While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

• MeSH
• adeninnukleotidy chemická syntéza   metabolismus   MeSH
• alosterické místo MeSH
• Bacillus subtilis MeSH
• bakteriální proteiny metabolismus   MeSH
• deoxyribonukleotidy MeSH
• Escherichia coli MeSH
• konformace proteinů MeSH
• ligasy metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

• MeSH
• bakteriální toxiny genetika   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   MeSH
• grampozitivní nesporulující tyčinky chemie   metabolismus   MeSH
• guanosinpentafosfát chemie   metabolismus   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• ligasy chemie   genetika   metabolismus   MeSH
• proteosyntéza účinky léků   fyziologie   MeSH
• pyrofosfatasy MeSH
• ribozomy metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

• MeSH
• adeninnukleotidy metabolismus   MeSH
• Bacteria růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• databáze genetické MeSH
• fyziologický stres fyziologie   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• ligasy metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• signální transdukce MeSH
• systémy toxin-antitoxin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FcεRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FcεRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.

• MeSH
• alergie imunologie   patologie   MeSH
• antigeny CD63 imunologie   MeSH
• bazofily imunologie   patologie   MeSH
• dospělí MeSH
• fosfodiesterasy imunologie   MeSH
• GPI-vázané proteiny imunologie   MeSH
• indoly chemie   MeSH
• jedy členovců toxicita   MeSH
• kousnutí a bodnutí hmyzem imunologie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• maleimidy chemie   MeSH
• pyrofosfatasy imunologie   MeSH
• receptory IgG imunologie   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

Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.

• MeSH
• deoxyguaninnukleotidy chemie   MeSH
• katalytická doména MeSH
• mutace MeSH
• mutageneze cílená MeSH
• pyrofosfatasy chemie   genetika   MeSH
• stabilita proteinů MeSH
• teplota MeSH
• thermolysin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Organophosphorus compounds (OP) nerve agents are among the most toxic chemical substances known. Their toxicity is due to their ability to bind to acetylcholinesterase. Currently, some enzymes, such as phosphotriesterase, human serum paraoxonase 1 and diisopropyl fluorophosphatase, capable of degrading OP, have been characterized. Regarding the importance of bioremediation methods for detoxication of OP, this work aims to study the interaction modes between the human human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and Sarin and VX, considering their Rp and Sp enantiomers, to evaluate the asymmetric catalysis of those compounds. In previous work, this enzyme has shown good potential to degrade phosphotriesters, and based on this characteristic, we have applied the human dUTPase to the OP degradation. Molecular docking, chemometrics and mixed quantum and molecular mechanics calculations have been employed, showing a good interaction between dUTPase and OP. Two possible reaction mechanisms were tested, and according to our theoretical results, the catalytic degradation of OP by dUTPase can take place via both mechanisms, beyond being stereoselective, that is, dUTPase cleaves one enantiomer preferentially in relation to other. Chemometric techniques provided excellent assistance for performing this theoretical investigation. The dUTPase study shows importance by the fact of it being a human enzyme. Communicated by Ramaswamy H. Sarma.

• MeSH
• analýza hlavních komponent MeSH
• biodegradace MeSH
• katalytická doména MeSH
• kvantová teorie * MeSH
• lidé MeSH
• nervová bojová látka chemie   metabolismus   MeSH
• organofosforové sloučeniny chemie   metabolismus   MeSH
• organothiofosforové sloučeniny chemie   metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• sarin chemie   metabolismus   MeSH
• simulace molekulového dockingu * MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční mikroskopie přístrojové vybavení   metody   MeSH
• mutageneze MeSH
• protozoální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• pyrofosfatasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• racionální návrh léčiv MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rezonanční přenos fluorescenční energie přístrojové vybavení   metody   MeSH
• Saccharomyces cerevisiae MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky * MeSH
• software MeSH
• zobrazení jednotlivé molekuly přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.

• MeSH
• amoxicilin metabolismus   farmakologie   MeSH
• antibakteriální látky metabolismus   farmakologie   MeSH
• bakteriální geny genetika   MeSH
• bakteriální léková rezistence genetika   MeSH
• fyziologická adaptace genetika   MeSH
• Helicobacter pylori účinky léků   genetika   růst a vývoj   MeSH
• mikrobiální testy citlivosti MeSH
• mikrobiální viabilita účinky léků   MeSH
• pyrofosfatasy genetika   MeSH
• regulace genové exprese u bakterií účinky léků   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.

• MeSH
• biopsie MeSH
• Charcotova-Marieova-Toothova nemoc krev   genetika   terapie   MeSH
• dospělí MeSH
• fosfodiesterasy genetika   MeSH
• genetická transkripce genetika   MeSH
• genetické markery genetika   MeSH
• glutathiontransferasa genetika   MeSH
• glykoproteiny genetika   MeSH
• kathepsin A genetika   MeSH
• kůže patologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• neuregulin-1 genetika   MeSH
• PPAR gama genetika   MeSH
• prognóza MeSH
• progrese nemoci * MeSH
• pyrofosfatasy genetika   MeSH
• senioři MeSH
• výsledek terapie * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: To identify the etiology of a novel, heritable encephalopathy in a small group of patients. METHODS: Magnetic resonance imaging (MRI) pattern analysis was used to select patients with the same pattern. Homozygosity mapping and whole exome sequencing (WES) were performed to find the causal gene mutations. RESULTS: Seven patients from 4 families (2 consanguineous) were identified with a similar MRI pattern characterized by T2 signal abnormalities and diffusion restriction in the posterior limb of the internal capsule, often also optic radiation, brainstem tracts, and cerebellar white matter, in combination with delayed myelination and progressive brain atrophy. Patients presented with early infantile onset encephalopathy characterized by progressive microcephaly, seizures, variable cardiac defects, and early death. Metabolic testing was unrevealing. Single nucleotide polymorphism array revealed 1 overlapping homozygous region on chromosome 20 in the consanguineous families. In all patients, WES subsequently revealed recessive predicted loss of function mutations in ITPA, encoding inosine triphosphate pyrophosphatase (ITPase). ITPase activity in patients' erythrocytes and fibroblasts was severely reduced. INTERPRETATION: Until now ITPA variants have only been associated with adverse reactions to specific drugs. This is the first report associating ITPA mutations with a human disorder. ITPase is important in purine metabolism because it removes noncanonical nucleotides from the cellular nucleotide pool. Toxicity of accumulated noncanonical nucleotides, leading to neuronal apoptosis and interference with proteins normally using adenosine triphosphate/guanosine triphosphate, probably explains the disease. This study confirms that combining MRI pattern recognition to define small, homogeneous patient groups with WES is a powerful approach for providing a fast diagnosis in patients with an unclassified genetic encephalopathy.

• MeSH
• geny recesivní genetika   MeSH
• kojenec MeSH
• lidé MeSH
• mutace genetika   MeSH
• nemoci mozku diagnóza   genetika   MeSH
• předškolní dítě MeSH
• pyrofosfatasy chemie   genetika   MeSH
• sekundární struktura proteinů MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH