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

Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research.

• Klíčová slova
• Adenylosuccinate lyase, Purine synthesis, RNA-seq, Transcriptome, adenosine monophosphate, (AMP), adenylosuccinate lyase, (ADSL), aminoimidazole carboxamide ribonucleotide, (AICAR), de novo purine synthesis, (DNPS), differentially expressed gene, (DEG), false discovery rate, (FDR), fetal calf macroserum, (FCM), fragments per kilobase of exon per million reads mapped, (FPKM), gene ontology, (GO), guanosine monophosphate, (GMP), minus adenine crADSL to minus adenine WT comparison, (MM), phosphoribosyl pyrophosphate, (PRPP), phosphoribosylaminoimidazolesuccinocarboxamide, (SAICAR), plus adenine crADSL to plus adenine WT comparison, (PP), succinyladenosine monophosphate, (SAMP),
• Publikační typ
• časopisecké články MeSH

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.

• MeSH
• adenylsukcinátlyasa nedostatek   genetika   MeSH
• autistická porucha MeSH
• deaminasy nukleotidů analýza   genetika   MeSH
• fibroblasty enzymologie   MeSH
• HeLa buňky MeSH
• hydroxymethyltransferasy a formyltransferasy analýza   genetika   MeSH
• keratinocyty enzymologie   MeSH
• kultivované buňky MeSH
• kůže cytologie   MeSH
• lidé MeSH
• multienzymové komplexy analýza   genetika   MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• poruchy metabolismu purinů a pyrimidinů enzymologie   genetika   MeSH
• puriny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• deaminasy nukleotidů MeSH
• hydroxymethyltransferasy a formyltransferasy MeSH
• inosine monophosphate synthase MeSH Prohlížeč
• multienzymové komplexy MeSH
• puriny MeSH

Succinylpurines accumulate in the body fluids of patients with adenylosuccinate lyase (ADSL) deficiency but their source in the cerebrospinal fluid remains obscure. Study based on the incorporation of 13C-stable isotope-labeled glycine into cultured oligodendroglia from ADSL-deficient patient and the measurement of labeled products by LC/MS/MS showed total intracellular concentrations of succinylpurines from 45 to 99μmol/l and so these results suggest that these cells can be the source of the compounds in vivo.

• MeSH
• adenosinmonofosfát analogy a deriváty   biosyntéza   MeSH
• adenylsukcinátlyasa nedostatek   MeSH
• aminoimidazolkarboxamid analogy a deriváty   MeSH
• fatální výsledek MeSH
• lidé MeSH
• novorozenec MeSH
• oligodendroglie metabolismus   MeSH
• ribonukleosidy biosyntéza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosinmonofosfát MeSH
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• ribonukleosidy MeSH
• succinyladenosine monophosphate MeSH Prohlížeč
• succinylaminoimidazole carboxamide riboside MeSH Prohlížeč

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

• MeSH
• adenylsukcinátlyasa biosyntéza   chemie   nedostatek   genetika   MeSH
• alternativní sestřih MeSH
• dítě MeSH
• Escherichia coli metabolismus   MeSH
• exony MeSH
• fenotyp MeSH
• fibroblasty metabolismus   MeSH
• genotyp MeSH
• kinetika MeSH
• klonování DNA MeSH
• kojenec MeSH
• komplementární DNA metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• předškolní dítě MeSH
• promotorové oblasti (genetika) MeSH
• protein - isoformy MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• teplota MeSH
• tkáňová distribuce MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• komplementární DNA MeSH
• protein - isoformy MeSH

Adenylosuccinate lyase (ADSL) deficiency is neurometabolic disease characterized by accumulation of dephosphorylated enzyme substrates SAICA-riboside (SAICAr) and succinyladenosine (S-Ado) in body fluids of affected individuals. The phenotypic severity differs considerably among patients: neonatal fatal, severe childhood, and moderate phenotypic forms correlating with different values for the ratio between S-Ado and SAICAr concentrations in cerebrospinal fluid have been distinguished. To reveal the biochemical and structural basis for this phenotypic heterogeneity, we expressed and characterized 19 ADSL mutant proteins identified in 16 patients representing clinically distinct subgroups. Respecting compound heterozygosity and considering the homotetrameric structure of ADSL, we used intersubunit complementation and prepared and characterized genotype-specific heteromeric mutant ADSL complexes. We correlated clinical phenotypes with biochemical properties of the mutant proteins and predicted structural impacts of the mutations. We found that phenotypic severity in ADSL deficiency is correlated with residual enzymatic activity and structural stability of the corresponding mutant ADSL complexes and does not seem to result from genotype-specific disproportional catalytic activities toward one of the enzyme substrates. This suggests that the S-Ado/SAICAr ratio is probably not predictive of phenotype severity; rather, it may be secondary to the degree of the patient's development (i.e., to the age of the patient at the time of sample collection).

• MeSH
• adenylsukcinátlyasa chemie   nedostatek   genetika   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fenotyp MeSH
• genetická heterogenita * MeSH
• lidé MeSH
• multienzymové komplexy chemie   nedostatek   genetika   MeSH
• mutace genetika   MeSH
• mutantní proteiny chemie   genetika   MeSH
• novorozenec MeSH
• sekundární struktura proteinů MeSH
• stabilita enzymů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• multienzymové komplexy MeSH
• mutantní proteiny 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.

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

Folate deficiency in the critical developmental period has been repeatedly associated with an increased risk of Autism spectrum disorders (ASD), but the key pathophysiological mechanism has not yet been identified. In this work, we focused on identifying genes whose defect has similar consequences to folate depletion in the metabolic network. Within the Flux Balance Analysis (FBA) framework, we developed a method of blocked metabolites that allowed us to define the metabolic consequences of various gene defects and folate depletion. We identified six genes (GART, PFAS, PPAT, PAICS, ATIC, and ADSL) whose blocking results in nearly the same effect in the metabolic network as folate depletion. All of these genes form the purine biosynthetic pathway. We found that, just like folate depletion, the blockade of any of the six genes mentioned above results in a blockage of purine metabolism. We hypothesize that this can lead to decreased adenosine triphosphate (ATP) and subsequently, an S-adenosyl methionine (SAM) pool in neurons in the case of rapid cell division. Based on our results, we consider the methylation defect to be a potential cause of ASD, due to the depletion of purine, and consequently S-adenosyl methionine (SAM), biosynthesis.

• Klíčová slova
• ADSL, ASD, ATIC, Flux Balance Analysis (FBA), GART, PAICS, PFAS, PPAT, autism, blocked metabolite, cerebral folate deficiency, folate, metabolic modeling, purine,
• Publikační typ
• časopisecké články MeSH

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

The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.

• MeSH
• adenosin analogy a deriváty   chemie   MeSH
• adenylsukcinátlyasa nedostatek   MeSH
• aminoimidazolkarboxamid analogy a deriváty   chemie   izolace a purifikace   MeSH
• biochemie metody   MeSH
• časové faktory MeSH
• chromatografie iontoměničová MeSH
• fosforylace MeSH
• kationty MeSH
• klinická chemie metody   MeSH
• klonování DNA MeSH
• komplementární DNA metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• poruchy metabolismu purinů a pyrimidinů diagnóza   metabolismus   MeSH
• rekombinantní proteiny chemie   MeSH
• ribonukleotidy chemie   izolace a purifikace   MeSH
• substrátová specifita MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• adenylsukcinátlyasa MeSH
• AICA ribonucleotide MeSH Prohlížeč
• aminoimidazolkarboxamid MeSH
• kationty MeSH
• komplementární DNA MeSH
• rekombinantní proteiny MeSH
• ribonukleotidy MeSH
• SAICAR MeSH Prohlížeč
• succinyladenosine MeSH Prohlížeč