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Overexpression of the Selective Autophagy Cargo Receptor NBR1 Modifies Plant Response to Sulfur Deficit
Get PDFPlants exposed to sulfur deficit elevate the transcription of NBR1 what might reflect an increased demand for NBR1 in such conditions. Therefore, we investigated the role of this selective autophagy cargo receptor in plant response to sulfur deficit (-S). Transcriptome analysis of the wild type and NBR1 overexpressing plants pointed out differences in gene expression in response to -S. Our attention focused particularly on the genes upregulated by -S in roots of both lines because of significant overrepresentation of cytoplasmic ribosomal gene family. Moreover, we noticed overrepresentation of the same family in the set of proteins co-purifying with NBR1 in -S. One of these ribosomal proteins, RPS6 was chosen for verification of its direct interaction with NBR1 and proven to bind outside the NBR1 ubiquitin binding domains. The biological significance of this novel interaction and the postulated role of NBR1 in ribosomes remodeling in response to starvation remain to be further investigated. Interestingly, NBR1 overexpressing seedlings have significantly shorter roots than wild type when grown in nutrient deficient conditions in the presence of TOR kinase inhibitors. This phenotype probably results from excessive autophagy induction by the additive effect of NBR1 overexpression, starvation, and TOR inhibition
Biotope and biocenosis of cryoconite hole ecosystems on Ecology Glacier in the maritime Antarctic
Get PDFAbstract: Despite recent great interest in glacier ecosystems in the continental Antarctic, little is known about their maritime counterparts. Our study presents descriptive data on cryoconite sediments and cryoconite holes on Ecology Glacier (King George Island) to accomplish three main objectives: (a) to identify main eukaryotic (algae, invertebrates) and prokaryotic (cyanobacteria) components of microbial communities; (b) to provide a “baseline” of community composition, organic matter and artificial contamination; and (c) identify key abiotic factors that might be important in community assembly. Cryoconite holes were sampled along an altitudinal gradient of Ecology Glacier in January, mid Austral Summer 2017. Cryoconite holes located in lower altitude were deeper than those located in the middle and the highest altitude. Seventeen species of algae and cyanobacteria with biomass of 0.79 to 5.37 µg/cm3 have been found in sediments. Dominant species were cyanobacterial Pseudanabaena frigida and Bacillariophyceae Microcostaus sp. Biomass of Bacillariophyceae was significantly higher than that of Chlorophyta and Cyanobacteria. We found three species of rotifers (two potentially new to science) and for the first time a glacier dwelling Acari (suspension feeder, Nanorchestes nivalis). Organic matter content ranged from 5.4% to 7.6%. Investigated artificial radionuclides included 137Cs, 238Pu, 239+240Pu and 241Am. 210Pb seems to be related to organic matter content. Overall, cryoconite holes on Ecology Glacier present unique habitats that serve as biodiversity hotspots of psychrophiles, source of organic matter, matrices for radioactivity tracking and model for observing changes in supraglacial ecosystems in Maritime Antarctic
Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum
Get PDFThe cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite
Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae
Get PDF: In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans
Polyprenol‑Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats
Get PDFDevelopment of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study,the effectiveness of two PTAI-based formulations (PTAI-6–8 and 10–14) was investigated
and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGFA)in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma
cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10–14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high
enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery
Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications
Get PDFContinuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism
Vimentin S‐glutathionylation at Cys328 inhibits filament elongation and induces severing of mature filaments in vitro
Get PDFVimentin intermediate filaments are a significant component of the
cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and
disassemble and thus participate in the dynamic processes of the cell. Posttranslational
modifications (PTMs) such as protein phosphorylation regulate
the multiphasic association of vimentin from soluble complexes to insoluble
filaments and the reverse processes. The thiol side chain of the single vimentin
cysteine at position 328 (Cys328) is a direct target of oxidative modifications
inside cells. Here, we used atomic force microscopy, electron
microscopy and a novel hydrogen–deuterium exchange mass spectrometry
(HDex-MS) procedure to investigate the structural consequences of S-nitrosylation
and S-glutathionylation of Cys328 for in vitro oligomerisation of
human vimentin. Neither modification affects the lateral association of tetramers
to unit-length filaments (ULF). However, S-glutathionylation of
Cys328 blocks the longitudinal assembly of ULF into extended filaments. Snitrosylation
of Cys328 does not hinder but slows down the elongation.
Likewise, S-glutathionylation of preformed vimentin filaments causes their
extensive fragmentation to smaller oligomeric species. Chemical reduction of
the S-glutathionylated Cys328 thiols induces reassembly of the small fragments
into extended filaments. In conclusion, our in vitro results suggest Sglutathionylation
as a candidate PTM for an efficient molecular switch in
the dynamic rearrangements of vimentin intermediate filaments, observed
in vivo, in response to changes in cellular redox status. Finally, we demonstrate
that HDex-MS is a powerful method for probing the kinetics of
vimentin filament formation and filament disassembly induced by PTMs
Glaucoma patients have an increased level of trimethylamine, a toxic product of gut bacteria, in the aqueous humor: a pilot study
Get PDFAβ5−xPeptides: N‑Terminal Truncation Yields Tunable Cu(II)Complexes
Get PDFThe Aβ5−x peptides (x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ5−x peptide models comprising the metal binding site: Aβ5−9, Aβ5−12, Aβ5−12Y10F, and Aβ5−16. Using CD and UV−vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M−1 at pH 7.4 for Aβ5−9 and Aβ5−12. This affinity was elevated 3-fold in Aβ5−16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ5−9 and Aβ5−12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ5−x complexes
could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ5−x peptides, which bind Cu(II) ions much more strongly than Aβ1−x peptides and only slightly weaker than Aβ4−x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains
BacSJ—Another Bacteriocin with Distinct Spectrum of Activity that Targets Man-PTS
Get PDFLactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L.lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L.lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ