In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

• MeSH
• GAR-transformylasa metabolismus   genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• metabolom * MeSH
• puriny * metabolismus   biosyntéza   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• GAR-transformylasa MeSH
• purine MeSH Prohlížeč
• puriny * MeSH

The cellular pool of purines is maintained by de novo purine synthesis (DNPS), recycling and degradation. Mutations in genes encoding DNPS enzymes cause their substrates to accumulate, which has detrimental effects on cellular division and organism development, potentially leading to neurological impairments. Unspecified neurological symptoms observed in many patients could not be elucidated even by modern techniques. It is presumable that some of these problems are induced by dysfunctions in DNPS enzymes. Therefore, we determined the concentrations of dephosphorylated DNPS intermediates by LC-MS/MS as markers of yet unpublished mutations in PFAS and PAICS genes connected with dysfunctions of carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) or phosphoribosylformylglycinamidine synthase (PFAS). We determined the criteria for normal values of metabolites and investigated 1,447 samples of urine and 365 dried blood spots of patients suffering from various forms of neurological impairment. We detected slightly elevated aminoimidazole riboside (AIr) concentrations in three urine samples and a highly elevated 5-formamidoimidazole-4-carboxamide riboside (FGAr) concentration in one urine sample. The accumulation of AIr or FGAr in body fluids can indicate PAICS or PFAS deficiency, respectively, which would be new disorders of DNPS caused by mutations in the appropriate genes. Measurement of DNPS intermediates in patients with neurological symptoms can uncover the cause of serious cellular and functional impairments that are otherwise inaccessible to detection. Further genetic and molecular analysis of these patients should establish the causal mutations for prenatal diagnosis, genetic consultation, and reinforce the DNPS pathway as a therapeutic target.

• MeSH
• lidé MeSH
• limita detekce MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• moč MeSH
• mutace genetika   MeSH
• nemoci nervového systému genetika   metabolismus   MeSH
• puriny biosyntéza   krev   chemie   moč   MeSH
• test suché kapky krve MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• purine MeSH Prohlížeč
• puriny MeSH

We report for the first time an autosomal recessive inborn error of de novo purine synthesis (DNPS)-PAICS deficiency. We investigated two siblings from the Faroe Islands born with multiple malformations resulting in early neonatal death. Genetic analysis of affected individuals revealed a homozygous missense mutation in PAICS (c.158A>G; p.Lys53Arg) that affects the structure of the catalytic site of the bifunctional enzyme phosphoribosylaminoimidazole carboxylase (AIRC, EC 4.1.1.21)/phosphoribosylaminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS). The mutation reduced the catalytic activity of PAICS in heterozygous carrier and patient skin fibroblasts to approximately 50 and 10% of control levels, respectively. The catalytic activity of the corresponding recombinant enzyme protein carrying the mutation p.Lys53Arg expressed and purified from E. coli was reduced to approximately 25% of the wild-type enzyme. Similar to other two known DNPS defects-adenylosuccinate lyase deficiency and AICA-ribosiduria-the PAICS mutation prevented purinosome formation in the patient's skin fibroblasts, and this phenotype was corrected by transfection with the wild-type but not the mutated PAICS. Although aminoimidazole ribotide (AIR) and aminoimidazole riboside (AIr), the enzyme substrates that are predicted to accumulate in PAICS deficiency, were not detected in patient's fibroblasts, the cytotoxic effect of AIr on various cell lines was demonstrated. PAICS deficiency is a newly described disease that enhances our understanding of the DNPS pathway and should be considered in the diagnosis of families with recurrent spontaneous abortion or early neonatal death.

• MeSH
• adenylsukcinátlyasa nedostatek   MeSH
• autistická porucha MeSH
• fatální výsledek MeSH
• fenotyp MeSH
• karboxylyasy genetika   metabolismus   MeSH
• lidé MeSH
• mnohočetné abnormality genetika   MeSH
• mutace MeSH
• novorozenec MeSH
• peptidsynthasy genetika   metabolismus   MeSH
• perinatální smrt MeSH
• poruchy metabolismu purinů a pyrimidinů MeSH
• puriny biosyntéza   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Dánsko MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• karboxylyasy MeSH
• peptidsynthasy MeSH
• phosphoribosylaminoimidazole carboxylase MeSH Prohlížeč
• phosphoribosylaminoimidazole-succinocarboxamide synthetase MeSH Prohlížeč
• puriny MeSH

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.

• MeSH
• chromatografie kapalinová MeSH
• CRISPR-Cas systémy MeSH
• DNA biosyntéza   chemie   MeSH
• editace genu MeSH
• HeLa buňky MeSH
• lidé MeSH
• puriny biosyntéza   chemie   MeSH
• RNA biosyntéza   chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• purine MeSH Prohlížeč
• puriny MeSH
• RNA MeSH

BACKGROUND: The enzymes involved in de novo purine synthesis (DNPS), one of the basic processes in eukaryotic cells, transiently and reversibly form a dynamic multienzyme complex called the purinosome in the cytoplasm. The purinosome has been observed in a broad spectrum of cells, but some studies claim that it is an artefact of the constructs used for visualization or stress granules resulting from the exposure of cells to nutrient-reduced growth media. Both may be true depending on the method of observation. To clarify this point, we combined two previously used methods, transfection and immunofluorescence, to detect purinosomes in purinosome-free cells deficient in particular DNPS steps (CR-DNPS cells) and in cells deficient in the salvage pathway, which resulted in construction of the purinosome regardless of purine level (CR-HGPRT cells). METHODS AND FINDINGS: To restore or disrupt purinosome formation, we transiently transfected CR-DNPS and CR-HGPRT cells with vectors encoding BFP-labelled wild-type (wt) proteins and observed the normalization of purinosome formation. The cells also ceased to accumulate the substrate(s) of the defective enzyme. The CR-DNPS cell line transfected with a DNA plasmid encoding an enzyme with zero activity served as a negative control for purinosome formation. No purinosome formation was observed in these cells regardless of the purine level in the growth medium. CONCLUSION: In conclusion, both methods are useful for the detection of purinosomes in HeLa cells. Moreover, the cell-based models prepared represent a unique system for the study of purinosome assembly with deficiencies in DNPS or in the salvage pathway as well as for the study of purinosome formation under the action of DNPS inhibitors. This approach is a promising step toward the treatment of purine disorders and can also provide targets for anticancer therapy.

• MeSH
• biologické modely * MeSH
• HeLa buňky MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• puriny biosyntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• multienzymové komplexy MeSH
• puriny MeSH

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.

• Klíčová slova
• AICA-ribosiduria, Adenylosuccinate lyase deficiency, CRISPR, De novo purine synthesis, Human cellular model, Purinosome,
• MeSH
• chromatografie kapalinová MeSH
• CRISPR-Cas systémy * MeSH
• HeLa buňky MeSH
• lidé MeSH
• multienzymové komplexy chemie   genetika   metabolismus   MeSH
• mutace MeSH
• puriny biosyntéza   metabolismus   MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• multienzymové komplexy MeSH
• purine MeSH Prohlížeč
• puriny MeSH

Comparison of the genomes of free-living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free-living to a parasitic life style has resulted in the loss of approximately 50% of protein-coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP-PFK which is homologous to the bacterial PPi -PFKs rather than to the canonical eukaryotic ATP-PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose-2,6-bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain-factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.

• Klíčová slova
• Leishmania, Leptomonas, Phytomonas, Trypanosoma, adaptation, lateral gene transfer, parasitism,
• MeSH
• aminokyseliny metabolismus   MeSH
• Bacteria genetika   metabolismus   MeSH
• dolichol metabolismus   MeSH
• ergosterol biosyntéza   MeSH
• Eukaryota genetika   metabolismus   MeSH
• fosfolipidy metabolismus   MeSH
• glukoneogeneze MeSH
• glykolýza MeSH
• Kinetoplastida enzymologie   genetika   metabolismus   MeSH
• koenzymy metabolismus   MeSH
• kyselina listová metabolismus   MeSH
• kyselina mevalonová metabolismus   MeSH
• metabolismus lipidů MeSH
• metabolismus sacharidů MeSH
• mikrotělíska metabolismus   MeSH
• mitochondrie enzymologie   metabolismus   MeSH
• močovina metabolismus   MeSH
• oxidoreduktasy metabolismus   MeSH
• pentózofosfátový cyklus MeSH
• peroxizomy metabolismus   MeSH
• polyaminy metabolismus   MeSH
• prenylace proteinů MeSH
• protozoální geny genetika   MeSH
• protozoální proteiny genetika   MeSH
• puriny biosyntéza   metabolismus   MeSH
• pyrimidiny biosyntéza   metabolismus   MeSH
• reaktivní formy kyslíku MeSH
• Trypanosomatina enzymologie   genetika   metabolismus   MeSH
• ubichinon metabolismus   MeSH
• vitaminy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aminokyseliny MeSH
• dolichol MeSH
• ergosterol MeSH
• fosfolipidy MeSH
• koenzymy MeSH
• kyselina listová MeSH
• kyselina mevalonová MeSH
• močovina MeSH
• oxidoreduktasy MeSH
• polyaminy MeSH
• protozoální proteiny MeSH
• purine MeSH Prohlížeč
• puriny MeSH
• pyrimidine MeSH Prohlížeč
• pyrimidiny MeSH
• reaktivní formy kyslíku MeSH
• ubichinon MeSH
• vitaminy MeSH

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.

• MeSH
• biologické modely MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• elektrochemické techniky MeSH
• karboxylyasy genetika   metabolismus   MeSH
• křečci praví MeSH
• metabolomika * MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• peptidsynthasy genetika   metabolismus   MeSH
• poruchy metabolismu purinů a pyrimidinů genetika   metabolismus   MeSH
• puriny biosyntéza   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• karboxylyasy MeSH
• peptidsynthasy MeSH
• phosphoribosylaminoimidazole carboxylase MeSH Prohlížeč
• phosphoribosylaminoimidazole-succinocarboxamide synthetase MeSH Prohlížeč
• puriny MeSH

• MeSH
• lidé MeSH
• poruchy metabolismu purinů a pyrimidinů klasifikace   diagnóza   metabolismus   MeSH
• puriny biosyntéza   MeSH
• zpětná vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• puriny MeSH

• MeSH
• dietoterapie MeSH
• dna (nemoc) * patologie   terapie   MeSH
• kolchicin terapeutické užití   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina močová krev   metabolismus   MeSH
• lidé MeSH
• puriny biosyntéza   MeSH
• tělesná námaha MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kolchicin MeSH
• kyselina močová MeSH
• puriny MeSH