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Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells, a Model of VPS13-Related Diseases
Get PDFIon homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson's diseases, respectively, and iron imbalance is observed in Parkinson's and Alzheimer's diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases
Utilization of cobalamin is ubiquitous in early-branching fungal phyla
Get PDFCobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all non-Dikarya fungi lineages. This observation is supported by the genomic presence of both B12-dependent enzymes and cobalamin modifying enzymes. Fungal cobalamin-dependent enzymes are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin usage with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms and which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and was likely present in the LECA
Charged amino acids may promote coronavirus SARS-CoV-2 fusion with the host ce
Get PDFThe charged amino acids in the spike protein of SARS-CoV-2 exhibit some specific
distribution. In the RBD region of the S1 unit the positive charge dominates in the protein interior and
the negative charge prevails on the surface exposed towards ACE2 receptor. The charged amino acids
in the S2 region preceding heptad repeats of the spike protein of SARS-CoV-2, exhibit a central
concentration, and the imbalance of a positive charge. The idea that both distributions of charged
amino acids may, in an electrostatic manner, facilitate the coronavirus infection of the cell is presented.
An evaluation of the virus-receptor binding energy, by docking the peptide resembling the human
receptor site to the virus RBD, and the estimation of electromechanical deformation of the cell
membrane by S2 during the prefusion process were included. Possible prevention of viral infection is
suggested
Evaluation of acidogenesis products’ effect on biogas production performed with metagenomics and isotopic approaches
No full textAbstract
Background
During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system.
Results
Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities.
Conclusions
In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock
Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
Get PDFE. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule
ANNEXIN1 mediates calcium‐dependent systemic defense in Arabidopsis plants upon herbivory and wounding
Get PDFCellular calcium (Ca) transients are endogenous signals involved in local and systemic sig-
naling and defense activation upon environmental stress, including wounding and herbivory.
Still, not all Ca 2+ channels contributing to the signaling have been identified, nor are their
modes of action fully known. Plant annexins are proteins capable of binding to anionic phos-
pholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is sug-
gested to contribute to Ca transport.
� Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca eleva-
tion was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for
a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera
littoralis larvae.
� Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on
ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feed-
ing on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced
defense-related responses on treated leaves, such as jasmonate accumulation and defense
gene expression. These responses remained local and were strongly reduced in systemic
leaves in ann1 plants.
� Our results indicate that ANN1 plays an important role in activation of systemic rather than
local defense in plants attacked by herbivorous insects
Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins
Get PDFBackground
DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.
Results
This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1 and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.
Conclusion
KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy
Partial Inhibition of Calcineurin Activity by Rcn2 as a Potential Remedy for Vps13 Deficiency
Get PDFRegulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13D), a model of VPS13-linked
neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13D mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13D cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2.
The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms
Bacterial Communities Associated with Poa annua Roots in Central European (Poland) and Antarctic Settings (King George Island)
Get PDFAbstract: Poa annua (annual bluegrass) is one of the most ubiquitous grass species in the world.
In isolated regions of maritime Antarctica, it has become an invasive organism threatening native
tundra communities. In this study, we have explored and compared the rhizosphere and rootendosphere
dwelling microbial community of P. annua specimens of maritime Antarctic and Central
European origin in terms of bacterial phylogenetic diversity and microbial metabolic activity with
a geochemical soil background. Our results show that the rhizospheric bacterial community was
unique for each sampling site, yet the endosphere communities were similar to each other. However,
key plant-associated bacterial taxa such as the Rhizobiaceae family were poorly represented in Antarctic
samples, probably due to high salinity and heavy metal concentrations in the soil. Metabolic activity
in the Antarctic material was considerably lower than in Central European samples. Antarctic
root endosphere showed unusually high numbers of certain opportunistic bacterial groups, which
proliferated due to low competition conditions. Thirteen bacterial families were recognized in this
study to form a core microbiome of the P. annua root endosphere. The most numerous were the
Flavobacteriaceae, suspected to be major contributors to the ecological success of annual bluegrass,
especially in harsh, Antarctic conditions