title: CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation.
abstract: Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors: Veronika
authors: V
authors: Baresova
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors: Matyas
authors: M
authors: Krijt
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors: Vaclava
authors: V
authors: Skopova
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors: Olga
authors: O
authors: Souckova
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic.
authors: Stanislav
authors: S
authors: Kmoch
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague, General University Hospital in Prague, Ke Karlovu 2, 128 08 Praha 2, Czech Republic. Electronic address: mzika@lf1.cuni.cz.
authors: Marie
authors: M
authors: Zikanova
mesh_terms: D064113:CRISPR-Cas Systems; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011687:Purines / Q000096:biosynthesis* / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D009097:Multienzyme Complexes; D011687:Purines; C030985:purine
keywords: AICA-ribosiduria; Adenylosuccinate lyase deficiency; CRISPR; De novo purine synthesis; Human cellular model; Purinosome