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Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O

Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

The hemoglobin concentration of 35 g/dl of human red blood cells is close to the solubility threshold. Using microwave dielectric spectroscopy, we have assessed the amount of water associated with hydration shells of methemoglobin as a function of its concentration in the presence or absence of ions. We estimated water-hemoglobin interactions to interpret the obtained data. Within the concentration range of 5-10 g/dl of methemoglobin, ions play an important role in defining the free-to-bound water ratio competing with hemoglobin to recruit water molecules for the hydration shell. At higher concentrations, hemoglobin is a major contributor to the recruitment of water to its hydration shell. Furthermore, the amount of bound water does not change as the hemoglobin concentration is increased from 15 to 30 g/dl, remaining at the level of ∼20% of the total intracellular water pool. The theoretical evaluation of the ratio of free and bound water for the hemoglobin concentration in the absence of ions corresponds with the experimental results and shows that the methemoglobin molecule binds about 1400 water molecules. These observations suggest that within the concentration range close to the physiological one, hemoglobin molecules are so close to each other that their hydration shells interact. In this case, the orientation of the hemoglobin molecules is most likely not stochastic, but rather supports partial neutralization of positive and negative charges at the protein surface. Furthermore, deformation of the red blood cell shape results in the rearrangement of these structures.

Alzheimer's disease (AD) is accompanied by the dysfunction of intracellular protein homeostasis systems, in particular the ubiquitin-proteasome system (UPS). Beta-amyloid peptide (Aβ), which is involved in the processes of neurodegeneration in AD, is a substrate of this system, however its effect on UPS activity is still poorly explored. Here we found that Aβ peptides inhibited the proteolytic activity of the antiapoptotic Arg/N-end rule pathway that is a part of UPS. We identified arginyltransferase Ate1 as a specific component of the Arg/N-end rule pathway targeted by Aβs. Aβ bearing the familial English H6R mutation, known to cause early-onset AD, had an even greater inhibitory effect on protein degradation through the Arg/N-end rule pathway than intact Aβ. This effect was associated with a significant decrease in Ate1-1 and Ate1-3 catalytic activity. We also found that the loss of Ate1 in neuroblastoma Neuro-2a cells eliminated the apoptosis-inducing effects of Aβ peptides. Together, our results show that the apoptotic effect of Aβ peptides is linked to their impairment of Ate1 catalytic activity leading to suppression of the Arg/N-end rule pathway proteolytic activity and ultimately cell death.

The functioning of the N, K-ATPase depends on the redox status of cells and its activity is inhibited by oxidative stress and hypoxia. We previously found that redox sensitivity of the Na,K-ATPase is mediated by glutathionylation of the α-subunit. An increase in the level of glutathionylation of cysteine residues in the Na,K-ATPase α-subunit under stressful conditions leads to a decrease in the activity of the enzyme and a change in its receptor function. The structure of the Na,K-ATPase undergoes significant conformational changes during functioning. The effects of enzyme conformation on its ability to undergo glutathionylation are not clear. Here we show that the highest level of glutathionylation in the α-subunit of Na,K-ATPase is achieved in the E1 (Na+-induced) conformation. The transition of the Na,K-ATPase to the E2 (K+-induced) conformation leads to a decrease in the efficiency of glutathionylation. The lowest efficiency of Na,K-ATPase glutathionylation was observed in the E2P and E2P ouabain states. According to molecular modelling data, the maximum number of cysteine residues available for glutathionylation are present in the E1P conformation. In the E2P conformation, the main functional cysteine residues (Cys204, Cys242, Cys452, and Cys456) are buried from the solvent, which makes them inaccessible for glutathionylation. Thus, the efficiency of α-subunit glutathionylation depends on enzyme conformation, which is altered by bound ligands and proteins. A shift in the E1/E2 equilibrium towards prevalence of E1 can lead to better access for the relevant ligands and proteins to the binding site located in the Na,K-ATPase α-subunit. Na,K-ATPase.

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, intracellular glutathione, 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.

This study focuses on assessing the possible impact of changes in hemoglobin (Hb) oxygenation on the state of water in its hydration shell as it contributes to red blood cell deformability. Microwave Dielectric Spectroscopy (MDS) was used to monitor the changes in interactions between water molecules and Hb, the number of water molecules in the protein hydration shell, and the dynamics of pre-protein water in response to the transition of Hb from the tense (T) to the relaxed (R) state, and vice versa. Measurements were performed for Hb solutions of different concentrations (5 g/dl-30 g/dl) in phosphate-buffered saline buffer. Cole-Cole parameters of the main water relaxation peak in terms of interactions of water molecules (dipole-dipole/ionic dipole) during the oxygenation-deoxygenation cycle were used to analyze the obtained data. The water mobility-represented by α as a function of ln τ-differed dramatically between the R (oxygenated) state and the T (deoxygenated) state of Hb at physiologically relevant concentrations (30 g/dl-35 g/dl or 4.5 mM-5.5 mM). At these concentrations, oxygenated hemoglobin was characterized by substantially lower mobility of water in the hydration shell, measured as an increase in relaxation time, compared to deoxyhemoglobin. This change indicated an increase in red blood cell cytosolic viscosity when cells were oxygenated and a decrease in viscosity upon deoxygenation. Information provided by MDS on the intraerythrocytic water state of intact red blood cells reflects its interaction with all of the cytosolic components, making these measurements powerful predictors of the changes in the rheological properties of red blood cells, regardless of the cause.

Na,K-ATPase is a redox-sensitive transmembrane protein. Understanding the mechanisms of Na,K-ATPase redox regulation can help to prevent impairment of Na,K-ATPase functioning under pathological conditions and reduce damage and death of cells. One of the basic mechanisms to protect Na,K-ATPase against stress oxidation is the glutathionylation reaction that is aimed to reduce several principal oxidized cysteines (244, 458, and 459) that are involved in Na,K-ATPase action regulation. In this study, we carried out in silico modeling to evaluate glutathione affinity on various stages of Na,K-ATPase action cycle, as well as to discover a reaction mechanism of disulfide bond formation between reduced glutathione and oxidized cysteine. To achieve this goal both glutathione and Na,K-ATPase conformer sampling was applied, the reliability of the protein-ligand complexes was examined by MD assay, the reaction mechanism was studied using semi-empirical PM6-D3H4 approach that could have a deal with large organic systems optimization.

We have shown that the inhibition of Na,K-ATPase during its long-term incubation with amyloid beta (Aβ42), 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.

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.

Exogenous RNases are selectively toxic to tumor cells. The reasons for this selectivity are not quite clear and should be searched for in the properties that distinguish malignant from normal cells. During onco-transformation, cells acquire properties allowing them to adapt to the altered microenvironment, such as resistance to hypoxia, changes in intracellular pH, disruption of ion transport, reduced adhesion and increased mobility, and production of specific exosomes. These adaptation mechanisms distinguish malignant cells from normal ones and give them a competitive advantage, ensuring survival and spread in the organism. Here, we analyze if the directed cytotoxic effect of exogenous RNases is linked to the disruption of the adaptive potential of tumor cells and how it can be used in anticancer therapy.

Severe hypoxia leads to decline in cardiac contractility and induces arrhythmic events in part due to oxidative damage to cardiomyocyte proteins including ion transporters. This results in compromised handling of Ca2+ ions that trigger heart contractile machinery. Here, we demonstrate that thiol-containing compounds such as N-acetylcysteine (NAC), glutathione ethyl ester (et-GSH), oxidized tetraethylglutathione (tet-GSSG), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) are capable of reducing negative effects of hypoxia on isolated rat cardiomyocytes. Preincubation of cardiomyocytes with 0.1 mM GSNO, 0.5 mM et-GSH, GSSG, tet-GSSG or with 10 mM NAC allows cells 5-times longer tolerate the hypoxic conditions and elicit regular Ca2+ transients in response to electric pacing. The shape of Ca2+ transients generated in the presence of GSNO, et-GSH and NAC was similar to that observed in normoxic control cardiomyocytes. The leader compound, GSNO, accelerated by 34% the recovery of normal contractile function of isolated rat heart subjected to ischemia-reperfusion. GSNO increased glutathionylation of Na,K-ATPase alpha-2 subunit, the principal ion-transporter of cardiac myocyte sarcolemma, which prevents irreversible oxidation of Na,K-ATPase and regulates its function to support normal Ca2+ ion handling in hypoxic cardiomyocytes. Altogether, GSNO appears effective cardioprotector in hypoxic conditions worth further studies toward its cardiovascular application.

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.

Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPaseα-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPaseα-subunit; however, basal glutathionylation ofα-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPaseα-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPaseα-subunit and provide evidence supporting the view that this modification is cotranslational.

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 mod-eling 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, glu-tathionylation 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.

The triggers of late-onset sporadic Alzheimer's disease (AD) are still poorly understood. Impairment of protein phosphorylation with age is well-known; however, the role of the phosphorylation in β-amyloid peptide (Aβ) is not studied sufficiently. Zinc-induced oligomerization of Aβ represents a potential seeding mechanism for the formation of neurotoxic Aβ oligomers and aggregates. Phosphorylation of Aβ by Ser8 (pS8-Aβ), localized inside the zinc-binding domain of the peptide, may significantly alter its zinc-induced oligomerization. Indeed, using dynamic light scattering, we have shown that phosphorylation by Ser8 dramatically reduces zinc-induced aggregation of Aβ, and moreover pS8-Aβ suppresses zinc-driven aggregation of non-modified Aβ in an equimolar mixture. We have further analyzed the effect of pS8-Aβ on the progression of cerebral amyloidosis with serial retro-orbital injections of the peptide in APPSwe/PSEN1dE9 murine model of AD, followed by histological analysis of amyloid burden in hippocampus. Unlike the non-modified Aβ that has no influence on the amyloidosis progression in murine models of AD, pS8-Aβ injections reduced the number of amyloid plaques in the hippocampus of mice by one-third. Recently shown inhibition of Na+,K+-ATPase activity by Aβ, which is thought to be a major contributor to neuronal dysfunction in AD, is completely reversed by phosphorylation of the peptide. Thus, several AD-associated pathogenic properties of Aβ are neutralized by its phosphorylation.

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids., Graphical abstract fx1

In this study, we determined whether binase, a ribonuclease from Bacillus pumilus, increases interferon sensitivity and apoptosis in SiHa cervical cancer cells infected with high-risk human papilloma virus (HPV) strain 16. Binase treatment increased SiHa cell apoptosis in a time- and concentration-dependent manner, as determined by flow cytometry, WST tests and real time xCelligence cell index analysis. Binase-treated SiHa cells showed reduced expression of E6 and E7 viral oncoproteins and increased expression of their intracellular targets, p53 and pRb. Combined treatment with binase and IFNα2b enhanced the interferon sensitivity of HPV-positive SiHa cells. By contrast, combined treatment with binase and IFNα2b in HPV-negative C33A cervical cancer cells, which do no expess E6 and E7, elicited no changes in interferon sensitivity or p53 and pRb expression. These findings suggest binase enhances interferon sensitivity and apoptosis in HPV-positive SiHa cervical cancer cells by suppressing E6 and E7 viral protein expression.

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit.

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS 40 and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1-5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS 40 and inhibits metastasis up to 50% in LLC and RLS 40 and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS 40-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its "oxygen-sensitivity" is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation, and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidized glutathione are the signaling messengers that make the Na,K-ATPase "oxygen-sensitive." This very ancient signaling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the "optimal" level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterize the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summary on (i) the sources of free radical production in hypoxic cells, (ii) localization of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzyme to a variety of stimuli (hypoxia, receptors' activation) (iii) redox-sensitive regulatory phosphorylation, and (iv) the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate. Better understanding of the processes underlying successful adaptation strategies will make it possible to harness them and use for treatment of patients with stroke and myocardial infarction, sleep apnoea and high altitude pulmonary oedema, and those undergoing surgical interventions associated with the interruption of blood perfusion., The review was funded by the grants of Swiss Nationa lScience Foundation IZK0Z3_157269/1 and 310030_124970/1 to AB and Russian Science Foundation (Grant#14-14-01152) to IP Spanish Government grants (partially funded by the European Union FEDER/EDRF) PI12/00875 and PI15/00107 and a grant from the Fundación Domingo Martínez are supporting AM, PH received a travel grant from the Instituto de Investigación Sanitaria Princesa (to PH),and by COST actions TD0901 (HypoxiaNet) and CM1001. PH is recipient of a pre-doctoral FPU fellowship from the Spanish Government and AM is supported by the I3SNS programme (ISCIII, Spanish Government, partially funded by FEDER/ERDF)

Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase--RNase from Bacillus intermedius--on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.

Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca2+-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase-induced RNA decrease did not correlate with apoptosis. Kit-transformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated. Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively. These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis.

Adjustment of the Na/K ATPase activity to changes in oxygen availability is a matter of survival for neuronal cells. We have used freshly isolated rat cerebellar granule cells to study oxygen sensitivity of the Na/K ATPase function. Along with transport and hydrolytic activity of the enzyme we have monitored alterations in free radical production, cellular reduced glutathione, and ATP levels. Both active K+ influx and ouabain-sensitive inorganic phosphate production were maximal within the physiological pO2 range of 3–5 kPa. Transport and hydrolytic activity of the Na/K ATPase was equally suppressed under hypoxic and hyperoxic conditions. The ATPase response to changes in oxygenation was isoform specific and limited to the α1-containing isozyme whereas α2/3-containing isozymes were oxygen insensitive. Rapid activation of the enzyme within a narrow window of oxygen concentrations did not correlate with alterations in the cellular ATP content or substantial shifts in redox potential but was completely abolished when NO production by the cells was blocked by l-NAME. Taken together our observations suggest that NO and its derivatives are involved in maintenance of high Na/K ATPase activity under physiological conditions.

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types – marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (K(d) = 1.9 microM) and this interaction depends only minimally on the Mg(2+) concentration; (ii) GTP binds to eRF3 (K(d) = 0.5 microM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1*eRF3*GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1*eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1*eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1*eRF3*GTP*Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3*eRF1 complex profoundly differ from those of prokaryotic RF3.