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GSK3 alpha and GSK3 beta phosphorylate arc and regulate its degradation
Get PDFThe selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases α and β (GSK3α, GSKβ; collectively named GSK3α/β) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3α and GSK3β contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/β substrate and showed that GSKβ promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3α/β inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3β-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3β terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/β-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function
Cut-and-paste transposons in fungi with diverse lifestyles
Get PDFTransposons (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighbourhoods and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a “cut and paste” fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole dataset and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species’ lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition
Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain.
Get PDFH1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species
A meiosis-specific Spt5 homolog involved in non-coding transcription
Get PDFSpt5 is a conserved and essential transcriptional regulator that binds directly to RNA polymerase and is involved in transcription elongation, polymerase pausing and various co-transcriptional processes. To investigate the role of Spt5 in non-coding transcription, we used the unicellular model Paramecium tetraurelia. In this ciliate, development is controlled by epigenetic mechanisms that use different classes of non-coding RNAs to target DNA elimination. We identified two SPT5 genes. One (STP5v) is involved in vegetative growth, while the other (SPT5m) is essential for sexual reproduction. We focused our study on SPT5m, expressed at meiosis and associated with germline nuclei during sexual processes. Upon Spt5m depletion, we observed absence of scnRNAs, piRNA-like 25 nt small RNAs produced at meiosis. The scnRNAs are a temporal copy of the germline genome and play a key role in programming DNA elimination. Moreover, Spt5m depletion abolishes elimination of all germline-limited sequences, including sequences whose excision was previously shown to be scnRNA-independent. This suggests that in addition to scnRNA production, Spt5 is involved in setting some as yet uncharacterized epigenetic information at meiosis. Our study establishes that Spt5m is crucial for developmental genome rearrangements and necessary for scnRNA production
Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain
Get PDFThe ability of Lactococcus lactis to adhere to the
intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells.
The results indicate that two plasmids, pIBB477a and b, are
involved in adhesion of the IBB477 strain. One of the genes
localised on plasmid pIBB477b (AJ89_14230), which encodes
cell wall-associated peptidase S8 (PrtP), mediates adhesion
of the IBB477 strain to bare, mucin- and fibronectincoated
polystyrene, as well as to HT29-MTX cells.
Interactions between bacteria and mucus secreted by HT29-
MTX cells were further investigated by fluorescent staining
and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus.
Finally, the ability of IBB477 strain and its ΔprtP deletion
mutant to colonise the gastrointestinal tract of conventional C57Bl/6mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut
Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa
Get PDFSimilarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein
ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but
results in increased frequency of anucleate cells production, longer division time, cell elongation,
altered colony morphology and defective swarming and swimming motility. Unlike in
other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting
that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism.
ParB overproduction affects growth rate, cell division and motility in a similar way as ParB
deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase
in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes
in growth rate and chromosome segregation, significantly alters the expression of 176 loci.
Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4
close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences
the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB- parS3/
parS4 interactions repress the transcription in this region. In addition, increased ParB level
brings about repression or activation of numerous genes including several transcriptional regulators
involved in SOS response, virulence and adaptation. Overall, our data support the
role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa
In vitro characteristics of Lactobacillus spp. strains isolated from the chicken digestive tract and their role in the inhibition of Campylobacter colonization
Get PDFCampylobacter jejuni/coli infections are the leading cause of bacterial diarrheal illnesses in humans. Many epidemiological studies indicate that improperly prepared meat from chickens that carry a high load of Campylobacter in their intestinal tracts is the key source of human infections. LAB, mainly members of the Lactococcus and Lactobacillus genera, increasingly have been tested as vehicles for the delivery of heterologous bacterial or viral antigens to animal mucosal immune systems. Thus, the objective of this study was to isolate, identify, and characterize Lactobacillus spp. strains isolated from chickens bred in Poland. Their ability to decrease the level of bird gut colonization by C. jejuni strain was also analyzed. First, the influence of the different chicken rearing systems was evaluated, especially the effect of diets on the Lactobacillus species that colonize the gut of chickens. Next, selected strains were analyzed in terms of their anti-Campylobacter activity in vitro; potential probiotic traits such as adhesion properties, bile and low pH tolerance; and their ability to grow on a defined carbon source. Given that improperly prepared chicken meat is the main source of human infection by Campylobacter, the selected strains were also assessed for their ability to inhibit Campylobacter colonization in the bird's intestine. These experiments revealed enormous physiological diversity among the Lactobacillus genus strains. Altogether, our results showed that L. plantarum strains isolated from the digestive tracts of chickens bred in Poland displayed some probiotic attributes in vitro and were able to decrease the level of bird gut colonization by C. jejuni strain. This suggests that they can be employed as vectors to deliver Campylobacter immunodominant proteins to the bird's immune system to strengthen the efficacy of in ovo vaccination
HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies
No full textDownregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors
A Strong Neutrophil Elastase Proteolytic Fingerprint Marks the Carcinoma Tumor Proteome
Get PDFProteolytic cascades are deeply involved in critical stages
of cancer progression. During the course of peptide-wise
analysis of shotgun proteomic data sets representative of
colon adenocarcinoma (AC) and ulcerative colitis (UC), we
detected a cancer-specific proteolytic fingerprint com-
posed of a set of numerous protein fragments cleaved
C-terminally to V, I, A, T, or C residues, significantly over-represented in AC. A peptide set linked by a common
VIATC cleavage consensus was the only prominent can-
cer-specific proteolytic fingerprint detected. This se-
quence consensus indicated neutrophil elastase as a
source of the fingerprint. We also found that a large frac-
tion of affected proteins are RNA processing proteins
associated with the nuclear fraction and mostly cleaved
within their functionally important RNA-binding domains.
Thus, we detected a new class of cancer-specific pep-
tides that are possible markers of tumor-infiltrating neu-
trophil activity, which often correlates with the clinical
outcome. Data are available via ProteomeXchange with
identifiers: PXD005274 (Data set 1) and PXD004249 (Data
set 2). Our results indicate the value of peptide-wise anal-
ysis of large global proteomic analysis data sets as op-
posed to protein-wise analysis, in which outlier differen-
tial peptides are usually neglected
Isolation and Characterization of Pseudomonas spp. Strains That Efficiently Decompose Sodium Dodecyl Sulfate
Get PDFDue to their particular properties, detergents are widely used in household cleaning products, cosmetics, pharmaceuticals, and in agriculture as adjuvants tailoring the features of pesticides or other crop protection agents. The continuously growing use of these various products means that water soluble detergents have become one of the most problematic groups of pollutants for the aquatic and terrestrial environments. Thus it is important to identify bacteria having the ability to survive in the presence of large quantities of detergent and efficiently decompose it to non-surface active compounds. In this study, we used peaty soil sampled from a surface flow constructed wetland in a wastewater treatment plant to isolate bacteria that degrade sodium dodecyl sulfate (SDS). We identified and initially characterized 36 Pseudomonas spp. strains that varied significantly in their ability to use SDS as their sole carbon source. Five isolates having the closest taxonomic relationship to the Pseudomonas jessenii subgroup appeared to be the most efficient SDS degraders, decomposing from 80 to 100% of the SDS present in an initial concentration 1 g/L in less than 24 h. These isolates exhibited significant differences in degree of SDS degradation, their resistance to high detergent concentration (ranging from 2.5 g/L up to 10 g/L or higher), and in chemotaxis toward SDS on a plate test. Mass spectrometry revealed several SDS degradation products, 1-dodecanol being dominant; however, traces of dodecanal, 2-dodecanol, and 3-dodecanol were also observed, but no dodecanoic acid. Native polyacrylamide gel electrophoresis zymography revealed that all of the selected isolates possessed alkylsulfatase-like activity. Three isolates, AP3_10, AP3_20, and AP3_22, showed a single band on native PAGE zymography, that could be the result of alkylsulfatase activity, whereas for isolates AP3_16 and AP3_19 two bands were observed. Moreover, the AP3_22 strain exhibited a band in presence of both glucose and SDS, whereas in other isolates, the band was visible solely in presence of detergent in the culture medium. This suggests that these microorganisms isolated from peaty soil exhibit exceptional capabilities to survive in, and break down SDS, and they should be considered as a valuable source of biotechnological tools for future bioremediation and industrial applications