Alterations of hydrogen peroxide (H2O2) levels have a profound impact on numerous signaling cascades orchestrating plant growth, development, and stress signaling, including programmed cell death. To expand the repertoire of known molecular mechanisms implicated in H2O2 signaling, we performed a forward chemical screen to identify small molecules that could alleviate the photorespiratory-induced cell death phenotype of Arabidopsisthaliana mutants lacking H2O2-scavenging capacity by peroxisomal catalase2. Here, we report the characterization of pakerine, an m-sulfamoyl benzamide from the sulfonamide family. Pakerine alleviates the cell death phenotype of cat2 mutants exposed to photorespiration-promoting conditions and delays dark-induced senescence in wild-type Arabidopsis leaves. By using a combination of transcriptomics, metabolomics, and affinity purification, we identified abnormal inflorescence meristem 1 (AIM1) as a putative protein target of pakerine. AIM1 is a 3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-oxidation that contributes to jasmonic acid (JA) and salicylic acid (SA) biosynthesis. Whereas intact JA biosynthesis was not required for pakerine bioactivity, our results point toward a role for β-oxidation-dependent SA production in the execution of H2O2-mediated cell death.

• MeSH
• Arabidopsis cytologie   účinky léků   genetika   metabolismus   MeSH
• buněčná smrt účinky léků   MeSH
• buněčné dýchání účinky léků   genetika   MeSH
• cyklopentany metabolismus   MeSH
• fotosyntéza účinky léků   genetika   MeSH
• fyziologický stres MeSH
• hydroponie metody   MeSH
• kyselina salicylová metabolismus   MeSH
• listy rostlin cytologie   účinky léků   metabolismus   MeSH
• meristém cytologie   účinky léků   metabolismus   MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• oxylipiny metabolismus   MeSH
• peroxid vodíku antagonisté a inhibitory   farmakologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné buňky účinky léků   metabolismus   MeSH
• semena rostlinná účinky léků   MeSH
• signální transdukce MeSH
• stanovení celkové genové exprese MeSH
• sulfonamidy chemická syntéza   farmakologie   MeSH
• transkriptom MeSH
• výpočetní biologie metody   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

Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.

• MeSH
• chromatografie kapalinová MeSH
• CRISPR-Cas systémy * MeSH
• HeLa buňky MeSH
• lidé MeSH
• multienzymové komplexy chemie   genetika   metabolismus   MeSH
• mutace MeSH
• puriny biosyntéza   metabolismus   MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

• MeSH
• endodeoxyribonukleasy chemie   genetika   metabolismus   MeSH
• fosfáty metabolismus   MeSH
• krystalizace MeSH
• molekulární sekvence - údaje MeSH
• multienzymové komplexy chemie   genetika   metabolismus   MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Solanum lycopersicum enzymologie   genetika   MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Myocardial infarction (MI) is the leading cause of death in industrialized countries. All the traditional risk factors for MI are responsible for approximately 50% of cases of MI cases. Attention therefore has recently focused on genetic variants that are not associated with conventional risk factors. One of them is the marker rs6922269, which has been suggested as a risk factor for development of MI in Western populations. We analyzed the relationship between rs6922269 variant on MTHFD1L gene and (i) risk of the acute coronary syndrome (ACS) in the Czech population and (ii) mortality in 7 years follow up. Rs6922269 (G>A) variant was analyzed (CR 99.3% for patients and 98.0% for controls) by PCR-RFLP in consecutively examined 1614 men and 503 women with ACS (age below 65 years) and in population-based controls--1191 men and 1368 women (aged up to 65 years). ANOVA and Chi square were used for statistical analysis. The genotype frequencies were almost identical (P=0.87) in the ACS patients and in controls and no differences were observed, if males (P=0.73) and females (P=0.93) were analysed separately. In addition, rs6922269 polymorphism was not associated with the classical risk factors (dyslipidemia, hypertension, obesity, smoking, diabetes) in control population. Cardiovascular mortality was significantly higher in males, carriers of the AA genotype (P<0.001, OR 2.52, 95% CI 1.40-4.55, for AA vs. +G). We conclude, that rs6922269 variant at MTHFD1L gene could be an important prognostic factor for cardiovascular mortality in patients after ACS.

• MeSH
• akutní koronární syndrom genetika   mortalita   MeSH
• aminohydrolasy genetika   MeSH
• dospělí MeSH
• formiáttetrahydrofolátligasa genetika   MeSH
• infarkt myokardu epidemiologie   genetika   mortalita   MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé středního věku MeSH
• lidé MeSH
• methylentetrahydrofolátdehydrogenasa (NADP) genetika   MeSH
• multienzymové komplexy genetika   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• rizikové faktory MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• enzymy opravy DNA genetika   metabolismus   MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• genetická epistáze fyziologie   MeSH
• HEK293 buňky MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• ubikvitin aktivující enzymy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

• MeSH
• Eukaryota genetika   metabolismus   MeSH
• exprese genu MeSH
• H-protein systému glycinového štěpení genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• organely metabolismus   MeSH
• oxidoreduktasy aminokyselin genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transferasy genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

• MeSH
• chromatin enzymologie   genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• F-box proteiny genetika   metabolismus   MeSH
• homologní rekombinace fyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• myši MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• 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) 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