It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.
- MeSH
- akaricidy farmakologie MeSH
- aminoacyl-tRNA-synthetasy antagonisté a inhibitory genetika MeSH
- Borrelia burgdorferi komplex MeSH
- klíšťata účinky léků mikrobiologie MeSH
- lidé MeSH
- lymeská nemoc farmakoterapie mikrobiologie přenos MeSH
- proteosyntéza účinky léků MeSH
- vyvíjení léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata 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
The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 μM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).
- MeSH
- antibakteriální látky toxicita MeSH
- antimycin A analogy a deriváty toxicita MeSH
- azidy toxicita MeSH
- buněčné dýchání účinky léků MeSH
- chloramfenikol toxicita MeSH
- citrátový cyklus účinky léků MeSH
- deoxyglukosa toxicita MeSH
- fluoracetáty toxicita MeSH
- glykolýza účinky léků MeSH
- malonáty toxicita MeSH
- proteosyntéza účinky léků MeSH
- pyruváty toxicita MeSH
- reprodukovatelnost výsledků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- stav beztíže MeSH
- streptomycin toxicita MeSH
- Synechococcus chemie účinky léků izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP+C10), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP+C10 causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid β-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP+C10 significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- doxycyklin farmakologie MeSH
- gentisáty chemie farmakologie MeSH
- heterocyklické sloučeniny chemie farmakologie MeSH
- ketoglutarátdehydrogenasový komplex antagonisté a inhibitory genetika MeSH
- lidé MeSH
- mitochondrie účinky léků patologie MeSH
- nádory prsu farmakoterapie genetika patologie MeSH
- organofosforové sloučeniny chemie farmakologie MeSH
- oxidativní fosforylace účinky léků MeSH
- proliferace buněk účinky léků MeSH
- proteinkinasy genetika MeSH
- proteosyntéza účinky léků MeSH
- ribozomy účinky léků MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stop codon readthrough-the decoding of a stop codon by a near-cognate tRNA-is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.
- MeSH
- aminoglykosidy farmakologie MeSH
- nukleotidy chemie MeSH
- proteosyntéza * účinky léků MeSH
- RNA transferová Gln MeSH
- RNA transferová Tyr MeSH
- RNA transferová metabolismus MeSH
- Saccharomyces cerevisiae genetika MeSH
- terminační kodon * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 μM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.
- MeSH
- antivirové látky chemie farmakologie MeSH
- deoxycukry chemie farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- plakové testy MeSH
- proteosyntéza účinky léků MeSH
- uridin analogy a deriváty chemie farmakologie MeSH
- viry klíšťové encefalitidy účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.
- MeSH
- elektronová kryomikroskopie MeSH
- Escherichia coli genetika metabolismus MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- kationické antimikrobiální peptidy farmakologie MeSH
- konformace proteinů MeSH
- peptidy - faktory ukončení metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- proteosyntéza účinky léků MeSH
- ribozomální proteiny metabolismus MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- simulace molekulového dockingu MeSH
- terminace translace peptidového řetězce MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- velké podjednotky ribozomu bakteriální metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-14C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.
- MeSH
- amoniak otrava MeSH
- citrátový cyklus účinky léků MeSH
- glutamin agonisté metabolismus MeSH
- hyperamonemie enzymologie metabolismus patofyziologie MeSH
- jaterní cirhóza etiologie metabolismus MeSH
- kyseliny ketoglutarové metabolismus MeSH
- methylhistidiny metabolismus MeSH
- orgánová specificita MeSH
- osmolární koncentrace MeSH
- oxidace-redukce MeSH
- potkani Wistar MeSH
- proteolýza účinky léků MeSH
- proteosyntéza účinky léků MeSH
- radioizotopy uhlíku MeSH
- svalová vlákna typu I účinky léků enzymologie metabolismus MeSH
- svalová vlákna typu II účinky léků enzymologie metabolismus MeSH
- svalové proteiny genetika metabolismus MeSH
- techniky in vitro MeSH
- větvené aminokyseliny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Rubroboletus satanas (Lenz) Kuan Zhao & Zhu L. Yang, known as Boletus satanas (Lenz) until 2014, and commonly known as the Devil´s bolete or Satan´s bolete, is a basidiomycete mushroom of the bolete family. Grows in mixed woodlands in the southern, warmer regions of Europe and North America. Satan´s bolete generally regarded as a poisonous mushroom, with predominantly gastrointestinal symptoms of nausea and violent vomiting occurring if eaten raw or isufficiently heat-treated. The toxicity of the mushroom corresponds to a toxic protein called bolesatine. Bolesatine is a toxic glycoprotein which has been shown to inhibit protein synthesis in cell-free systems and cell culture and is toxic to rodents. Biology, chemistry, pharmacology and toxicology of bolesatine is discussed in this article.
- Klíčová slova
- Rubroboletus,
- MeSH
- Agaricales * chemie klasifikace MeSH
- antitumorózní látky terapeutické užití MeSH
- fungální proteiny * chemie izolace a purifikace toxicita MeSH
- gastroenteritida chemicky indukované komplikace terapie MeSH
- glykoproteiny chemie otrava toxicita MeSH
- houby chemie klasifikace MeSH
- lektiny chemie imunologie MeSH
- lidé MeSH
- mykotoxiny * chemie otrava toxicita MeSH
- otrava houbami klasifikace komplikace terapie MeSH
- proteosyntéza genetika imunologie účinky léků MeSH
- ricin chemie otrava toxicita MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed in vivo and in vitro experiments to examine the effects of PB on glutamine (GLN), branched-chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5 mM). L-[1-(14) C] leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched-chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.
- MeSH
- fenylbutyráty farmakologie MeSH
- glutamin metabolismus MeSH
- kosterní svaly účinky léků metabolismus MeSH
- leucin metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- potkani Wistar MeSH
- proteosyntéza účinky léků MeSH
- svalové proteiny metabolismus MeSH
- techniky tkáňových kultur MeSH
- větvené aminokyseliny metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
