The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA * chemie   metabolismus   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce * MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• Thermosynechococcus metabolismus   MeSH
• transkripční faktory metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is used in traditional medicine for various purposes. Previous phytochemical studies focused on phenolic compounds, but triterpenoids were almost overlooked. OBJECTIVE: The study focused on the isolation of compounds with dual antidiabetic activity from the aerial parts of Z. clinopodioides subsp. bungeana. MATERIALS AND METHODS: Separation of CHCl3-soluble fraction by silica gel column chromatography using different mobile phases and purification of compounds by semi-preparative HPLC or preparative TLC. The structures of pure compounds were elucidated by 1D and 2D NMR experiments along with HRMS. Compound 1 was additionally identified by the single crystal X-ray diffraction method. α-Glucosidase inhibitory assay and GLUT4 expression and translocation in C2C12 myotubes were conducted to evaluate antidiabetic potential of isolated compounds. RESULTS: This phytochemical study led to the isolation of 20 compounds, including a unique monoterpene diperoxy dimer (1). Compounds 7 and 9-11 displayed more potent α-glucosidase inhibitory activity (IC50 45.3-135.3 μM) than acarbose used as a positive control (IC50 264.7 μM), while only pomolic acid (5) increased GLUT4 translocation in C2C12 myotubes in a significant manner. CONCLUSION: Extensive chromatographic separation led to the isolation and identification of a unique monoterpene diperoxy dimer (1) from aerial parts of Z. clinopodioides subsp. bungeana. Some triterpenes inhibited α-glucosidase, another increased GLUT4 translocation. Although none of the isolated compounds demonstrated dual antidiabetic activity, selected triterpenes proved to be potent antidiabetic agents in vitro.

• MeSH
• alfa-glukosidasy metabolismus   MeSH
• buněčné linie MeSH
• hluchavkovité * chemie   MeSH
• hypoglykemika * farmakologie   chemie   izolace a purifikace   MeSH
• inhibitory glykosidových hydrolas farmakologie   izolace a purifikace   chemie   MeSH
• myši MeSH
• nadzemní části rostlin chemie   MeSH
• přenašeč glukosy typ 4 metabolismus   MeSH
• rostlinné extrakty chemie   farmakologie   MeSH
• triterpeny * farmakologie   chemie   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

G-quadruplexes (G4s) formed within RNA are emerging as promising targets for therapeutic intervention in cancer, neurodegenerative disorders and infectious diseases. Sequences containing a succession of short GG blocks, or uneven G-tract lengths unable to form three-tetrad G4s (GG motifs), are overwhelmingly more frequent than canonical motifs involving multiple GGG blocks. We recently showed that DNA is not able to form stable two-tetrad intramolecular parallel G4s. Whether RNA GG motifs can form intramolecular G4s under physiological conditions and play regulatory roles remains a burning question. In this study, we performed a systematic analysis and experimental evaluation of a number of biologically important RNA regions involving RNA GG motifs. We show that most of these motifs do not form stable intramolecular G4s but need to dimerize to form stable G4 structures. The strong tendency of RNA GG motif G4s to associate may participate in RNA-based aggregation under conditions of cellular stress.

• MeSH
• dimerizace MeSH
• G-kvadruplexy * MeSH
• genetická transkripce MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• RNA * chemie   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt 'big' protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt 'small' model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the 'small' protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity.

• MeSH
• konformace nukleové kyseliny MeSH
• molekulární modely MeSH
• peptidy chemie   metabolismus   MeSH
• peptidyltransferasy metabolismus   chemie   MeSH
• ribozomální proteiny * metabolismus   chemie   MeSH
• ribozomy * metabolismus   MeSH
• RNA metabolismus   chemie   MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH

Interactions between living cells and nanoparticles are extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness, and surface charge are regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigates the role of cellular mechanosensitive components in cell-nanoparticle interactions. It is demonstrated that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, the study proposes targeting YAP may sensitize triple-negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.

• MeSH
• lidé MeSH
• nanočástice * MeSH
• signální proteiny YAP MeSH
• signální transdukce fyziologie   MeSH
• triple-negativní karcinom prsu * farmakoterapie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

• MeSH
• Bacillus subtilis genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• bakteriální RNA * metabolismus   genetika   MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• konformace nukleové kyseliny MeSH
• Mycobacterium smegmatis genetika   metabolismus   enzymologie   MeSH
• Mycobacterium tuberculosis genetika   metabolismus   MeSH
• nekódující RNA MeSH
• regulace genové exprese u bakterií MeSH
• sigma faktor * metabolismus   genetika   MeSH
• Streptomyces coelicolor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Five putrescine and spermidine derivatives (1-5) together with five rotenoids (6-10) were isolated from a methanolic extract of the flowers of A. fruticosa that displayed promising inhibition of 76.0 ± 1.9% for AChE and 90.0 ± 4.0% for BuChE at a concentration of 1 mg/mL. Although the anticholinesterase activities of the isolated compounds did not reach that of galantamine, molecular docking revealed that all-trans-tri-p-coumaroylspermidine and trans-trans-cis-tri-p-coumaroylspermidine showed binding poses mimicking the known inhibitor galantamine and thus could serve as model molecules in future searches for new AChE and BuChE inhibitors.

• Publikační typ
• časopisecké články MeSH

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

• MeSH
• chřipka lidská * farmakoterapie   MeSH
• hemaglutininové glykoproteiny viru chřipky metabolismus   MeSH
• hemaglutininy farmakologie   MeSH
• kyselina N-acetylneuraminová farmakologie   metabolismus   MeSH
• kyseliny neuraminové MeSH
• lidé MeSH
• neuraminidasa metabolismus   MeSH
• virus chřipky A, podtyp H1N1 * MeSH
• virus chřipky A, podtyp H3N2 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

• Publikační typ
• časopisecké články MeSH