title: Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders.
abstract: Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.
authors: Eleanor Roosevelt Institute and Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Denver, CO 80208, USA.
authors: Nathan
authors: N
authors: Duval
authors: Eleanor Roosevelt Institute and Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Denver, CO 80208, USA.
authors: Kyleen
authors: K
authors: Luhrs
authors: Eleanor Roosevelt Institute and Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Denver, CO 80208, USA.
authors: Terry G
authors: TG
authors: Wilkinson
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, 120 00 Prague 2, Czech Republic.
authors: Veronika
authors: V
authors: Baresova
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, 120 00 Prague 2, Czech Republic.
authors: Vaclava
authors: V
authors: Skopova
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, 120 00 Prague 2, Czech Republic.
authors: Stanislav
authors: S
authors: Kmoch
authors: Eleanor Roosevelt Institute and Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Denver, CO 80208, USA.
authors: Guido N
authors: GN
authors: Vacano
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, 120 00 Prague 2, Czech Republic.
authors: Marie
authors: M
authors: Zikanova
authors: Eleanor Roosevelt Institute and Department of Biological Sciences, University of Denver, 2101 E. Wesley Ave., Denver, CO 80208, USA.
authors: David
authors: D
authors: Patterson
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D055664:Electrochemical Techniques; D055432:Metabolomics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism; D011687:Purines / Q000096:biosynthesis*; D016415:Sequence Alignment
chemical_list: D011687:Purines; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D010453:Peptide Synthases; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase
references: Gene. 2009 Jan 15;429(1-2):23-30
references: 19007868
references: Hum Mol Genet. 2012 Apr 1;21(7):1534-43
references: 22180458
references: Biochemistry. 1992 Jun 2;31(21):5022-32
references: 1534690
references: Cell. 1980 Jul;20(3):797-805
references: 7418008
references: Cell. 2008 Aug 8;134(3):405-15
references: 18674809
references: Anal Biochem. 1993 Sep;213(2):323-8
references: 8238909
references: Bioinformatics. 2009 Oct 1;25(19):2537-43
references: 19654118
references: Hum Mutat. 2010 Apr;31(4):445-55
references: 20127976
references: Mol Genet Metab. 2008 Aug;94(4):435-42
references: 18524658
references: BMC Bioinformatics. 2011 May 13;12:151
references: 21569468
references: Methods Mol Biol. 2007;358:159-74
references: 17035686
references: J Biotechnol. 2011 Dec 10;156(3):227-35
references: 21945585
references: J Mol Biol. 2007 Mar 9;366(5):1603-14
references: 17224163
references: Bioorg Med Chem. 2009 May 1;17(9):3317-23
references: 19362848
references: J Mol Biol. 1990 Oct 5;215(3):403-10
references: 2231712
references: J Clin Invest. 1984 Oct;74(4):1422-7
references: 6480832
references: Hum Mol Genet. 1997 Nov;6(12):2043-50
references: 9328467
references: Gene. 1999 Nov 1;239(2):381-91
references: 10548741
references: Infect Immun. 1999 Jun;67(6):2867-73
references: 10338493
references: Cell. 2011 Feb 18;144(4):499-512
references: 21315436
references: Biochem Genet. 1977 Feb;15(1-2):195-210
references: 849250
references: Genes Dev. 2009 Jun 15;23(12):1399-407
references: 19528318
references: J Inherit Metab Dis. 2009 Apr;32(2):247-63
references: 19291420
references: J Biol Chem. 2005 Nov 25;280(47):39582-93
references: 16176927
references: Cancer Lett. 2004 Aug 20;212(1):83-93
references: 15246564
references: J Inherit Metab Dis. 2010 Feb;33(1):1-7
references: 20049533
references: J Biol Chem. 2012 Oct 19;287(43):36201-7
references: 22955281
references: Blood. 2004 Sep 1;104(5):1435-41
references: 15142881
references: Mol Genet Metab. 2011 Jan;102(1):61-8
references: 20884265
references: Development. 2009 Aug;136(15):2601-11
references: 19570845
references: Biochemistry. 1994 Feb 22;33(7):1850-60
references: 8110788
references: Science. 2008 Apr 4;320(5872):103-6
references: 18388293
references: Somatic Cell Genet. 1975 Jan;1(1):91-110
references: 1235902
references: Clin Chem. 1984 Sep;30(9):1477-88
references: 6147209