De novo purine synthesis Dotaz Zobrazit nápovědu
- Publikační typ
- abstrakt z konference MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Many patients are examined in the Institute of Inherited Metabolic Disorders with a diagnostic efficiency of approximately 2%. High numbers of them suffer with neurological symptoms that do not fit into any currently known metabolic disorder. Therefore, it is highly probable that some of them can suffer from defects of de novo purine synthesis (DNPS). To date, there is no sufficient screening method for the detection of such defects. The aim of this project is to study the prevalence of DNPS disorders in a population of patients with neurological impairment that has not yet been diagnosed. Screening will be performed by determining the levels of DNPS intermediates by LC-MS/MS in the body fluids. Based on our experiences having established a detection method for SAICA-riboside, one of the DNPS intermediates, that provided a diagnosis in 40% of all the patients worldwide diagnosed with adenylosuccinate lyase deficiency, we suggest that the screening of the other intermediates can help to define new types of DNPS defects, as well as establish the diagnosis in some patients.
V metabolických laboratořích je každoročně vyšetřeno velké množství pacientů s nespecifickým neurologickým postižením, u kterých se nepodaří stanovit diagnózu. Je velmi pravděpodobné, že u části z nich by se mohlo jednat o dosud nevyšetřované geneticky podmíněné poruchy de novo syntézy purinů (DNPS). Cílem tohoto projektu je studium prevalence těchto poruch v populaci dětských pacientů s nespecifickým neurologickým onemocněním stanovením substrátů pro enzymy DNPS metodou LC-MS/MS v močích a suchých krevních kapkách. Na základě naší zkušenosti se stanovením SAICA-ribosidu, jednoho ze substrátů enzymů DNPS, kdy se podařilo diagnostikovat 40% všech celosvětově popsaných pacientů s deficitem adenylosukcinátlyázy, předpokládáme, že analýzy dalších substrátů DNPS povedou k definici nových poruch de novo syntézy purinů a stanovení diagnózy alespoň u části postižených pacientů.
- MeSH
- adenylsukcinátlyasa MeSH
- analýza moči MeSH
- biochemická analýza krve MeSH
- dítě MeSH
- genetická predispozice k nemoci MeSH
- genetické testování MeSH
- nemoci nervového systému diagnóza MeSH
- poruchy metabolismu purinů a pyrimidinů MeSH
- puriny biosyntéza MeSH
- Check Tag
- dítě MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- neurologie
- biochemie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
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.
- Publikační typ
- časopisecké články MeSH
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.
- Publikační typ
- časopisecké články 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
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
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
