S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Regulation of Serotonin 1A Receptor SUMOylation by SENP2 and PIASxα

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17β-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated.

p-STAT3 is a PDC-E2 interacting partner in human cholangiocytes and hepatocytes with potential pathobiological implications

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

A Proliferation-Inducing Ligand Regulation in Polymorphonuclear Neutrophils by Panax ginseng

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor superfamily that was first identified as a factor favoring tumorigenesis. APRIL is important fitness and survival factors for B cells and plasma cells in the periphery. Considering this, as well as the quantitative predominance of neutrophils among the peripheral blood leukocytes, we carried out the first study assessing the influence of the transforming growth factor (TGF)-β signaling pathway on APRIL expression in these cells. Furthermore, as the Rb1 ginsenoside is known to exhibit multiple pharmacological activities, we verified if the saponin is capable of modulating the process. The present study shows that TGF-β increased the expression of APRIL and the level of phospho-p38, phospho-Akt(T308), and phospho-Akt(S473) in the cytoplasmic fraction, as well as the expression of Fra1, c-Fos, and c-Jun in the nuclear fraction, of neutrophils. However, exposure of these cells to Rb1 reduced the expression and level of the investigated proteins. No changes were found in the expression of APRIL and the level of p-p38 in the cytoplasmic fraction of neutrophils following the application of Rb1 alone, as well as in the neutrophils incubated first with Rb1 and then with TGF-β, whereas a higher level of phosphorylation was observed for Akt and PI3 kinases in the cells. Moreover, a higher expression of all the studied transcription factors was observed in the nuclear fraction of neutrophils. Based on the observed changes, it may be assumed that the expression of APRIL molecule in TGF-β-induced neutrophils and its regulation by Rb1 are associated with PI3K/AKT signaling pathways and transcription factors Fra-1, Fra-2, c-Jun, and c-Fos. Rb1 appears to be a favorable factor that may be potentially used in the modulation of tumor-promoting APRIL expression.

Identification and localization of proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms

Background:To successfully colonize the oral cavity, bacteria must adhere directly or indirectly to the oral surfaces available. Fusobacterium nucleatum plays an important role in the development of the oral biofilm community due to its broad adhesion capabilities, serving as a bridge between the members of the oral biofilm community that cannot be directly joined together. The purpose of this study was to identify and localize the proteins associated with the formation of biofilms of Streptococcus gordonii and F. nucleatum. Methods: Multispecies biofilms were identified by amplification of the srtA and radD genes by real-time PCR. Biofilm cells cultured with sucrose were counted. The protein concentrations in the membrane and cytoplasmic fractions were quantified by western blot. Results: The proteins HSP40 and GAPDH were detected in the cytoplasmic fraction of biofilm and F. nucleatum, respectively. The available anti-GAPDH antibody is specific for GAPDH produced by F. nucleatum, which indicated the coaggregation of F. nucleatum on S. gordonii. Conclusions: HSP40 was only detected in the cytoplasmic fraction of the biofilms, making it one of the essential proteins for adherence. This complex set of interactions could have critical implications for the formation and maturation of oral biofilms in vivo and could provide clues to the mechanism behind the distribution of organisms within the human oral cavity.

The effect of arginine on the activity and compartmentalization of lysosomal cysteine proteinases of parenchymatous organs in oxidative stress on the background of experimental hyperhomocysteinemia

Aim. Evaluation of the effect of arginine on the activity and intracellular distribution of cathepsins B, L, H in liver, kidney and lung tissue in experimental hyperhomocysteinemia and developing on its background oxidative damage of proteins. Materials and Methods. Hyperhomocysteinemia in male rats of the Wistar line was formed by daily oral administration of a suspension of methionine at a dose of 1.5 g/kg 2 times a day for 21 days, to study the action of arginine the substance was used orally from 12 to 21 days of methionine administration at a dose of 500 mg/kg. In tissue homogenates measurements were carried out in cytoplasmic and lysosomal fractions. The state of oxidative modification of proteins was evaluated by analysis of the absorption spectrum of carbonyl derivatives, the activity of cathepsins B, L, H was determined by spectrofluorometric method, the activity of acid phosphatase – by the unified method «at the end point». Results. In the cytoplasmic (nonsedimentary) fraction of the liver and kidney reduced activity of cathepsin L (in both tissues), cathepsin B (in the kidney), cathepsin H (in the liver) was found on the background of the increase in the products of proteins oxidative modification in experimental hyperhomocysteinemia. The introduction of arginine in experimental hyperhomocysteinemia completely eliminated the manifestations of oxidative damage of proteins, partially correcting the activity of enzymes: there was an increase in the activity in non sedimentary (cytoplasmic) fraction due to intracellular redistribution of enzymes. There is found inverse correlation between the content of oxidative carbonylation products of proteins and the activity of cathepsins in the non sedimentary fraction, as well as the proportion of their non sedimentary activity. Conclusions. 1. Arginine at a dose of 500 mg/kg at a 10day administration completely corrects the increase in the products of oxidative protein carbonylation that develops in experimental hyperhomocysteinemia. 2. Under the influence of arginine there is an increase in the reduced due to isolated hyperhomocysteinemia activity of cathepsins B, L, H in the cytoplasmic fraction of the liver and kidney due to intracellular redistribution of enzymes. 3. Arginine administration causes nonselective increase of the lysosomal membrane permeability, and as a result, changes in the compartmentalization of lysosomal cysteine proteases. 4. The inverse correlation of the level of protein oxidative modification products with the activity of cathepsins in cytoplasmic (nonsedimentary) fractions, and the proportions of their nonsedimentary activity, suggesting the presence of contribution of changes in the compartmentalization of lysosomal cysteine proteinases in developing under the action of arginine compensation of oxidative stress in experimental hyperhomocysteinemia.

The effect of arginine on the activity and compartmentalization of lysosomal cysteine proteinases of parenchymatous organs in oxidative stress on the background of experimental hyperhomocysteinemia

Aim. Evaluation of the effect of arginine on the activity and intracellular distribution of cathepsins B, L, H in liver, kidney and lung tissue in experimental hyperhomocysteinemia and developing on its background oxidative damage of proteins. Materials and Methods. Hyperhomocysteinemia in male rats of the Wistar line was formed by daily oral administration of a suspension of methionine at a dose of 1.5 g/kg 2 times a day for 21 days, to study the action of arginine the substance was used orally from 12 to 21 days of methionine administration at a dose of 500 mg/kg. In tissue homogenates measurements were carried out in cytoplasmic and lysosomal fractions. The state of oxidative modification of proteins was evaluated by analysis of the absorption spectrum of carbonyl derivatives, the activity of cathepsins B, L, H was determined by spectrofluorometric method, the activity of acid phosphatase – by the unified method «at the end point». Results. In the cytoplasmic (nonsedimentary) fraction of the liver and kidney reduced activity of cathepsin L (in both tissues), cathepsin B (in the kidney), cathepsin H (in the liver) was found on the background of the increase in the products of proteins oxidative modification in experimental hyperhomocysteinemia. The introduction of arginine in experimental hyperhomocysteinemia completely eliminated the manifestations of oxidative damage of proteins, partially correcting the activity of enzymes: there was an increase in the activity in non sedimentary (cytoplasmic) fraction due to intracellular redistribution of enzymes. There is found inverse correlation between the content of oxidative carbonylation products of proteins and the activity of cathepsins in the non sedimentary fraction, as well as the proportion of their non sedimentary activity. Conclusions. 1. Arginine at a dose of 500 mg/kg at a 10day administration completely corrects the increase in the products of oxidative protein carbonylation that develops in experimental hyperhomocysteinemia. 2. Under the influence of arginine there is an increase in the reduced due to isolated hyperhomocysteinemia activity of cathepsins B, L, H in the cytoplasmic fraction of the liver and kidney due to intracellular redistribution of enzymes. 3. Arginine administration causes nonselective increase of the lysosomal membrane permeability, and as a result, changes in the compartmentalization of lysosomal cysteine proteases. 4. The inverse correlation of the level of protein oxidative modification products with the activity of cathepsins in cytoplasmic (nonsedimentary) fractions, and the proportions of their nonsedimentary activity, suggesting the presence of contribution of changes in the compartmentalization of lysosomal cysteine proteinases in developing under the action of arginine compensation of oxidative stress in experimental hyperhomocysteinemia.

Structure of cortical cytoskeleton in fibers of mouse muscle cells after being exposed to a 30-day space flight on board the BION-M1 biosatellite

The aim of the work was to analyze changes in the organization of the cortical cytoskeleton in fibers of the mouse soleus muscle, tibialis anterior muscle and left ventricular cardiomyocytes after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles did not differ significantly within the study groups compared with the vivarium control group. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes did not differ significantly within all study groups and correlated with the transversal stiffness. A similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues compared with the control levels in the postflight group, with lowered beta-actin gene expression rates in the postflight group. After completion of the space flight, the content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins from the mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this protein were decreased at the time of dissection (it was started after 13 h after landing). At the same time, the content of alpha-actinin-1 decreased in the membranous fraction and increased in the cytoplasmic fraction of proteins from the soleus muscle fibers.