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). 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. 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.

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

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.