Irina Yu. Petrushanko

S-glutathionylation of the Na,K-ATPase catalytic α subunit is a determinant of the enzyme redox-sensitivity

Background: Na,K-ATPase activity is extremely sensitive to changes in the redox state. Results: Binding of glutathione to the regulatory cysteine residues of the catalytic subunit completely inhibits the Na,K-ATPase by blocking the ATP-binding site. Conclusion: S-Glutathionylation of the catalytic subunit is revealed as a mechanism controlling the Na,K-ATPase function. Significance: Regulatory S-glutathionylation adjusts Na,K-ATPase activity to the changes in intracellular redox state and ATP levels. Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.

Termination of translation in eukaryotes is mediated by the quaternary eRF1•eRF3•GTP•Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

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 (Kd = 1.9 μM) and this interaction depends only minimally on the Mg2+ concentration; (ii) GTP binds to eRF3 (Kd = 0.5 μM) 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.

Oxygen-induced Regulation of Na/K ATPase in Cerebellar Granule Cells

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.

Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes.

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.

The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form

Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the γ-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an “activity chimera,” with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its “ratcheted state,” with hybrid tRNA binding sites.

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca(2+) and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.

HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

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 TGFβ1-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 1α. 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.

Oncogenic c-kit transcript is a target for binase.

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.

Binase cleaves cellular noncoding RNAs and affects coding mRNAs

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.

Binding of ouabain and marinobufagenin leads to different structural changes in Na,K‐ATPase and depends on the enzyme conformation

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 sameK i 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.