[Heat-shock protein HSP70 reduces the secretion of TNFα by neuroblastoma cells and human monocytes induced with beta-amyloid peptides].

The progress of neurodegeneration in Alzheimer's disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aß). In this paper, we showed that Aß(1-42) and isoAß(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAß(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aß, which leads to a decrease in neuronal cell damage at the organism level.

[Heat-shock protein HSP70 protects neuroblastoma cells SK-N-SH from the neurotoxic effects hydrogen peroxide and the ß-amyloid peptide].

Neuronal cell death in Alzheimer's disease is associated with the development of oxidative stress caused by the reactive oxygen species (ROS), which can be generated as a result of the effect of beta-amyloid peptides. One of the sources of ROS is hydrogen peroxide, inducing the apoptosis and necrosis of neural tissue cells. The mechanism of hydrogen peroxide apoptotic action includes launching signaling pathways that involve protein kinases PI3K, p38MAPK, JNK and ERK. Oxidative stress leads to increased synthesis of heat-shock proteins in the cells including HSP70. It was shown that the exogenous HSP70 could reduce generation of ROS in cells. In this study, we determined how HSP70 affected apoptosis and necrosis in human neuroblastoma cells SK-N-SH, induced by hydrogen peroxide and ß-amyloid peptide Aß(1-42). It was shown that HSP70 reduces the cytotoxic effects of hydrogen peroxide and beta-amyloid, and protein kinases PI3K and JNK play an important role in the mechanism of HSP70 protective effect on the peroxide induced apoptosis in SK-N-SH cells.

[The ability of cells to adjust to the low oxigen content associated with Na,K-ATPase glutationilation].

Decreasing the amount of oxygen in the tissues under hypoxic and ischemic conditions, observed at a number of pathologic processes, inevitably leads to their damage. One of the main causes of cell damage and death is a violation of the systems maintaining ionic balance. Na,K-ATPaseis a basic ion-transporting protein of animal cell plasma membrane and inhibition of the Na,K-ATPase activity at lower concentrations of oxygen is one of the earliest and most critical events for cell viability. Currently there is an active search for modulators of Na,K-ATPase activity. For this purpose traditionally used cardiac glycosides but the existence of serious adverse effects forced to look for alternative inhibitors of Na,K-ATPase. Previously we have found that the glutathionylation of Na,K-ATPase catalytic subunit leads to a complete-inhibition of the enzyme. In this paper it is shown that the agents which increase the level of Na,K-ATPase glutathionylation: ethyl glutathione (et-GSH), oxidized glutathione (GSSG) and N-acetyl cysteine (NAC), increase cell survival under oxygen deficiency conditions, prevent decline of ATP in the cells and normalize their redox status. Concentration range in which these substances have a maximum protective effect, and does not exhibit cytotoxic properties was defined: for et-GSH 0.2-0.5 mM, for GSSG 0.2-1 mM, for NAC 10 to 15 mM. The results show prospects for development of methods for tissues protection from damage caused by oxygen starvation by varying the degree of Na,K-ATPase glutathionylation.

[Cellular targets of antitumor ribonucleases].

Some ribonucleases (RNases) produce selective toxic effect on the cancer cells. The mechanism of this antitumor activity remains largely unclear. The subject of this review is the RNases interaction with cellular components, resulting in the induction of apoptosis of tumor cells. Cell surface structures, which are potential acceptors of the exogenous RNase are discussed: acidic lipids and glycoproteins, heparansulfate-containing proteoglycans, actin, and RNA-associated proteins. Cell membranes of normal and malignant cells differ according to the composition of these components, which largely determines the selectivity of RNases for the latter. Different types of RNA are examined as intracellular targets of the RNases activity, evidence is presented demonstrating the possibility of exogenous RNases intervening in the process of RNA interference. The role of potassium channels, NF-kappaB-dependent.signaling pathway and various caspases in apoptosis induced by exogenous RNases is discussed. Evidence is also presented showing that the sensitivity of cells to exogenous RNases is linked to the expression of certain oncogenes, namely RAS, KIT, AML1-ETO. It is suggested that discovering the details of the mechanisms of RNases cytotoxic effect in malignant cells susceptible to their activity, will in the future serve as a foundation to developing new tools of targeted anticancer therapy.

[Expression of FLT3-ITD oncogene confers mice progenitor B-cells BAF3 sensitivity to the ribonuclease binase cytotoxic action].

Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.

[X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase].

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 A resolution. The kinetic parameters of the hydrolysis of substrates of different lengths (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease in the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.

[The interaction of NS3 protein of hepatitis C virus with polymethylen derivatives of nucleic bases].

NS3 protein of hepatitis C virus plays the key role in the virus functioning. It possesses three enzymatic activities, namely protease activity, associated with N-terminal domain of the protein, and helicase/NTPase activities specific for C-terminal domain. Here, the effect of some polimethylenic derivatives of the nucleic bases on helicase and ATPase enzyme activities has been studied. Several of compounds tested displayed inhibitory activity towards NS3 helicase. However, most compounds demonstrated strong activating effect on ATPase activity of the enzyme as well as several other ATPases. The ATPase activating mechanism was not described earlier. The activation potency of the compounds depended on substrate/activator concentration ratio, and was maximal at the 1000:1. The activation mechanism scheme that allows us to explain phenomena observed is proposed.

[Different domain organization of two main conformational states of Na+/K+-ATPase].

Na+/K+-ATPase generates an electrochemical gradient of Na+ and K+, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two ground conformational states, E1 and E2. To characterize the domain organization of the protein in these conformations, the thermal denaturation of Na+/K+-ATPase from duck salt glands and rabbit kidneys has been studied in the presence of Na+ and K+, which induce the transition of the enzyme to the conformation E1 or E2. The melting curves for the apoforms of Na+/K+-ATPases have different shapes: the curve for the enzyme from the rabbit shows one transition at 56.1 degrees C, whereas the denaturation of Na+/K+-ATPase from the duck is characterized by two transitions, at 49.8 and 56.9 degrees C. Sodium and potassium ions abolish the difference in the domain organization of Na+/K+-ATPases. The melting curves for Na+/K+-ATPases in conformation E2 in both cases exhibit a single peak of thermal absorption at about 63 degrees C. The melting curves for the enzymes in conformation E1 show three peaks of thermal absorption, indicating the denaturation of three domains. The difference in the domain organization of Na+/K+-ATPase in conformations E1 and E2 may be of importance in different sensitivity of these conformations of the enzyme to temperature, proteolytic enzymes, and oxidative stress.

[Binase possesses a selective cytotoxic action on kit-transformed precursors of myeloid cells].

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.

[Thermal denaturation of eukaryotic class 1 translation termination factor eRF1. Relationship between stability of the eRF1 molecule and alteration of functional activity of its mutants].

Thermal denaturation of eukaryotic class-1 translation termination factor eRF1 and its mutants was examined using differential scanning microcalorimetry (DSK). Changes of free energy caused by mutants in the N domain of human eRF1 were calculated. Melting of eRF1 molecule composed of three individual domains is cooperative. Some amino acid substitutions did not affect protein thermostability and in some other cases even slightly stabilize the protein globule. These imply that these amino acid residues are not involved in maintenance of the 3D structure of human eRF1. Thus, in Glu55Asp, Tyr125Phe, Asn61Ser, Glu55Arg, Glu55A1a, Asn61Ser + Ser64Asp, Cys127Ala and Ser64Asp mutants selective inactivation of release activity is not caused by a destabilization of protein 3D structure and, most likely, is associated with local stereochemical changes introduced by substitutions of amino acid side chains in the functionally essential sites of N-domain molecule. Some residues (Asn129, Phe131) as shown by calorimetric measurements are essential for preservation of stable protein structure, but at the same time they affect selective stop codon recognition probably via their neighboring amino acids. Recognition of UAG and UAA stop codons in vitro is more sensitive to preservation of protein stability than the UGA recognition.

[Inhibitors of ribonucleases].

RNases are the most important enzymes of cellular metabolism. They influence gene expression, cell growth and differentiation, participate in cell protection against pathogens and induction of apoptosis. Since intracellular RNases exist mainly in complexes with their inhibitors, the latter are also involved in all above-mentioned processes. The review describes natural protein inhibitors of animal, plant, and bacterial RNases along with synthetic low molecular-weight inhibitors. Special attention is paid to the perspectives of application of RNase inhibitors to therapy of oncological and allergic diseases. Despite wide distribution of RNases and their numerous studies, the number of available natural and synthetic inhibitors of these enzymes remains limited. Creation of highly efficient low-molecular inhibitors of RNase activity of angiogenin and eosinophil-associated RNases, aimed at the therapy of oncological and allergic diseases, still remains quite actual.

[I87E mutation prevents barstar dimerization]. / Mutatsiia I87E prepiatstvuet dimerizatsii barstara.

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate]. / Ingibirovanie neorganicheskoi pirofosfatazy Escherichia coli neorganicheskim fosfatom.

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.