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
• 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

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.

• 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

• MeSH
• lékařská počítačová informatika MeSH
• ordinace MeSH
• technologie MeSH

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.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

• MeSH
• adenylsukcinátlyasa genetika   nedostatek   MeSH
• dítě MeSH
• fenotyp MeSH
• klonování DNA MeSH
• komplementární DNA analýza   biosyntéza   MeSH
• lidé MeSH
• mutace MeSH
• nemoci centrálního nervového systému enzymologie   patologie   MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• předškolní dítě 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

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

Nepostradatelná kniha pro každého správce sítě a pro každého uživatele, který chce proniknout problematice počítačových sítí skutečně pod kůži. Velmi fundovaně popisuje základní principy fungování sítí, protokoly sítí LAN a WAN a velkou rodinu internetových protokolů TCP/IP. Značný prostor věnuje i problematice síťového managementu. Je doplněna celou řadou užitečných praktických rad a příkladů. Může se stát cennou příručkou každého, kdo přichází s uvedenými technologiemi do styku a chce si v tomto oboru rozšířit znalosti.

• MeSH
• počítačové komunikační sítě MeSH
• počítačové systémy MeSH

• Konspekt
• Počítačové sítě

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.

• Publikační typ
• časopisecké články MeSH