiency – 442 35.1.11 Deoxyguanosine Kinase Deficiency – 442 35.2 Inborn Errors of Pyrimidine Metabolism – 445 35.2.1 UMP Synthase Deficiency (Hereditary Orotic Aciduria) – 445 35.2.2 Dihydropyrimidine Dehydrogenase Deficiency – 445 35.2.3 Dihydropyrimidinase Deficiency – 446 35.2.4 Ureidopropionase Deficiency – 446 35.2.5 Pyrimidine 5’-Nucleotidase Deficiency – 446 35.2.6 Cytosolic 5’-Nucleotidase Superactivity – 447 35.2.7 Thymidine Phosphorylase Deficiency – 447 35.2.8 Thymidine Kinase Deficiency – 447 References – 447 434 Chapter 35 · Disorders of Purine and Pyrimidine Metabolism Purine Metabolism Purine nucleotides are essential cellular constituents 4 The catabolic pathway starts from GMP, IMP and which intervene in energy transfer, metabolic regula- AMP, and produces uric acid, a poorly soluble tion, and synthesis of DNA and RNA. Purine metabo- compound, which tends to crystallize once its lism can be divided into three pathways: plasma concentration surpasses 6.5–7 mg/dl (0.38– 4 The biosynthetic pathway, often termed de novo, 0.47 mmol/l). starts with the formation of phosphoribosyl pyro- 4 The salvage pathway utilizes the purine bases, gua- phosphate (PRPP) and leads to the synthesis of nine, hypoxanthine and adenine, which are pro- inosine monophosphate (IMP).
    [Show full text]
  • Purine Metabolism in Cultured Endothelial Cells
    PURINE METABOLISM IN MAN-III Biochemical, Immunological, and Cancer Research Edited by Aurelio Rapado Fundacion Jimenez Diaz Madrid, Spain R.W.E. Watts M.R.C. Clinical Research Centre Harrow, England and Chris H.M.M. De Bruyn Department of Human Genetics University of Nijmegen Faculty of Medicine Nijmegen, The Netherlands PLENUM PRESS · NEW YORK AND LONDON Contents of Part Β I. PURINE METABOLISM PATHWAYS AND REGULATION A. De Novo Synthesis; Precursors and Regulation De Novo Purine Synthesis in Cultured Human Fibroblasts 1 R.B. Gordon, L. Thompson, L.A. Johnson, and B.T. Emmerson Comparative Metabolism of a New Antileishmanial Agent, Allopurinol Riboside, in the Parasite and the Host Cell 7 D. J. Nelson, S.W. LaFon, G.B. Elion, J.J. Marr, and R.L. Berens Purine Metabolism in Rat Skeletal Muscle 13 E. R. Tully and T.G. Sheehan Alterations in Purine Metabolism in Cultured Fibroblasts with HGPRT Deficiency and with PRPPP Synthetase Superactivity 19 E. Zoref-Shani and 0. Sperling Purine Metabolism in Cultured Endothelial Cells 25 S. Nees, A.L. Gerbes, B. Willershausen-Zönnchen, and E. Gerlach Determinants of 5-Phosphoribosyl-l-Pyrophosphate (PRPP) Synthesis in Human Fibroblasts 31 K.0, Raivio, Ch. Lazar, H. Krumholz, and M.A. Becker Xanthine Oxidoreductase Inhibition by NADH as a Regulatory Factor of Purine Metabolism 35 M.M. Jezewska and Z.W. Kaminski vii viii CONTENTS OF PART Β Β. Nucleotide Metabolism Human Placental Adenosine Kinase: Purification and Characterization 41 CM. Andres, T.D. Palella, and I.H. Fox Long-Term Effects of Ribose on Adenine Nucleotide Metabolism in Isoproterenol-Stimulated Hearts .
    [Show full text]
  • Metabolomics Identifies Pyrimidine Starvation As the Mechanism of 5-Aminoimidazole-4-Carboxamide-1- Β-Riboside-Induced Apoptosis in Multiple Myeloma Cells
    Published OnlineFirst April 12, 2013; DOI: 10.1158/1535-7163.MCT-12-1042 Molecular Cancer Cancer Therapeutics Insights Therapeutics Metabolomics Identifies Pyrimidine Starvation as the Mechanism of 5-Aminoimidazole-4-Carboxamide-1- b-Riboside-Induced Apoptosis in Multiple Myeloma Cells Carolyne Bardeleben1, Sanjai Sharma1, Joseph R. Reeve3, Sara Bassilian3, Patrick Frost1, Bao Hoang1, Yijiang Shi1, and Alan Lichtenstein1,2 Abstract To investigate the mechanism by which 5-aminoimidazole-4-carboxamide-1-b-riboside (AICAr) induces apoptosis in multiple myeloma cells, we conducted an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26-fold increase in orotate associated with a decrease in uridine monophosphate (UMP) levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and 5-phosphoribosyl- a-pyrophosphate (PRPP). As all pyrimidine nucleotides can be synthesized from UMP, this suggested that the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine, and thymidine, but not purines adenosine or guanosine, rescued multiple myeloma cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan, or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back.
    [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]
  • Adenosine Deaminase (ADA) Isoenzymes ADA1 and ADA2 in Biological Fluids
    Eur Respir J 1997; 10: 2186–2187 Copyright ERS Journals Ltd 1997 DOI: 10.1183/09031936.97.10092186 European Respiratory Journal Printed in UK - all rights reserved ISSN 0903 - 1936 CORRESPONDENCE Adenosine deaminase (ADA) isoenzymes ADA1 and ADA2 in biological fluids To the Editor: readily obtained by measuring total ADA and the ADA2 which is not inhibited by 100 mmol·L-1 of added EHNA. We read with great interest the editorial by GAKIS [1] In fact, this method is being increasingly used by acquired about the extreme importance of the adenosine deami- immune deficiency syndrome (AIDS) researchers since nase (ADA) isoenzymes ADA1 and ADA2 on the some authors have reported a higher serum ADA activ- homeostasis of 2' deoxyadenosine and adenosine, espe- ity in human immunodeficiency virus (HIV)-1 positive cially when monocytes and macrophages are infected patients [4]. By the method we propose, some authors by intracellular microorganisms. Serum ADA activity have found that ADA2 isoenzyme activity is of con- is increased in various conditions such as liver disease, siderable prognostic value in AIDS and adult T-cell tuberculosis, typhoid, infective mononucleosis and cer- leukaemia (ATL) cases [5, 6]. tain malignancies, especially those of haematopoietic origin. The origin of serum ADA and the mechanisms by which serum activities are increased have not been References fully elucidated. 1. Gakis C. Adenosine deaminase (ADA) isoenzymes ADA1 Whatever the biological role of ADA1 and ADA2, it and ADA2: diagnostic and biological role. Eur Respir has been demonstrated that the presence (low or high) J 1996; 632–633. of these isoenzymes in biological fluids has diagnostic 2.
    [Show full text]
  • Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme
    biomolecules Review Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme Francesca Ronca * and Antonio Raggi Laboratory of Biochemistry, Department of Pathology, University of Pisa, via Roma 55, 56126 Pisa, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-050-2218-273; Fax: +39-050-2218-660 Received: 19 July 2018; Accepted: 20 August 2018; Published: 23 August 2018 Abstract: Multiple muscle-specific isoforms of the Zn2+ metalloenzyme AMP deaminase (AMPD) have been identified based on their biochemical and genetic differences. Our previous observations suggested that the metal binding protein histidine-proline-rich glycoprotein (HPRG) participates in the assembly and maintenance of skeletal muscle AMP deaminase (AMPD1) by acting as a zinc chaperone. The evidence of a role of millimolar-strength phosphate in stabilizing the AMPD-HPRG complex of both AMPD1 and cardiac AMP deaminase (AMPD3) is suggestive of a physiological mutual dependence between the two subunit components with regard to the stability of the two isoforms of striated muscle AMPD. The observed influence of the HPRG content on the catalytic behavior of the two enzymes further strengthens this hypothesis. Based on the preferential localization of HPRG at the sarcomeric I-band and on the presence of a Zn2+ binding motif in the N-terminal regions of fast TnT and of the AMPD1 catalytic subunit, we advance the hypothesis that the Zn binding properties of HPRG could promote the association of AMPD1 to the thin filament. Keywords: AMP deaminase (AMPD); histidine-proline-rich glycoprotein (HPRG); striated muscle; Troponin T (TnT) 1.
    [Show full text]
  • STRIPE2 Encodes a Putative Dcmp Deaminase That Plays an Important Role in Chloroplast Development in Rice
    Available online at www.sciencedirect.com ScienceDirect JGG Journal of Genetics and Genomics 41 (2014) 539e548 ORIGINAL RESEARCH STRIPE2 Encodes a Putative dCMP Deaminase that Plays an Important Role in Chloroplast Development in Rice Jing Xu a, Yiwen Deng a, Qun Li a, Xudong Zhu b,*, Zuhua He a,* a National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China b China National Rice Research Institute, Hangzhou 31006, China Received 31 March 2014; revised 8 May 2014; accepted 9 May 2014 Available online 19 June 2014 ABSTRACT Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the g-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination.
    [Show full text]
  • ADAR RNA Editing Below the Backbone
    Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW ADAR RNA editing below the backbone LIAM KEEGAN, ANZER KHAN, DRAGANA VUKIC, and MARY O’CONNELL CEITEC at Masaryk University Brno, Pavilion A35, Brno CZ-62500, Czech Republic ABSTRACT ADAR RNA editing enzymes (adenosine deaminases acting on RNA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster, which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain–RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems. Keywords: ADAR; Drosophila melanogaster; RNA editing; dsRNA; RNA modification; epitranscriptome INTRODUCTION tion detected by standard RNA-seq. Therefore, studies on ADAR RNA editing began much earlier and they now also ADARs: promiscuous and site-specific RNA editing lead the way toward understanding the effects of a range of other enzymatic modifications that have been found more re- ADARs (adenosine deaminases acting on RNA) were dis- cently in mRNA (O’Connell et al.
    [Show full text]
  • Identification of ADAR1 Adenosine Deaminase Dependency in a Subset
    ARTICLE https://doi.org/10.1038/s41467-018-07824-4 OPEN Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells Hugh S. Gannon1,2, Tao Zou1,2, Michael K. Kiessling3,4, Galen F. Gao 2, Diana Cai1,2, Peter S. Choi1,2, Alexandru P. Ivan 1, Ilana Buchumenski5, Ashton C. Berger2, Jonathan T. Goldstein2, Andrew D. Cherniack 1,2, Francisca Vazquez 2, Aviad Tsherniak 2, Erez Y. Levanon 5, William C. Hahn 1,2,6,7 & Matthew Meyerson 1,2,7,8 1234567890():,; Systematic exploration of cancer cell vulnerabilities can inform the development of novel cancer therapeutics. Here, through analysis of genome-scale loss-of-function datasets, we identify adenosine deaminase acting on RNA (ADAR or ADAR1) as an essential gene for the survival of a subset of cancer cell lines. ADAR1-dependent cell lines display increased expression of interferon-stimulated genes. Activation of type I interferon signaling in the context of ADAR1 deficiency can induce cell lethality in non-ADAR1-dependent cell lines. ADAR deletion causes activation of the double-stranded RNA sensor, protein kinase R (PKR). Disruption of PKR signaling, through inactivation of PKR or overexpression of either a wild- type or catalytically inactive mutant version of the p150 isoform of ADAR1, partially rescues cell lethality after ADAR1 loss, suggesting that both catalytic and non-enzymatic functions of ADAR1 may contribute to preventing PKR-mediated cell lethality. Together, these data nominate ADAR1 as a potential therapeutic target in a subset of cancers. 1 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
    [Show full text]
  • Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells'
    [CANCER RESEARCH 33, 94-103, January 1973] Identification of the Enzymatic Pathways of Nucleotide Metabolism in Human Lymphocytes and Leukemia Cells' E. M. Scholar and P. Calabresi Department of Medicine, The Roger Williams General Hospital, Providence, Rhode Island 02908, and The Division of Biological and Medical Sciences,Brown University, Providence,Rhode Island 02912 SUMMARY monocytes, and lymphocytes, it is difficult to know in what particular fraction an enzyme activity is present. It would therefore be advantageous to separate out the different Extracts of lymphocytes from normal donors and from components of the leukocyte fraction before investigating patients with chronic lymphocytic leukemia (CLL) and acute their enzymatic activities. All previously reported work on the lymphoblastic leukemia were examined for a variety of enzymes of purine nucleotide metabolism was done at best in enzymes with activity for purine nucleotide biosynthesis, the whole white blood cell fraction. Those enzymes found to interconversion, and catabolism as well as for a selected be present included adenine and guanine phosphoribosyltrans number of enzymes involved in pyrimidine nucleotide ferase (2, 32), PNPase2 (7), deoxyadenosine deaminase (7), metabolism. Lymphocytes from all three donor types (normal, ATPase (4), and inosine kinase (21). CLL, acute lymphocytic leukemia) contained the following A detailed knowledge of the enzymatic pathways of purine enzymatic activities: adenine and guanine phosphoribosyl and pyrimidine nucleotide metabolism in normal lymphocytes transferase , adenosine kinase , nucieoside diphosphate kinase, and leukemia cells is important in elucidating any biochemical adenylate kinase, guanylate kinase, cytidylate kinase, uridylate differences that may exist. Such differences may be exploited kinase, adenosine deaminase, purine nucleoside phosphorylase, in chemotherapy and in gaining and understanding of the and adenylate deaminase (with ATP).
    [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]
  • Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases
    molecules Review Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases Barbara Kutryb-Zajac * , Paulina Mierzejewska, Ewa M. Slominska and Ryszard T. Smolenski * Department of Biochemistry, Medical University of Gdansk, Debinki 1 St., 80-211 Gdansk, Poland; [email protected] (P.M.); [email protected] (E.M.S.) * Correspondence: [email protected] (B.K.-Z); [email protected] (R.T.S.); Tel.: +48-58-349-14-64 (B.K.-Z.); +48-58-349-14-60 (R.T.S.) Academic Editors: Luigi A. Agrofoglio, Vincent Roy and Cyril Nicolas Received: 31 August 2020; Accepted: 7 October 2020; Published: 12 October 2020 Abstract: Adenosine deaminase (ADA) is an enzyme of purine metabolism that irreversibly converts adenosine to inosine or 20deoxyadenosine to 20deoxyinosine. ADA is active both inside the cell and on the cell surface where it was found to interact with membrane proteins, such as CD26 and adenosine receptors, forming ecto-ADA (eADA). In addition to adenosine uptake, the activity of eADA is an essential mechanism that terminates adenosine signaling. This is particularly important in cardiovascular system, where adenosine protects against endothelial dysfunction, vascular inflammation, or thrombosis. Besides enzymatic function, ADA protein mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. Furthermore, alteration in ADA activity was demonstrated in many cardiovascular pathologies such as atherosclerosis, myocardial ischemia-reperfusion injury, hypertension, thrombosis, or diabetes. Modulation of ADA activity could be an important therapeutic target. This work provides a systematic review of ADA activity and anchoring inhibitors as well as summarizes the perspectives of their therapeutic use in cardiovascular pathologies associated with increased activity of ADA.
    [Show full text]