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A protein architecture guided screen for modification dependent restriction endonucleases
Get PDFModification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI), and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5 methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in E. coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases
SNF1-Related Protein Kinases SnRK2.4 and SnRK2.10 Modulate ROS Homeostasis in Plant Response to Salt Stress
Get PDFIn response to salinity and various other environmental stresses, plants accumulate reactive oxygen species (ROS). The ROS produced at very early stages of the stress response act as signaling molecules activating defense mechanisms, whereas those produced at later stages in an uncontrolled way are detrimental to plant cells by damaging lipids, DNA, and proteins. Multiple systems are involved in ROS generation and also in ROS scavenging. Their level and activity are tightly controlled to ensure ROS homeostasis and protect the plant against the negative effects of the environment. The signaling pathways responsible for maintaining ROS homeostasis in abiotic stress conditions remain largely unknown. Here, we show that in Arabidopsis thaliana, two abscisic acid- (ABA)-non-activated SNF1-releted protein kinases 2 (SnRK2) kinases, SnRK2.4 and SnRK2.10, are involved in the regulation of ROS homeostasis in response to salinity. They regulate the expression of several genes responsible for ROS generation at early stages of the stress response as well as those responsible for their removal. Moreover, the SnRK2.4 regulate catalase levels and its activity and the level of ascorbate in seedlings exposed to salt stress
Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors
Get PDFBackground: Interactions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation.
Results: Microbial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed.
Conclusions: Studies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion
Comparison of α-Helix and β-Sheet Structure Adaptation to a Quantum Dot Geometry: Toward the Identification of an Optimal Motif for a Protein Nanoparticle Cover
Get PDFWhile quantum dots (QDs) are useful as fluorescent labels, their application in biosciences is limited due to the stability and hydrophobicity of their surface. In this study, we tested two types of proteins for use as a cover for spherical QDs, composed of cadmium selenide. Pumilio homology domain (Puf), which is mostly α-helical, and leucine-rich repeat (LRR) domain, which is rich in β-sheets, were selected to determine if there is a preference for one of these secondary structure types for nanoparticle covers. The protein sequences were optimized to improve their interaction with the surface of QDs. The solubilization of the apoproteins and their assembly with nanoparticles required the application of a detergent, which was removed in subsequent steps. Finally, only the Puf-based cover was successful enough as a QD hydrophilic cover. We showed that a single polypeptide dimer of Puf, PufPuf, can form a cover. We characterized the size and fluorescent properties of the obtained QD:protein assemblies. We showed that the secondary structure of the Puf proteins was not destroyed upon contact with the QDs. We demonstrated that these assemblies do not promote the formation of reactive oxygen species during illumination of the nanoparticles. The data represent advances in the effort to obtain a stable biocompatible cover for QDs
Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans
Get PDFThe essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant
The Role of Dimethyl Sulfoxide (DMSO) in Gene Expression Modulation and Glycosaminoglycan Metabolism in Lysosomal Storage Disorders on an Example of Mucopolysaccharidosis
Get PDFObstacles to effective therapies for mucopolysaccharidoses (MPSs) determine the need
for continuous studies in order to enhance therapeutic strategies. Dimethyl sulfoxide (DMSO) is
frequently utilised as a solvent in biological studies, and as a vehicle for drug therapy and the in vivo
administration of water-insoluble substances. In the light of the uncertainty on the mechanisms of
DMSO impact on metabolism of glycosaminoglycans (GAGs) pathologically accumulated in MPSs,
in this work, we made an attempt to investigate and resolve the question of the nature of GAG
level modulation by DMSO, the isoflavone genistein solvent employed previously by our group in
MPS treatment. In this work, we first found the cytotoxic effect of DMSO on human fibroblasts at
concentrations above 3%. Also, our results displayed the potential role of DMSO in the regulation of
biological processes at the transcriptional level, then demonstrated a moderate impact of the solvent
on GAG synthesis. Interestingly, alterations of lysosomal ultrastructure upon DMSO treatment were
visible. As there is growing evidence in the literature that DMSO can affect cellular pathways leading
to numerous changes, it is important to expand our knowledge concerning this issue
Ancient Function of Teneurins in Tissue Organization and Neuronal Guidance in the Nematode Caenorhabditis elegans
Get PDFIndividual variation in migratory movements of chinstrap penguins leads to widespread occupancy of ice-free winter habitats over the continental shelf and deep ocean basins of the Southern Ocean
Get PDFA goal of tracking migratory animals is to characterize the habitats they use and to interpret population processes with respect to conditions experienced en route to, and within, overwintering areas. For migratory seabirds with broad breeding ranges, inferring population-level effects of environmental conditions that are experienced during migratory periods would benefit by directly comparing how birds from different breeding aggregations disperse, characterizing the physical conditions of areas they use, and determining whether they occupy shared foraging areas. We therefore tracked 41 adult and juvenile chinstrap penguins (Pygoscelis antarctica) from three breeding locations in the northern Antarctic Peninsula region during the austral winter of 2017. The satellite tracking data revealed overlap of individuals over continental shelf areas during autumn months (MarMay), shared outbound corridors that track the southern Antarctic circumpolar current front, followed by occupancy of progressively colder, deeper, and ice-free waters that spanned the entire western hemisphere south of the Polar Front. Despite broadly similar physical environments used by individuals from different colonies, the proportion of birds from each colony that remained within 500km of their colony was positively correlated with their local population trends. This suggests that local migration strategies near the Antarctic Peninsula may benefit breeding populations. However, the magnitude of intercolony and intra-colony overlap was generally low given the broad scale of habitats occupied. High individual variation in winter movements suggests that habitat selection among chinstrap penguins is more opportunistic, without clear colony-specific preference for fine-scale foraging hotspots. Mixing of individuals from multiple colonies across broad regions of the Southern Ocean would expose chinstrap penguins from the Antarctic Peninsula to a shared environmental experience that helps explain the regional decline in their abundance
Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity
Get PDFThe DXO family of proteins participates in eukaryotic mRNA 5'-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5'-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties
