In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFβ and other cellular processes.

Department of Biological Sciences University of Denver Denver CO 80210 USA

Eleanor Roosevelt Institute University of Denver Denver CO 80210 USA

Knoebel Institute for Healthy Aging University of Denver 2155 E Wesley Avenue Denver CO 80210 USA

Molecular and Cellular Biophysics Program University of Denver Denver CO 80210 USA

Research Unit for Rare Diseases Department of Pediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

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Saxton R.A., Sabatini D.M. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–976. doi: 10.1016/j.cell.2017.02.004. PubMed DOI PMC

Jeon S.-M. Regulation and function of AMPK in physiology and diseases. Exp. Mol. Med. 2016;48:e245. doi: 10.1038/emm.2016.81. PubMed DOI PMC

Meares G.P., Qin H., Liu Y., Holdbrooks A.T., Benveniste E.N. AMP-activated protein kinase restricts IFN-γ signaling. J. Immunol. 2013;190:372–380. doi: 10.4049/jimmunol.1202390. PubMed DOI PMC

Garcia D., Shaw R.J. AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance. Mol. Cell. 2017;66:789–800. doi: 10.1016/j.molcel.2017.05.032. PubMed DOI PMC

Hardie D.G. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. 2011;25:1895–1908. doi: 10.1101/gad.17420111. PubMed DOI PMC

Cantó C., Jiang L.Q., Deshmukh A.S., Mataki C., Coste A., Lagouge M. Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle. Cell Metab. 2010;11:213–219. doi: 10.1016/j.cmet.2010.02.006. PubMed DOI PMC

Hawley S.A., Selbert M.A., Goldstein E.G., Edelman A.M., Carling D., Hardie D.G. 5’-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J. Biol. Chem. 1995;270:27186–27191. doi: 10.1074/jbc.270.45.27186. PubMed DOI

Davies S.P., Helps N.R., Cohen P.T., Hardie D.G. 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett. 1995;377:421–425. doi: 10.1016/0014-5793(95)01368-7. PubMed DOI

Corton J.M., Gillespie J.G., Hawley S.A., Hardie D.G. 5-Aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur. J. Biochem. 1995;229:558–565. doi: 10.1111/j.1432-1033.1995.tb20498.x. PubMed DOI

Douillet D.C., Pinson B., Ceschin J., Hürlimann H.C., Saint-Marc C., Laporte D. Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR. J. Biol. Chem. 2018;294 doi: 10.1074/jbc.RA118.004964. jbc.RA118.004964–815. PubMed DOI PMC

Kirchner J., Brüne B., Namgaladze D. AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages. Sci. Rep. 2018;8:7801. doi: 10.1038/s41598-018-26102-3. PubMed DOI PMC

Guigas B., Bertrand L., Taleux N., Foretz M., Wiernsperger N., Vertommen D. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase–independent effect on glucokinase translocation. Diabetes. 2006;55:865–874. doi: 10.2337/diabetes.55.04.06.db05-1178. PubMed DOI

Spurr I.B., Birts C.N., Cuda F., Benkovic S.J., Blaydes J.P., Tavassoli A. Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization. Chembiochem. 2012;13:1628–1634. doi: 10.1002/cbic.201200279. PubMed DOI PMC

Asby D.J., Cuda F., Beyaert M., Houghton F.D., Cagampang F.R., Tavassoli A. AMPK activation via modulation of de novo purine biosynthesis with an inhibitor of ATIC Homodimerization. Chem. Biol. 2015;22:838–848. doi: 10.1016/j.chembiol.2015.06.008. PubMed DOI

Marie S., Heron B., Bitoun P., Timmerman T., Van Den Berghe G., Vincent M.-F. AICA-ribosiduria: a novel, neurologically devastating inborn error of purine biosynthesis caused by mutation of ATIC. Am. J. Hum. Genet. 2004;74:1276–1281. doi: 10.1086/421475. PubMed DOI PMC

Baresova V., Krijt M., Skopova V., Souckova O., Kmoch S., Zikánová M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. Mol. Genet. Metab. 2016;119:270–277. doi: 10.1016/j.ymgme.2016.08.004. PubMed DOI

Holmes E.W., McDonald J.A., McCord J.M., Wyngaarden J.B., Kelley W.N. Human glutamine phosphoribosylpyrophosphate amidotransferase. Kinetic and regulatory properties. J. Biol. Chem. 1973;248:144–150. PubMed

Tu A.S., Patterson D. Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity. J. Cell. Physiol. 1978;96:123–132. doi: 10.1002/jcp.1040960115. PubMed DOI

Mazzarino R.C., Baresova V., Zikánová M., Duval N., Wilkinson T.G., Patterson D. The CRISPR-Cas9 crADSL HeLa transcriptome: a first step in establishing a model for ADSL deficiency and SAICAR accumulation. Mol. Genet. Metab. Rep. 2019;21:100512. doi: 10.1016/j.ymgmr.2019.100512. PubMed DOI PMC

Mlecnik B., Galon J., Bindea G. Comprehensive functional analysis of large lists of genes and proteins. J. Proteome. 2018;171:2–10. doi: 10.1016/j.jprot.2017.03.016. PubMed DOI

Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. doi: 10.1101/gr.1239303. PubMed DOI PMC

Ashburner M., Ball C.A., Blake J.A., Botstein D., Butler H., Cherry J.M. Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat. Genet. 2000;25:25–29. doi: 10.1038/75556. PubMed DOI PMC

Fabregat A., Jupe S., Matthews L., Sidiropoulos K., Gillespie M., Garapati P. The reactome pathway knowledgebase. Nucleic Acids Res. 2018;46:D649–D655. doi: 10.1093/nar/gkx1132. PubMed DOI PMC

Maere S., Heymans K., Kuiper M. BiNGO: a cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005;21:3448–3449. doi: 10.1093/bioinformatics/bti551. PubMed DOI

Silva C.H.T.P., Silva M., Iulek J., Thiemann O.H. Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism. J. Biomol. Struct. Dyn. 2008;25:589–597. doi: 10.1080/07391102.2008.10507205. PubMed DOI

Watts R.W. Molecular variation in relation to purine metabolism. J. Clin. Pathol. Suppl. (R Coll Pathol.) 1974;8:48–63. PubMed PMC

Reznick R.M., Zong H., Li J., Morino K., Moore I.K., Yu H.J. Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis. Cell Metab. 2007;5:151–156. doi: 10.1016/j.cmet.2007.01.008. PubMed DOI PMC

Vingtdeux V., Davies P., Dickson D.W., Marambaud P. AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer’s disease and other tauopathies. Acta Neuropathol. 2010;121:337–349. doi: 10.1007/s00401-010-0759-x. PubMed DOI PMC

Erol A. An integrated and unifying hypothesis for the metabolic basis of sporadic Alzheimer’s disease. J. Alzheimers Dis. 2008;13:241–253. doi: 10.3233/jad-2008-13302. PubMed DOI

Cai Z., Yan L.-J., Li K., Quazi S.H., Zhao B. Roles of AMP-activated protein kinase in Alzheimer’s disease. NeuroMolecular Med. 2012;14:1–14. doi: 10.1007/s12017-012-8173-2. PubMed DOI

Caberlotto L., Lauria M., Nguyen T.-P., Scotti M. The central role of AMP-kinase and energy homeostasis impairment in Alzheimer’s disease: a multifactor network analysis. PLoS One. 2013;8 doi: 10.1371/journal.pone.0078919. PubMed DOI PMC

Kinney J.W., Bemiller S.M., Murtishaw A.S., Leisgang A.M., Salazar A.M., Lamb B.T. Inflammation as a central mechanism in Alzheimer's disease. Alzheimers Dement (N. Y.) 2018;4:575–590. doi: 10.1016/j.trci.2018.06.014. PubMed DOI PMC

Thomas M.H., Pelleieux S., Vitale N., Olivier J.L. Arachidonic acid in Alzheimer's disease. J. Neurol. Neuromed. 2016;1:1–6.

Wang X., Zhu M., Hjorth E., Cortés-Toro V., Eyjolfsdottir H., Graff C. Resolution of inflammation is altered in Alzheimer's disease. Alzheimers Dement. 2015;11:40–50. doi: 10.1016/j.jalz.2013.12.024. e1–2. PubMed DOI PMC

Whittington R.A., Planel E., Terrando N. Impaired resolution of inflammation in Alzheimer’s disease: a review. Front. Immunol. 2017;8:1464. doi: 10.3389/fimmu.2017.01464. PubMed DOI PMC

Serhan C.N., Chiang N., Dalli J., Levy B.D. Lipid mediators in the resolution of inflammation. Cold Spring Harb. Perspect. Biol. 2014;7:a016311. doi: 10.1101/cshperspect.a016311. PubMed DOI PMC

Di Scala C., Fantini J., Yahi N., Barrantes F.J., Chahinian H. Anandamide revisited: how cholesterol and ceramides control receptor-dependent and receptor-independent signal transmission pathways of a lipid neurotransmitter. Biomolecules. 2018;8 doi: 10.3390/biom8020031. PubMed DOI PMC

Ritter J.K., Li G., Xia M., Boini K. Anandamide and its metabolites: what are their roles in the kidney? Front. Biosci. (Schol. Ed.) 2016;8:264–277. doi: 10.2741/s461. PubMed DOI PMC

Hanna V.S., Hafez E.A.A. Synopsis of arachidonic acid metabolism: a review. J. Adv. Res. 2018;11:23–32. doi: 10.1016/j.jare.2018.03.005. PubMed DOI PMC

Stephenson D.T., Lemere C.A., Selkoe D.J., Clemens J.A. Cytosolic phospholipase A2 (cPLA2) immunoreactivity is elevated in Alzheimer’s disease brain. Neurobiol. Dis. 1996;3:51–63. doi: 10.1006/nbdi.1996.0005. PubMed DOI

Ho L., Purohit D., Haroutunian V., Luterman J.D., Willis F., Naslund J. Neuronal cyclooxygenase 2 expression in the hippocampal formation as a function of the clinical progression of Alzheimer disease. Arch. Neurol. 2001;58:487–492. doi: 10.1001/archneur.58.3.487. PubMed DOI

Cui J.-G., Kuroda H., Chandrasekharan N.V., Pelaez R.P., Simmons D.L., Bazan N.G. Cyclooxygenase-3 gene expression in Alzheimer hippocampus and in stressed human neural cells. Neurochem. Res. 2004;29:1731–1737. doi: 10.1023/b:nere.0000035809.70905.8a. PubMed DOI

Mohri I., Kadoyama K., Kanekiyo T., Sato Y., Kagitani-Shimono K., Saito Y. Hematopoietic prostaglandin D synthase and DP1 receptor are selectively upregulated in microglia and astrocytes within senile plaques from human patients and in a mouse model of Alzheimer disease. J. Neuropathol. Exp. Neurol. 2007;66:469–480. doi: 10.1097/01.jnen.0000240472.43038.27. PubMed DOI

McGeer P.L., McGeer E.G. NSAIDs and Alzheimer disease: epidemiological, animal model and clinical studies. Neurobiol. Aging. 2007;28:639–647. doi: 10.1016/j.neurobiolaging.2006.03.013. PubMed DOI

Wu Y., Zhai H., Wang Y., Li L., Wu J., Wang F. Aspirin-triggered Lipoxin A4 attenuates lipopolysaccharide-induced intracellular ROS in BV2 microglia cells by inhibiting the function of NADPH oxidase. Neurochem. Res. 2012;37:1690–1696. doi: 10.1007/s11064-012-0776-3. PubMed DOI

Decker Y., McBean G., Godson C. Lipoxin A4 inhibits IL-1beta-induced IL-8 and ICAM-1 expression in 1321N1 human astrocytoma cells. Am. J. Phys. Cell Physiol. 2009;296 doi: 10.1152/ajpcell.00380.2008. C1420–7. PubMed DOI

Medeiros R., Kitazawa M., Passos G.F., Baglietto-Vargas D., Cheng D., Cribbs D.H. Aspirin-triggered lipoxin A4 stimulates alternative activation of microglia and reduces Alzheimer disease-like pathology in mice. Am. J. Pathol. 2013;182:1780–1789. doi: 10.1016/j.ajpath.2013.01.051. PubMed DOI PMC

Zhu M., Wang X., Hjorth E., Colas R.A., Schroeder L., Granholm A.-C. Pro-resolving lipid mediators improve neuronal survival and increase Aβ42 phagocytosis. Mol. Neurobiol. 2016;53:2733–2749. doi: 10.1007/s12035-015-9544-0. PubMed DOI PMC

Kettenmann H., Hanisch U.-K., Noda M., Verkhratsky A. Physiology of microglia. Physiol. Rev. 2011;91:461–553. doi: 10.1152/physrev.00011.2010. PubMed DOI

Carniglia L., Ramírez D., Durand D., Saba J., Turati J., Caruso C. Neuropeptides and microglial activation in inflammation, pain, and neurodegenerative diseases. Mediat. Inflamm. 2017;2017:1–23. doi: 10.1155/2017/5048616. PubMed DOI PMC

Koellhoffer E.C., McCullough L.D., Ritzel R.M. Old maids: aging and its impact on microglia function. Int. J. Mol. Sci. 2017;18 doi: 10.3390/ijms18040769. PubMed DOI PMC

Paglinawan R., Malipiero U., Schlapbach R., Frei K., Reith W., Fontana A. TGFbeta directs gene expression of activated microglia to an anti-inflammatory phenotype strongly focusing on chemokine genes and cell migratory genes. Glia. 2003;44:219–231. doi: 10.1002/glia.10286. PubMed DOI

Lieb K., Engels S., Fiebich B.L. Inhibition of LPS-induced iNOS and NO synthesis in primary rat microglial cells. Neurochem. Int. 2003;42:131–137. doi: 10.1016/s0197-0186(02)00076-1. PubMed DOI

Wyss-Coray T., Lin C., Yan F., Yu G.Q., Rohde M., McConlogue L. TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat. Med. 2001;7:612–618. doi: 10.1038/87945. PubMed DOI

Ritzel R.M., Patel A.R., Pan S., Crapser J., Hammond M., Jellison E. Age- and location-related changes in microglial function. Neurobiol. Aging. 2015;36:2153–2163. doi: 10.1016/j.neurobiolaging.2015.02.016. PubMed DOI

Ramírez G., Rey S., von Bernhardi R. Proinflammatory stimuli are needed for induction of microglial cell-mediated AbetaPP_{244-C} and Abeta-neurotoxicity in hippocampal cultures. J. Alzheimers Dis. 2008;15:45–59. doi: 10.3233/jad-2008-15104. PubMed DOI

von Bernhardi R., Ramírez G., Toro R., Eugenín J. Pro-inflammatory conditions promote neuronal damage mediated by amyloid precursor protein and decrease its phagocytosis and degradation by microglial cells in culture. Neurobiol. Dis. 2007;26:153–164. doi: 10.1016/j.nbd.2006.12.006. PubMed DOI

Colangelo V., Schurr J., Ball M.J., Pelaez R.P., Bazan N.G., Lukiw W.J. Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: transcription and neurotrophic factor down-regulation and up-regulation of apoptotic and pro-inflammatory signaling. J. Neurosci. Res. 2002;70:462–473. doi: 10.1002/jnr.10351. PubMed DOI

Tichauer J.E., Flores B., Soler B., Eugenín-von Bernhardi L., Ramírez G., von Bernhardi R. Age-dependent changes on TGFβ1 Smad3 pathway modify the pattern of microglial cell activation. Brain Behav. Immun. 2014;37:187–196. doi: 10.1016/j.bbi.2013.12.018. PubMed DOI PMC

Derynck R., Zhang Y.E. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–584. doi: 10.1038/nature02006. PubMed DOI

Flores B., von Bernhardi R. Transforming growth factor beta 1 modulates amyloid beta-induced glial activation through the Smad3-dependent induction of MAPK phosphatase-1. J. Alzheimers Dis. 2012;32:417–429. PubMed

Massé K., Dale N. Purines as potential morphogens during embryonic development. Purinergic Signal. 2012;8:503–521. doi: 10.1007/s11302-012-9290-y. PubMed DOI PMC

Zimmermann H. Purinergic signaling in neural development. Semin. Cell Dev. Biol. 2011;22:194–204. doi: 10.1016/j.semcdb.2011.02.007. PubMed DOI

Fumagalli M., Lecca D., Abbracchio M.P., Ceruti S. Pathophysiological role of purines and pyrimidines in neurodevelopment: unveiling new pharmacological approaches to congenital brain diseases. Front. Pharmacol. 2017;8:941. doi: 10.3389/fphar.2017.00941. PubMed DOI PMC

Rodrigues R.J., Marques J.M., Cunha R.A. Purinergic signalling and brain development. Semin. Cell Dev. Biol. 2019;95:34–41. doi: 10.1016/j.semcdb.2018.12.001. PubMed DOI

Chan C.Y., Zhao H., Pugh R.J., Pedley A.M., French J., Jones S.A. Purinosome formation as a function of the cell cycle. Proc. Natl. Acad. Sci. 2015 doi: 10.1073/pnas.1423009112. PubMed DOI PMC

Zhao H., Chiaro C.R., Zhang L., Smith P.B., Chan C.Y., Pedley A.M. Quantitative analysis of purine nucleotides indicates purinosomes increase de novo purine biosynthesis. J. Biol. Chem. 2015 doi: 10.1074/jbc.M114.628701. PubMed DOI PMC

Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis

Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity