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Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase.
Get PDFAtopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation play a role in pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and while CD1a-expressing Langerhans cells are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. Here we observed that house dust mite (HDM) generates neolipid antigens for presentation by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth and showed rapid effector function, consistent with antigen-driven maturation. To define the underlying mechanisms, we analyzed HDM-challenged human skin and observed allergen-derived phospholipase (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2 and such cells infiltrated the skin after allergen challenge. Filaggrin insufficiency is associated with atopic dermatitis, and we observed that filaggrin inhibits PLA2 activity and inhibits CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity, such as Gell and Coombs are predicated on the idea that non-peptide stimulants of T cells act as haptens that modify peptides or proteins. However our results point to a broader model that does not posit haptenation, but instead shows that HDM proteins generate neolipid antigens which directly activate T cells. Specifically, the data identify a pathway of atopic skin inflammation, in which house dust mite-derived phospholipase A2 generates antigenic neolipids for presentation to CD1a-reactive T cells, and define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach
Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/ Cxcr7 axis
Get PDFThe skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite
cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and
remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as
extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies
aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous
studies we showed that Sdf-1 (stromal derived factor ¡1) increased migration of stem cells and
their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the
expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study
we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1
receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary
myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the
cells of different migration and myogenic potential. We showed that Sdf-1 altered actin
organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-
Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the
transcription profile of genes encoding factors engaged in cells adhesion and migration. As the
result, cells such as primary myoblasts or embryonic stem cells, became characterized by more
effective migration when transplanted into regenerating muscle
An electrochemical immunosensor based on a 4,4′-thiobisbenzenethiol self-assembled monolayer for the detection of hemagglutinin from avian influenza virus H5N1
Get PDFAn electrochemical immunosensor for the detection of hemagglutinin from avian influenza virus H5N1 is presented in this paper. The following steps lead up to the construction of immunosensor: (i) modification of gold electrodes with 4,4′-thiobisbenzenethiol, (ii) modification of self-assembled monolayer of 4,4′-thiobisbenzenethiol with gold colloidal nanoparticles, (iii) immobilization of single chain variable fragments of antibodies (scFv) against hemagglutinin H5 via Ssingle bondAu covalent bonds, (iv) blocking of the remaining free space with bovine serum albumin. The interactions between the scFv and hemagglutinin variants have been explored with electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3−/4− as an electroactive marker. The immunosensor was able to detect two different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: the short fragment (17–340 residues) of A/swan/Poland/305-135V08/2006 and the long (17–530 residues) of A/Bar-headed Goose/Qinghai/12/2005. The strongest response has been observed for the long variant with a detection limit of 0.6 pg/mL and a dynamic range from 4.0 to 20.0 pg/mL. The recombinant hemagglutinin (17–527 residues) from A/chicken/Netherlands/1/03 (H7N7), used as the negative control generated a weak response. This confirms the selectivity of the immunsensor proposed. A miniaturized version of the immunosensor, based on screen-printed gold electrodes, was tested with the same set of recombinant hemagglutinins and it achieved a linear range from 1 to 8 pg/mL with a detection limit of 0.9 pg/mL for the long fragment of hemagglutinin
Phosphorylation of spore coat proteins by a family of atypical protein kinases
Get PDFThe modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology
Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications
Get PDFβ-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-αÐinduced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications
Maf1-mediated regulation of yeast RNA polymerase III is correlated with CCA addition at the 3′ end of tRNA precursors
Get PDFIn eukaryotic cells tRNA synthesis is negatively regulated by the protein Maf1, conserved from yeast to humans.
Maf1 from yeast Saccharomyces cerevisiae mediates repression of trna transcription when cells are transferred
from medium with glucose to medium with glycerol, a non-fermentable carbon source. The strain with deleted
gene encoding Maf1 (maf1Δ) is viable but accumulates tRNA precursors. In this study tRNA precursors were analysed by RNA-Seq and Northern hybridization in wild type strain and maf1Δ mutant grown in glucose medium or upon shift to repressive conditions. A negative effect of maf1Δ mutant on the addition of the auxiliary CCA nucleotides to the 3′ end of pre-tRNAs was observed in cells shifted to unfavourable growth conditions. This effectwas reduced by overexpression of the yeast CCA1 gene encoding ATP(CTP):tRNA nucleotidyltransferase. The CCA sequence at the 3′ end is important for export of tRNA precursors from the nucleus and essential for tRNA charging with amino acids. Data presented here indicate that CCA-addition to intron-containing end-processed tRNA precursors is a limiting step in tRNA maturation when there is no Maf1 mediated RNA polymerase III (Pol III) repression. The correlation between CCA synthesis and Pol III regulation by Maf1 could be important in coordination of tRNA transcription, processing and regulation of translation
Control of Saccharomyces cerevisiae pre-tRNA processing by environmental conditions.
Get PDFtRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3' termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNAUAU (Ile) and pre-tRNAUUU (Lys) these aberrant forms are unprocessed at the 5' ends, but they possess extended 3' termini. Sequencing analyses showed that partial 3' processing precedes 5' processing for pre-tRNAUAU (Ile). An aberrant pre-tRNA(Tyr) that accumulates also possesses extended 3' termini, but it is processed at the 5' terminus. Similar forms of these aberrant pre-tRNAs are detected in the rex1Δ strain that is defective in 3' exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3' end protection. We further show direct correlation between the inhibition of 3' end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3' end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3' processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3' end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions
Halogen bonds involved in binding of halogenated ligands by protein kinases.
Get PDFAnalysis of 664 known structures of protein kinase complexes with halogenated ligands revealed 424 short contacts between a halogen atom and a potential protein X-bond acceptor, the topology and geometry of which were analyzed according to the type of a halogen atom (X = Cl, Br, I) and a putative protein X-bond acceptor. Among 236 identified halogen bonds, the most represented ones are directed to backbone carbonyls of the hinge region and may replace the pattern of ATP-like hydrogen bonds. Some halogen-π interactions with either aromatic residues or peptide bonds, that accompany the interaction with the hinge region, may possibly enhance ligand selectivity. Interestingly, many of these halogen-π interactions are bifurcated. Geometrical preferences identify iodine as the strongest X-bond donor, less so bromine, while virtually no such preferences were observed for chlorine; and a backbone carbonyl as the strongest X-bond acceptor. The presence of a halogen atom in a ligand additionally affects the properties of proximal hydrogen bonds, which according to geometrical parameters get strengthened, when a nitrogen of a halogenated ligand acts as the hydrogen bond donor
The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase, considered as a risk factor for stroke due to increased homocysteine level, can be protected and reactivated by heat shock proteins
Get PDFThe A222V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients
A Multiplexed Cytokeratin Analysis Using Targeted Mass Spectrometry Reveals Specific Profiles in Cancer-Related Pleural Effusions
Get PDFPleural effusion (PE), excess fluid in the pleural space, is often observed in lung cancer patients and also forms due to many benign ailments. Classifying it quickly is critical, but this remains an analytical challenge often lengthening the diagnosis process or exposing patients to unnecessary risky invasive procedures. We tested the analysis of PE using a multiplexed cytokeratin (CK) panel with targeted mass spectrometry–based quantitation for its rapid classification. CK markers are often assessed in pathological examinations for cancer diagnosis and guiding treatment course. We developed methods to simultaneously quantify 33 CKs in PE using peptide standards for increased analytical specificity and a simple CK enrichment method to detect their low amounts. Analyzing 121 PEs associated with a variety of lung cancers and noncancerous causes, we show that abundance levels of 10 CKs can be related to PE etiology. CK-6, CK-7, CK-8, CK-18, and CK-19 were found at significantly higher levels in cancer-related PEs. Additionally, elevated levels of vimentin and actin differentiated PEs associated with bacterial infections. A classifier algorithm effectively grouped PEs into cancer-related or benign PEs with 81% sensitivity and 79%specificity. A set of undiagnosed PEs showed that our method has potential to shorten PE diagnosis time. For the first time, we show that a cancer-relevant panel of simple-epithelial CK markers currently used in clinical assessment can also be quantitated in PEs. Additionally, while requiring less invasive sampling, our methodology demonstrated a significant ability to identify cancer-related PEs in clinical samples and thus could improve patient care in the future