[Low-Molecular Thiols as a Factor Improving the Sensitivity of Escherichia coli Mutants with Impaired ADP-Heptose Synthesis to Antibiotics].

Low molecular-weight thiols as glutathione and cysteine are an important part of the cell's redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (ΔcysB and ΔcysE) and inhibition of glutathione synthesis (ΔgshAB) lead to a decrease in the sensitivity of the ΔgmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (ΔeamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine-cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ΔgmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ΔgmhA strain to antibiotics.

[Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation].

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.

[Blood-Brain Barrier Transwell Modeling].

In vitro blood-brain barrier (BBB) modeling with the use of the brain endothelial cells grown on a transwell membrane is widely used to investigate BBB disorders and factors intended to ameliorate these pathologies. Endothelial cells, due to tight junction proteins, ensure selective permeability for a number of substances. The low integrity (i.e., high permeability) of the BBB model, as compared to the physiological one, complicates evaluation of the effects caused by different agents. Thus, the selection of conditions to improve barrier integrity is an essential task. In this study, mouse brain endothelial cells bEnd.3 are used in experiments on transwell modeling. To determine which factors enhance BBB integrity, the effects of the cultivation medium, the number of cells during seeding, the state of the transwell membrane, and cultivation in the presence or in the absence of primary mouse neurons and matrigel as a matrix on the passage of a fluorescent label through the cell monolayer were assessed. The effect of fetal bovine serum on the tight junction protein claudin-5 was analyzed by immunocytochemistry. The obtained cultivation parameter data facilitate the solution to the problem of low integrity of the BBB transwell model and bring the model closer to the physiologically relevant indicators.

[Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells].

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.

[Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue].

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aß) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aß metal-binding domain (Aß(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aß(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aß(1-16). In contrast, the Aß(1-16) isoform with the English mutation (H6R) and the Aß(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

[Bacillus pumilus Ribonuclease Inhibits Migration of Human Duodenum Adenocarcinoma HuTu 80 Cells].

Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 µg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48-72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein.

BACKGROUND: We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. METHODS: Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. RESULTS: S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. CONCLUSIONS: S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. GENERAL SIGNIFICANCE: The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

[Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid ß].

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the ß-amyloid peptide (Aß) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Aß42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Aß42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Aß MBD play an essential role in the interaction of zinc-induced Aß aggregates with heparin.

[Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides].

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Anti-amyloid Therapy of Alzheimer's Disease: Current State and Prospects.

Drug development for the treatment of Alzheimer's disease (AD) has been for a long time focused on agents that were expected to support endogenous ß-amyloid (Aß) in a monomeric state and destroy soluble Aß oligomers and insoluble Aß aggregates. However, this strategy has failed over the last 20 years and was eventually abandoned. In this review, we propose a new approach to the anti-amyloid AD therapy based on the latest achievements in understanding molecular causes of cerebral amyloidosis in AD animal models.

Glutathionylation of Na,K-ATPase Alpha-Subunit Alters Enzyme Conformation and Sensitivity to Trypsinolysis.

We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the enzyme conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different conformations with different extent of glutathionylation. Incubation of the protein in the E1 conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the enzyme in both E1 and E2 conformations. The pattern of tryptic digestion of the α-subunit in E2 conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 conformation is similar to that of the native enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated enzymes. The data obtained were confirmed using molecular modeling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN conformation than in the E1- and E2-conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glutathionylation affects enzyme conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of enzyme glutathionylation and increase in the enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena are discussed.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

[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.

Amyloid-ß with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation.

Neuronal dysfunction and loss associated with the accumulation of amyloid-ß (Aß) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aß42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aß42 with isomerised Asp7 (isoAß42) which is one of the most abundant Aß isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAß42 rather than intact Aß42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAß42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAß42, being to date the only known synthetic Aß species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAß42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

[The Effect of Beta-Amyloid Peptides and Main Stress Protein HSP70 on Human SH-SY5Y Neuroblastoma Proteome].

The accumulation and aggregation of ß-amyloids are major molecular events underlying the progression of Alzheimer's disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aß and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aß42 and isomerized Asp7 (isoAß42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aß-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aß peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aß peptides.

Alzheimer's disease Aß42 peptide induces an increase in Na,K-ATPase glutathionylation.

We have shown that the inhibition of Na,K-ATPase during its long-term incubation with amyloid beta (Aß42), an Alzheimer's disease protein, is caused by the change in the thiol redox status of cells leading to induction of glutathionylation α-subunit of Na,K-ATPase. To restore the activity of Na,K-ATPase, it is proposed to use reducing agents, which promote normalization of the redox status of cells and deglutathionylation of the protein.

[Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells treated by amyloid-beta 1-42 with isomerized Asp7].

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-ß (Aß42) and its more toxic form with isomerized Asp7 (isoAß42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAß42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAß42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aß peptides, proteasomes, and HSP70.

[Changes in the receptor function of Na,K-ATPase during hypoxia and ischemia].

Na,K-ATPase maintains sodium and potassium homeostasis. It is the only known receptor for cardiotonic steroids such as ouabain. Binding of ouabain to Na,K-ATPase leads to the activation of Src kinase and the subsequent initiation of intracellular signaling pathways, including the induction of apoptosis. Changes in Na,K-ATPase activity is one of the earliest responses to hypoxia and is most critical for cell survival. However, it is not known how the hypoxia affects the functioning of Na,K-ATPase as a receptor. We have shown that, under the conditions of hypoxia and ischemia, ouabain is less toxic for murine fibroblast cells (SC-1 cell line) and ouabain does not cause an increase in the level of reactive oxygen species, which is typically observed at 20% pO2. Under hypoxia, the treatment of cells with ouabain also does not lead to the activation of Na,K-ATPase-associated Src kinase. Thus, at low oxygen content, the receptor function of Na,K-ATPase is altered, and cells become less sensitive to cardiotonic steroids. The decrease in sensitivity to cardiotonic steroids, which is evident at hypoxic conditions, should be taken into account in clinical practice. At the same time, in the presence of ouabain the cells are less sensitive to hypoxia, which indicates that cardiotonic steroids can be protective in acute ischemia.

[Isomerization of Asp7 increases the toxic effects of amyloid ß and its phosphorylated form in SH-SY5Y neuroblastoma cells].

The generation of amyloid ß (Aß) toxic oligomers during the formation of senile plaques and amyloid fibrils is thought to play a central role in the onset and progression of Alzheimer's disease. Aß production is a physiological process, but the factors that trigger a transition to pathogenic Aß aggregation remain unknown. Posttranslational modifications of Aß could potentially induce the transition. The effects of Aß and its modified forms containing isomerized Asp7, phosphorylated Ser8, or both, were studied in SH-SY5Y human neuroblastoma cells. Asp7 isomerization of was shown to increase cytotoxicity of both the intact and phosphorylated Aß. An increase in cytotoxicity was not associated with an increased internalization of the isomerized Asp7-containing Aß or an influence on the function of mitochondria or reduced glutathione and reactive oxygen species levels. The nitric oxide (NO) level was identified as a determinant of the cytotoxic effect of isomerized Asp7-containing peptides, a decrease in NO level correlating with an increase in cytotoxicity.

[Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis].

Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.