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.

• Klíčová slova
• ADSL, AICAR, AIR, ATIC, FGAR, PAICS, PFAS, SAICAR, cytotoxicity, purine synthesis,
• Publikační typ
• časopisecké články MeSH

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

• Klíčová slova
• ADSL, ADSLD, DNA damage, SAICAR, cell biology, chicken, cilia, developmental biology, human, microcephaly, zebrafish,
• MeSH
• adenylsukcinátlyasa nedostatek   metabolismus   MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   MeSH
• autistická porucha metabolismus   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• ciliopatie metabolismus   MeSH
• dánio pruhované metabolismus   MeSH
• fenotyp MeSH
• fosfoproteiny metabolismus   MeSH
• kur domácí metabolismus   MeSH
• lidé MeSH
• mikrocefalie metabolismus   MeSH
• neurogeneze * MeSH
• poruchy autistického spektra metabolismus   MeSH
• poruchy metabolismu purinů a pyrimidinů metabolismus   MeSH
• poškození DNA MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• puriny metabolismus   MeSH
• ribonukleotidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• CCP110 protein, human MeSH Prohlížeč
• fosfoproteiny MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• purine MeSH Prohlížeč
• puriny MeSH
• ribonukleotidy MeSH
• SAICAR MeSH Prohlížeč

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

• MeSH
• adenosin metabolismus   MeSH
• adenylsukcinátlyasa genetika   metabolismus   MeSH
• karcinogeneze MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• proliferace buněk MeSH
• prolyl-4-hydroxylasy HIF genetika   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• triple-negativní karcinom prsu enzymologie   genetika   patofyziologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosin MeSH
• adenylsukcinátlyasa MeSH
• EGLN2 protein, human MeSH Prohlížeč
• mikro RNA MeSH
• MIRN22 microRNA, human MeSH Prohlížeč
• MYC protein, human MeSH Prohlížeč
• prolyl-4-hydroxylasy HIF MeSH
• protoonkogenní proteiny c-myc 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