Proteomic and transcriptomic analyses of Cutibacterium acnes biofilms and planktonic cultures in presence of epinephrine.

Transcriptomic and proteomic analysis were performed on 72 h biofilms of the acneic strain Cutibacterium acnes and planktonic cultures in the presence of epinephrine. Epinephrine predominantly downregulated genes associated with various transporter proteins. No correlation was found between proteomic and transcriptomic profiles. In control samples, the expression of 51 proteins differed between planktonic cultures and biofilms. Addition of 5 nM epinephrine reduced this number, and in the presence of 5 µM epinephrine, the difference in proteomic profiles between planktonic cultures and biofilms disappeared. According to the proteomic profiling, epinephrine itself was more effective in the case of C. acnes biofilms and potentially affected the tricarboxylic acid cycle (as well as alpha-ketoglutarate decarboxylase Kgd), biotin synthesis, cell division, and transport of different compounds in C. acnes cells. These findings are consistent with recent research on Micrococcus luteus, suggesting that the effects of epinephrine on actinobacteria may be universal.

[Characteristics of δ-Aminolevulinic Acid Dehydratase of the Cold-Water Sponge Halisarca dujardinii].

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals.

Data on nucleoid-associated proteins isolated from Mycoplasma gallisepticum after intracellular infection.

Mycoplasma gallisepticum (M. gallisepticum) belongs to the class of Mollicutes. It causes chronic respiratory disease in avian species. It is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, M. gallisepticum has a limited variety of DNA-binding proteins (DBP) and transcription factors. Consequently, the diversity of DNA-binding proteins and transcription factors (TF) in M. gallisepticum is limited in comparison with related bacteria such as Bacillus subtilis. Studies have shown, however, that mycoplasmas demonstrate a wide range of differential expression of genes in response to various stress factors, which promotes effective adaptation to unfavorable conditions. We assume that in the case of mycoplasmas, which are characterized by a combination of the reduction of known gene expression regulation systems and a high adaptive potential, the coordination of gene expression can be provided due to local changes in the structure and spatial organization of the chromosome. The study of the dynamic changes of the proteomic profile of M. gallisepticum nucleoid may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation including its changes upon invasion of the host cells.

LC-MS/MS analysis of lesional and normally looking psoriatic skin reveals significant changes in protein metabolism and RNA processing.

BACKGROUND: Plaque psoriasis is a chronic autoimmune disorder characterized by the development of red scaly plaques. To date psoriasis lesional skin transcriptome has been extensively studied, whereas only few proteomic studies of psoriatic skin are available. AIM: The aim of this study was to compare protein expression patterns of lesional and normally looking skin of psoriasis patients with skin of the healthy volunteers, reveal differentially expressed proteins and identify changes in cell metabolism caused by the disease. METHODS: Skin samples of normally looking and lesional skin donated by psoriasis patients (n = 5) and samples of healthy skin donated by volunteers (n = 5) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After protein identification and data processing, the set of differentially expressed proteins was subjected to protein ontology analysis to characterize changes in biological processes, cell components and molecular functions in the patients' skin compared to skin of the healthy volunteers. The expression of selected differentially expressed proteins was validated by ELISA and immunohistochemistry. RESULTS: The performed analysis identified 405 and 59 differentially expressed proteins in lesional and normally looking psoriatic skin compared to healthy control. In normally looking skin of the patients, we discovered decreased expression of KNG1, APOE, HRG, THBS1 and PLG. Presumably, these changes were needed to protect the epidermis from spontaneous activation of kallikrein-kinin system and delay the following development of inflammatory response. In lesional skin, we identified several large groups of proteins with coordinated expression. Mainly, these proteins were involved in different aspects of protein and RNA metabolism, namely ATP synthesis and consumption; intracellular trafficking of membrane-bound vesicles, pre-RNA processing, translation, chaperoning and degradation in proteasomes/immunoproteasomes. CONCLUSION: Our findings explain the molecular basis of metabolic changes caused by disease in skin lesions, such as faster cell turnover and higher metabolic rate. They also indicate on downregulation of kallikrein-kinin system in normally looking skin of the patients that would be needed to delay exacerbation of the disease. Data are available via ProteomeXchange with identifier PXD021673.

Optimization of Method for Human Sex Determination Using Peptidome Analysis of Teeth Enamel from Teeth of Different Biological Generation, Archeological Age, and Degrees of Taphonomic Preservation.

Determination of biological sex to human remains is a fundamental requirement in anthropological, archeological, and forensic anthropological studies. Sex determination based on morphological criteria is significantly limited in the cases of juvenile remains and adult skeletons in a poor state of preservation. Regular attempts have been made to use alternative techniques to resolve this issue, including analysis of tooth enamel peptides by liquid chromatography/mass spectrometry. Optimization of this method involving acid etching of tooth enamel for 10 min followed by desalting of the products of etching on SDB-RPS StageTips microcolumns and analysis of desalted sample (1/3) by liquid chromatography/mass spectrometry allowed reliable sex determination to fossil remains within a wide range of archeological and biological ages without destructing analyzed teeth. Increasing the duration of enamel etching ensured a 2 to 3-fold increase in the total number of identified peptides and, more importantly, in the number of identified fragments of amelogenin Y isoform specific for male teeth, which facilitated reliable sex determination of fossil remains. The suggested technique was tested with 8 permanent and 15 deciduous teeth of different archaeological age and different degree of preservation. Two amelogenin Y-specific peptide sequences were identified. One of these peptides [SM(+15.99)IRPPYS)] was found in all male-derived samples without exception; the other peptide [IRPPYSS(+79.97)], which contained phosphorylated Ser66 residue, was found only in the enamel from deciduous teeth, which suggests that phosphorylation of Ser66 plays a role in the enamel formation in deciduous teeth.

GPI-Modified Proteins Non-covalently Attached to Saccharomyces cerevisiae Yeast Cell Wall.

Yeast cell wall GPI-anchored proteins lack the lipid part of the anchor and are covalently bound to the high-molecular-weight polysaccharides glucan and/or chitin through the mannose residues. They perform many functions, including participation in the cell wall molecular ensemble formation and providing cell resistance to stress. In this work, we identified a pool of GPI-modified proteins firmly bound to the cell wall by non-covalent interactions with the high-molecular-weight structural polysaccharides. We believe that the detected proteins are intermediate forms in the processing of the cell wall GPI-proteins, since they had already lost the lipid part of the GPI anchor and are absent in the lipoprotein fraction extracted according to Folch, but were not yet incorporated into the cell wall by the covalent binding to high-molecular-weight polysaccharides because they could be extracted into water by heating of delipidized cell walls. This group of previously unknown proteins might be present in the cell wall in a form of lipid-associated microcompartments represented by transport vesicles recently found in yeast. GPI-modified proteins non-covalently attached to the high-molecular-weight polysaccharides were found in the cell walls of both the parent strain and yeast devoid of glucanosyltransglycosylase Bgl2, which indicates that the pathway of their incorporation into the cell wall is independent on this enzyme.

Dimeric Disintegrins from the Steppe Viper V. ursinii Venom.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the αVß1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa. The method of peptide mapping established the structure of a new heterodimeric disintegrin weighing 13 995.5 Da and shows that it belongs to the class of RGD/KGD-containing disintegrins.

Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ±â€¯0.7) × 106 M-1s-1 and an SI of 4.7 ±â€¯0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

[Enhancement of Na,K-ATPase Activity as a Result of Removal of Redox Modifications from Cysteine Residues of the al Subunit: the Effect of Reducing Agents].

Na,K-ATPase is a transmembrane enzyme that creates a gradient of sodium and potassium, which is necessary for the viability of animal cells. The activity of Na,K-ATPase depends on the redox status of the cell, decreasing with oxidative stress and hypoxia. Previously, we have shown that the key role in the redox sensitivity of Na,K-ATPase is played by the regulatory glutathionylation of cysteine residues of the catalytic alpha subunit, which leads to the inhibition of the enzyme. In this study, the effect of reducing agents (DTT, ME, TCEP) on the level of glutathionylation of the alpha subunit of Na,K-ATPase from rabbit kidneys and the enzyme activity has been evaluated. We have found that the reducing agents partially deglutathionylate the protein, which leads to its activation. It was impossible to completely remove glutathionylation from the native rabbit kidney protein. The treatment of a partially denatured protein on the PVDF membrane with reducing agents (TCEP, NaBH4) also does not lead to the complete deglutathionylation of the protein. The obtained data indicate that Na,K-ATPase isolated from rabbit kidneys has both regulatory and basal glutathionylation, which appears to play an important role in the redox regulation of the function of Na, K-ATPase in mammalian tissues.

New paradoxical three-finger toxin from the cobra Naja kaouthia venom: Isolation and characterization.

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of "orphan" toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.

Mediators and Biomarkers of Inflammation in Meningitis: Cytokine and Peptidome Profiling of Cerebrospinal Fluid.

Differential diagnosis of bacterial and viral meningitis is an urgent problem of the modern clinical medicine. Early and accurate detection of meningitis etiology largely determines the strategy of its treatment and significantly increases the likelihood of a favorable outcome for the patient. In the present work, we analyzed the peptidome and cytokine profiles of cerebrospinal fluid (CSF) of 17 patients with meningitis of bacterial and viral etiology and of 20 neurologically healthy controls. In addition to the identified peptides (potential biomarkers), we found significant differences in the cytokine status of the CSF of the patients. We found that cut-off of 100 pg/ml of IL-1ß, TNF, and GM-CSF levels discriminates bacterial and viral meningitis with 100% specificity and selectivity. We demonstrated for the first time the reduction in the level of two cytokines, IL-13 and GM-CSF, in the CSF of patients with viral meningitis in comparison with the controls. The decrease in GM-CSF level in the CSF of patients with viral meningitis can be explained by a disproportionate increase in the levels of cytokines IL-10, IFN-γ, and IL-4, which inhibit the GM-CSF expression, whereas IL-1, IL-6, and TNF activate it. These observations suggest an additional approach for differential diagnosis of bacterial and viral meningitis based on the normalized ratio IL-10/IL-1ß and IL-10/TNF > 1, as well as on the ratio IFN-γ/IL-1ß and IFN-γ/TNF < 0.1. Our findings extend the panel of promising clinical and diagnostic biomarkers of viral and bacterial meningitis and reveal opposite changes in the cytokine expression in meningitis due to compensatory action of pro- and antiinflammatory factors.

Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

Changes in the serum protein composition in mice with transplanted Ehrlich's carcinoma.

Injection of blood serum from mice with Ehrlich's carcinoma stimulates the growth of transplanted tumor, which proves the presence of tumor-specific factors in the serum. Experiments on (CBA×C57Bl/6)F1 male mice with transplanted Ehrlich's carcinoma demonstrated the appearance of new proteins in the serum, some of them are identified. The authors suggest continuing the search for tumor-associated factor by combining proteomic analytical methods and testing of identified candidate proteins for their effects on tumor growth.

[Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin ß-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the ß-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

[Analysis of extracellular proteome of Staphylococcus aureus 6 at the end of exponential growth phase].

AIM: Determine protein specter that Staphylococcus aureus synthesizes and secretes at early growth phase--the exponential phase. MATERIALS AND METHODS: Proteins secreted by S. aureus strain 6 into cultivation medium at the end of exponential growth phase (4.5 hours) were studied. 11 proteins were identified by liquid chromatography--mass-spectrometry method. RESULTS: Only in 3 of these proteins the presence of signal peptides was predicted, which indicates their extracellular localization; the rest of the proteins were localized predominantly in bacterial cytoplasm. 5 of 11 proteins function as enzymes of carbohydrate metabolism. Other extracellular proteins that could indicate its contamination with proteins from disrupted bacterial cells were not detected in S. aureus cultural liquid filtrate. It has been suggested that enzymes of carbohydrate metabolism can provide bacterial cells with energy necessary for passage from lag-phase into exponential growth phase. Superoxide dismutase enzyme probably provides the course of oxidation-reduction processes. Synthesis of other proteolytic enzymes and toxins is carried out at later stages of development of bacterial population. Immunization of mice with a mixture of 11 identified proteins showed their protective properties after infection by S. aureus 6 strain. CONCLUSION: Based on the above-mentioned, the complex of isolated proteins may be perspective in development of a new strategy of prophylaxis and therapy of staphylococcus infections.

[Proteomic technologies for identification of serum potential biomarkers of autoimmune demyelinating polyneuropathies].

Time-of-flight MALDI mass spectrometry (MALDI-TOF-MS) profiling of blood serum of patients with Guillain-Barré syndrome (GBS, 36 samples), chronic inflammatory demyelinating polyneuropathy (CIDP, 24 samples) and practically healthy donors (HD) (35 samples) was carried out in order to identify potential biomarkers of autoimmune demyelinating polyneuropathies (ADP). To simplify the peptide-protein mixture of serum prior to MALDI-TOF-MS analysis samples were pre-fractionated on magnetic microparticles with a weak cation-exchange (MB-WCX) surface. Comparative analysis of mass spectrometric data using the classification algorithms (genetic and neural network-controlled) revealed a characteristic set of peaks, agreed change area with a high specificity and sensitivity of the differentiated mass spectrometry profiles of the blood serum of patients with DPNP and healthy donors (for GBS values of these characteristics reached 100 and 100, and for CIDP 94.1 and 100% respectively). Comparative analysis of mass spectrometric profiles of serum samples obtained from patients with GBS and CIDP, allowed to build a classification model to differentiate these diseases from each other, with a specificity of 88.9 and a sensitivity of 80%.

[Exploration and identification of Physcomitrella patens moss peptides].

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.

Growth-dependent release of carbohydrate metabolism-related and antioxidant enzymes from Staphylococcus aureus strain 6 as determined by proteomic analysis.

Proteins released into the culture medium by Staphylococcus aureus (S. aureus) strain 6 were determined at the end of the exponential growth phase (4.5 h). Eleven proteins were identified by liquid chromatography coupled with mass spectrometry. Three proteins were predicted to have signal peptides indicating their extracellular localization. The other proteins were presumably located in the cytoplasm of the bacteria. Five out of the 11 proteins were involved in carbohydrate metabolism. Other intracellular proteins of S. aureus were not detected in the culture medium. This indicates that the release of these 11 proteins was specific and that unspecific protein release due to damaged or dying bacteria did not play a role. It is suggested that enzymes associated with carbohydrate metabolism may provide the energy necessary for the transition of bacteria from a resting to a proliferative state. Another enzyme released by S. aureus, superoxide dismutase, may catalyze redox reactions in this context. The production of other proteolytic enzymes and toxins may take place at later stages of bacterial growth. A cocktail of these 11 proteins was used for the immunization of mice. Indeed, vaccination with these proteins prolonged the survival times of mice upon infection with S. aureus strain 6. Therefore, these proteins may have implications for the development of novel strategies for the prevention and therapy of S. aureus infections.