Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

• Klíčová slova
• HeLa cells, mass spectrometry, metabolomics, purine de novo synthesis, rare metabolic disorders,
• Publikační typ
• časopisecké články MeSH

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

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.

• Klíčová slova
• 5-aminoimidazole-4-carboxamide ribonucleoside, (AICAr), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase, (ATIC), 5-aminoimidazole-4-carboxamide ribonucleotide, (ZMP), 5-formamido-4-imidazolecarboxamide ribonucleotide, (FAICAR), AICA-ribosiduria, AMP-activated protein kinase, (AMPK), Alzheimer's disease, Development, Purine synthesis, RNA-seq, Tuberous Sclerosis Complex 1 and 2, (TSC1 and TSC2), adenine phosphoribosyltransferase, (APRT), adenosine monophosphate, (AMP), adenosine triphosphate, (ATP), adenylosuccinate lyase, (ADSL), arachidonic acid, (AA), cyclooxygenase, (COX), cytochrome, P450 (CYP), cytosolic phospholipase A2, (cPLA2), de novo purine synthesis, (DNPS), differentially expressed gene, (DEG), false discovery rate, (FDR), fatty acid amide hydrolase, (FAAH), fetal calf macroserum, (FCM), fetal calf serum, (FCS), fragments per kilobase of exon per million reads mapped, (FPKM), gene ontology, (GO), guanosine monophosphate, (GMP), inosine monophosphate, (IMP), interferon, (INF), lipoxygenase, (LOX), mammalian Target of Rapamycin, (mTOR), minus adenine crATIC to minus adenine WT comparison, (MM), phospholipase, (PLA), phosphoribosyl pyrophosphate, (PRPP), phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase, (PAICS), plus adenine crATIC to plus adenine WT comparison, (PP), xanthine monophosphate, (XMP),
• Publikační typ
• časopisecké články MeSH