Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.
- MeSH
- buněčný cyklus * MeSH
- konformace proteinů MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie metody MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- proteiny chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny MeSH
Polyketide synthases (PKSs) are crucial multidomain enzymes in diverse natural product biosynthesis. Parrots use a type I PKS to produce a unique pigment called psittacofulvin in their feathers. In domesticated budgerigars and lovebirds, the same amino acid substitution (R644W) within malonyl/acetyltransferase (MAT) domain of this enzyme has been shown to cause the blue phenotype with no psittacofulvin pigmentation, proposing a strong evolutionary constraint on the mechanism. Here, we identified seven previously unreported variants in PKS associated with defective psittacofulvin production in four diverse species, including three nonsense mutations. Intriguingly, three of the remaining nonsynonymous substitutions reside within the ketoacyl synthase (KS) domain, whereas one at MAT domain. The heterologous expression of these PKS variants in yeast confirmed complete or partial loss of psittacofulvin production. These findings establish PKS as a functionally conserved key-enzyme determining psittacofulvin-based hues among diverse parrots, highlighting multiple conserved domains essential for the PKS function.
- MeSH
- mutace * MeSH
- papouškovití * genetika MeSH
- peří metabolismus MeSH
- pigmentace genetika MeSH
- polyketidsynthasy * genetika metabolismus chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- polyketidsynthasy * MeSH
Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol 4-phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to the presence of charged lipids and to membrane fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.
- MeSH
- biologický transport MeSH
- buněčná membrána * metabolismus MeSH
- fluidita membrány MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- fosfatidylseriny metabolismus MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus genetika MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- steroidní receptory metabolismus MeSH
- unilamelární lipozómy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfatidylinositolfosfáty MeSH
- fosfatidylseriny MeSH
- Oxysterol Binding Proteins MeSH
- phosphatidylinositol 4-phosphate MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny * MeSH
- steroidní receptory MeSH
- unilamelární lipozómy MeSH
Nanostructured materials with antibacterial activity face the same threat as conventional antibiotics - bacterial resistance, which reduces their effectiveness. However, unlike antibiotics, research into the emergence and mechanisms of bacterial resistance to antibacterial nanomaterials is still in its early stages. Here we show how Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria develop resistance to silver nanoparticles, resulting in an increase in the minimum inhibitory concentration from 1.69 mg/L for S. aureus and 3.38 mg/L for E. coli to 54 mg/L with repeated exposure over 12 and 6 cultivation steps, respectively. The mechanism of resistance is the same for both types of bacteria and involves the aggregation of silver nanoparticles leading to the formation of black precipitates. However, the way in which Gram-positive and Gram-negative bacteria induce aggregation of silver nanoparticles is completely different. Chemical analysis of the surface of the silver precipitates shows that aggregation is triggered by flagellin production in E. coli and by bacterial biofilm formation in S. aureus. However, resistance in both types of bacteria can be overcome by using pomegranate rind extract, which inhibits both flagellin and biofilm production, or by stabilizing the silver nanoparticles by covalently binding them to a composite material containing graphene sheets, which protects the silver nanoparticles from aggregation induced by the bacterial biofilm produced by S. aureus. This research improves the understanding of bacterial resistance mechanisms to nanostructured materials, which differ from resistance mechanisms to conventional antibiotics, and provides potential strategies to combat bacterial resistance and develop more effective antimicrobial treatments.
- MeSH
- antibakteriální látky * farmakologie chemie MeSH
- bakteriální léková rezistence * účinky léků MeSH
- biofilmy účinky léků růst a vývoj MeSH
- Escherichia coli * účinky léků MeSH
- kovové nanočástice * chemie MeSH
- mikrobiální testy citlivosti * MeSH
- Staphylococcus aureus * účinky léků MeSH
- stříbro * farmakologie chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- stříbro * MeSH
Investigating microorganisms in metal-enriched environments holds the potential to revolutionize the sustainable recovery of critical metals such as lanthanides (Ln3+). We observe Hyphomicrobium spp. as part of a Fe2+/Mn2+-oxidizing consortia native to the ferruginous bottom waters of a Ln3+-enriched lake in Czechia. Notably, one species shows similarities to recently discovered bacteria expressing proteins with picomolar Ln3+ affinity. This finding was substantiated by developing an in-silico ionic competition model and recombinant expression of a homolog protein (Hm-LanM) from Hyphomicrobium methylovorum. Biochemical assays validate Hm-LanM preference for lighter Ln3+ ions (from lanthanum to gadolinium). This is comparable to established prototypes. Bioinformatics analyses further uncover additional H. methylovorum metabolic biomolecules in genomic proximity to Hm-LanM analogously dependent on Ln3+, including an outer membrane receptor that binds Ln3+-chelating siderophores. These combined observations underscore the remarkable strategy of Hyphomicrobium spp. for thriving in relatively Ln3+ enriched zones of metal-polluted environments.
- MeSH
- bakteriální proteiny * metabolismus genetika chemie MeSH
- jezera mikrobiologie MeSH
- lanthanoidy metabolismus chemie MeSH
- počítačová simulace MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny * MeSH
- lanthanoidy MeSH
- železo MeSH
Membrane penetration by non-enveloped viruses is diverse and generally not well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. Enteroviruses enter cells by receptor-mediated endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing enteroviruses deform, rupture, and release the virus particles into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of particles that released their genomes, but did not prevent them from reaching the cytoplasm. Inhibiting post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. The rupture of endosomes also occurs in control cells and after the endocytosis of very low-density lipoprotein. In summary, our results show that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus particles into the cytoplasm to initiate infection. Since the studied enteroviruses employ different receptors for cell entry but are delivered into the cytoplasm by cell-mediated endosome disruption, it is likely that most if not all enteroviruses, and probably numerous other viruses from the family Picornaviridae, can utilize endosome rupture to infect cells.
- MeSH
- buněčná membrána ultrastruktura virologie MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cytoplazma virologie MeSH
- elektronová kryomikroskopie MeSH
- endocytóza * MeSH
- endozomy * patologie virologie MeSH
- HeLa buňky MeSH
- lidé MeSH
- makrolidy farmakologie MeSH
- pikornavirové infekce * virologie MeSH
- Rhinovirus * genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bafilomycin A1 MeSH Prohlížeč
- makrolidy MeSH
Termites digest wood using Carbohydrate-Active Enzymes (CAZymes) produced by gut bacteria with whom they have cospeciated at geological timescales. Whether CAZymes were encoded in the genomes of their ancestor's gut bacteria and transmitted to modern termites or acquired more recently from bacteria not associated with termites is unclear. We used gut metagenomes from 195 termites and one Cryptocercus, the sister group of termites, to investigate the evolution of termite gut bacterial CAZymes. We found 420 termite-specific clusters in 81 bacterial CAZyme gene trees, including 404 clusters showing strong cophylogenetic patterns with termites. Of the 420 clusters, 131 included at least one bacterial CAZyme sequence associated with Cryptocercus or Mastotermes, the sister group of all other termites. Our results suggest many bacterial CAZymes have been encoded in the genomes of termite gut bacteria since termite origin, indicating termites rely upon many bacterial CAZymes endemic to their guts to digest wood.
- MeSH
- Bacteria * enzymologie genetika MeSH
- bakteriální proteiny metabolismus genetika MeSH
- fylogeneze * MeSH
- Isoptera * mikrobiologie enzymologie MeSH
- metabolismus sacharidů MeSH
- metagenom MeSH
- molekulární evoluce MeSH
- střevní mikroflóra MeSH
- švábi mikrobiologie enzymologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
The genomic signature of an organism captures the characteristics of repeated oligonucleotide patterns in its genome 1, such as oligomer frequencies, GC content, and differences in codon usage. Viruses, however, are obligate intracellular parasites that are dependent on their host cells for replication, and information about genomic signatures in viruses has hitherto been sparse.Here, we investigate the presence and specificity of genomic signatures in 2,768 eukaryotic viral species from 105 viral families, aiming to illuminate dependencies and selective pressures in viral genome evolution. We demonstrate that most viruses have highly specific genomic signatures that often also differ significantly between species within the same family. The species-specificity is most prominent among dsDNA viruses and viruses with large genomes. We also reveal consistent dissimilarities between viral genomic signatures and those of their host cells, although some viruses present slight similarities, which may be explained by genetic adaptation to their native hosts. Our results suggest that significant evolutionary selection pressures act upon viral genomes to shape and preserve their genomic signatures, which may have implications for the field of synthetic biology in the construction of live attenuated vaccines and viral vectors.
Photosynthetic organisms harvest light for energy. Some eukaryotic algae have specialized in harvesting far-red light by tuning chlorophyll a absorption through a mechanism still to be elucidated. Here, we combined optically detected magnetic resonance and pulsed electron paramagnetic resonance measurements on red-adapted light-harvesting complexes, rVCP, isolated from the freshwater eustigmatophyte alga Trachydiscus minutus to identify the location of the pigments responsible for this remarkable adaptation. The pigments have been found to belong to an excitonic cluster of chlorophylls a at the core of the complex, close to the central carotenoids in L1/L2 sites. A pair of structural features of the Chl a403/a603 binding site, namely the histidine-to-asparagine substitution in the magnesium-ligation residue and the small size of the amino acid at the i-4 position, resulting in a [A/G]xxxN motif, are proposed to be the origin of this trait. Phylogenetic analysis of various eukaryotic red antennae identified several potential LHCs that could share this tuning mechanism. This knowledge of the red light acclimation mechanism in algae is a step towards rational design of algal strains in order to enhance light capture and efficiency in large-scale biotechnology applications.
- MeSH
- chlorofyl a * metabolismus chemie MeSH
- chlorofyl metabolismus MeSH
- elektronová paramagnetická rezonance MeSH
- fylogeneze MeSH
- světlo MeSH
- světlosběrné proteinové komplexy * metabolismus genetika chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chlorofyl a * MeSH
- chlorofyl MeSH
- světlosběrné proteinové komplexy * MeSH
Pheromones convey rich ethological information and guide insects' search behavior. Insects navigating in turbulent environments are tasked with the challenge of coding the temporal structure of an odor plume, obliging recognition of the onset and offset of whiffs of odor. The coding mechanisms that shape odor offset recognition remain elusive. We designed a device to deliver sharp pheromone pulses and simultaneously measured the response dynamics from pheromone-tuned olfactory receptor neurons (ORNs) in male moths and Drosophila. We show that concentration-invariant stimulus duration encoding is implemented in moth ORNs by spike frequency adaptation at two time scales. A linear-nonlinear model fully captures the underlying neural computations and offers an insight into their biophysical mechanisms. Drosophila use pheromone cis-vaccenyl acetate (cVA) only for very short distance communication and are not faced with the need to encode the statistics of the cVA plume. Their cVA-sensitive ORNs are indeed unable to encode odor-off events. Expression of moth pheromone receptors in Drosophila cVA-sensitive ORNs indicates that stimulus-offset coding is receptor independent. In moth ORNs, stimulus-offset coding breaks down for short ( < 200 ms) whiffs. This physiological constraint matches the behavioral latency of switching from the upwind surge to crosswind cast flight upon losing contact with the pheromone.
- MeSH
- acetáty MeSH
- čich fyziologie MeSH
- čichové buňky * fyziologie MeSH
- čichové dráhy fyziologie MeSH
- Drosophila melanogaster fyziologie MeSH
- Drosophila fyziologie MeSH
- feromony * MeSH
- kyseliny olejové MeSH
- můry * fyziologie MeSH
- odoranty analýza MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetáty MeSH
- cis-vaccenyl acetate MeSH Prohlížeč
- feromony * MeSH
- kyseliny olejové MeSH
