Biochemistry (Moscow) (v.77, #1)

Molecular structure of phospholipase D and regulatory mechanisms of its activity in plant and animal cells by Y. S. Kolesnikov; K. P. Nokhrina; S. V. Kretynin; I. D. Volotovski; J. Martinec; G. A. Romanov; V. S. Kravets (1-14).
Phospholipase D (PLD) catalyzes hydrolysis of phospholipids with production of phosphatidic acid, which often acts as secondary messenger of transduction of intracellular signals. This review summarizes data of leading laboratories on specific features of organization and regulation of PLD activity in plant and animal cells. The main structural domains of PLD (C2, PX, PH), the active site, and other functionally important parts of the enzyme are discussed. Regulatory mechanisms of PLD activity are characterized in detail. Studies associated with molecular design, analysis, and synthesis of new nontoxic substances capable of inhibiting different PLD isoenzymes in vivo are shown to be promising for biotechnology and medicine.
Keywords: phospholipase D; domains; calcium; lipids; G-proteins; protein kinases; protein-protein interactions

Noncanonical functions of aminoacyl-tRNA synthetases by E. V. Smirnova; V. A. Lakunina; I. Tarassov; I. A. Krasheninnikov; P. A. Kamenski (15-25).
Aminoacyl-tRNA synthetases, together with their main function of covalent binding of an amino acid to a corresponding tRNA, also perform many other functions. They take part in regulation of gene transcription, apoptosis, translation, and RNA splicing. Some of them function as cytokines or catalyze different reactions in living cells. Noncanonical functions can be mediated by additional domains of these proteins. On the other hand, some of the noncanonical functions are directly associated with the active center of the aminoacylation reaction. In this review we summarize recent data on the noncanonical functions of aminoacyl-tRNA synthetases and on the mechanisms of their action.
Keywords: aminoacyl-tRNA synthetases; noncanonical functions

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.
Keywords: Chp/RhoV GTPase; Pak6 protein kinase; protein-protein interactions

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein by N. V. Ravin; R. Y. Kotlyarov; E. S. Mardanova; V. V. Kuprianov; A. I. Migunov; L. A. Stepanova; L. M. Tsybalova; O. I. Kiselev; K. G. Skryabin (33-40).
Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.
Keywords: influenza; vaccine; plant biofactories; M2 protein; HBc antigen; viral vector; virus-like particle

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.
Keywords: nitrite; oxoferryl forms of hemoproteins; hydrogen peroxide; thiamine; thiochrome; thiamine disulfide; oxodihydrothiochrome

Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes by O. B. Shevelev; V. I. Rykova; L. A. Fedoseeva; E. Yu. Leberfarb; G. M. Dymshits; N. G. Kolosova (56-61).
Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days). Wistar rats of the same age were used as controls. Expression levels of the genes Ext1, Ext2, and Hpse in the brain of rats of both strains were maximal during the two first weeks of life, and the contents of mRNAs of all genes in the brain of newborn and 7-day-old OXYS rats were significantly higher than in Wistar rats. By the 14th day of life the differences leveled, but at the age of 30 days on the background of a decrease in the contents of mRNAs of Ext1, Ext2, and Hpse in OXYS rats they became more pronounced (three-, four-, and twofold, respectively). Differences between the strains were absent at the age of 60 days and 14 months, and expression of all the genes was significantly lower than in the newborn animals. A strong positive correlation was found between contents of mRNAs of all the studied genes, and this suggested that heparanase should be involved in HSPG metabolism together with Ext1 and Ext2. Based on these and earlier findings, we conclude that development of the OXYS rat brain occurs on the background of significant alterations in HSPG metabolism that precede the development of neurodegenerative manifestations recently detected by magnetic resonance imaging.
Keywords: brain aging; heparan sulfate proteoglycans; expression of Ext1, Ext2, and Hpse genes; senescence-accelerated OXYS rats

Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold by L. E. Petrovskaya; L. N. Shingarova; E. A. Kryukova; E. F. Boldyreva; S. A. Yakimov; S. V. Guryanova; V. N. Novoseletsky; D. A. Dolgikh; M. P. Kirpichnikov (62-70).
Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin (10Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC50 of TNF.
Keywords: tumor necrosis factor; TNF-binding proteins; fibronectin domain; CDR grafting; ELISA; cytotoxicity test

Site-directed mutations were introduced into PsbO protein of photosystem 2 to study the role of two lysine residues, 223 and 226 (LGAKPPK), in the green alga Chlamydomonas reinhardtii. Lysines 223 and 226 homologous to His228 and His231 from cyanobacteria are located on the protein side facing the lumen and can participate in formation of a channel connecting the Mn cluster with the intrathylakoid space. The K223E and K226E mutants were generated on the basis of the ΔpsbO strain of C. reinhardtii with the substitution of glutamic acid for the lysine residues. The K226E mutation leads to a decrease in stability of the protein and development of the ΔpsbO phenotype (the absence of both photosynthetic activity of photosystem 2 and photoautotrophic growth), with substantially decreased PsbO content in the cells. In the case of K223E, the mutant strain accumulated the normal level of PsbO protein and was able to grow photoautotrophically and to evolve oxygen. However, the rate of oxygen evolution and the F v/F m ratio were reduced by 15–20% compared to the control. Also, the time of the dark decay of F v in the presence of DCMU in the cells of the K223E mutant was increased, indicating impairment in the water-oxidizing complex. In general, our study shows the importance of amino acids K223 and K226 located at the lumenal surface of PsbO protein for the activity of the water-oxidizing complex.
Keywords: PsbO protein; site-directed mutagenesis; photosystem 2; water-oxidizing complex; Chlamydomonas reinhardtii

Effect of amiodarone on thermotolerance and Hsp104p synthesis in the yeast Saccharomyces cerevisiae by I. V. Fedoseeva; D. V. Pjatricas; N. N. Varakina; T. M. Rusaleva; A. V. Stepanov; E. G. Rikhvanov; G. B. Borovskii; V. K. Voinikov (78-86).
Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca2+ level in cells of the yeast Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca2+ level. It is supposed that the change in intracellular Ca2+ concentration plays an important role in activation of the HSP104 gene expression and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium homeostasis is discussed.
Keywords: amiodarone; Saccharomyces cerevisiae ; thermotolerance; Hsp104p; mitochondrion

The O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Arenibacter palladensis type strain KMM 3961T and studied by chemical methods and 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H,13C HSQC, and HMBC experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units containing two mannose residues (Man), one 2-acetamido-2-deoxy-D-galactose residue (D-GalNAc), and one 2-acetamido-2-deoxy-L-galacturonic acid residue (L-GalNAcA) and having the following structure: $$ o 2) - alpha - D - Manp - (1 o 6) - alpha - D - Manp - (1 o 4) - alpha - L - GalpNAcA - (1 o 3) - eta - D - GalpNAc - (1 o$$ .
Keywords: Arenibacter palladensis ; marine bacteria; O-specific polysaccharide; structure; NMR spectroscopy; 2-acetamido-2-deoxy-L-galacturonic acid

Nitric oxide — an activating factor of adenosine deaminase 2 in vitro by Ye. G. Sargisova; N. A. Andreasyan; H. L. Hayrapetyan; H. A. Harutyunyan (92-97).
In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.
Keywords: adenosine deaminase 2; enzyme activation; NO; conformation; allostery; heparin

Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.
Keywords: Erwinia carotovora ; aromatic aminotransferase; purification; subunit content; isoelectric point; substrate specificity

Increased PARP-1 levels in nuclear matrix isolated from heat shock treated rat liver by G. Zaalishvili; E. Zaldastanishvili; M. Karapetian; T. Zaalishvili (105-110).
Poly(ADP-ribose) polymerase-1 (PARP-1), a chromatin-associated enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers onto a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. PARP-1 levels in the nuclear matrix vary depending on the matrix isolation method used. The nuclear matrix appears to be the most thermosensitive nuclear structure during heat shock. Here we provide evidence for the extensive translocation of PARP-1 from chromatin to the nuclear matrix during heat shock. This translocation is accompanied by inhibition of PARP activity in the nucleus and elevation of PARP activity in the nuclear matrix. Our data suggest that thermal destabilization of the nuclear matrix is less likely to contribute to the translocation of PARP-1 to the nuclear matrix.
Keywords: PARP-1; nuclear matrix; heat shock

Here we attempt to supplement the metastatic niche concept with a stage of “preniche” that determines the site of development of a premetastatic niche and of a subsequent metastasis. The “preniche” includes all cellular and molecular events in the site of a prospective metastasis preceding the entrance of myeloid progenitor cells. The “preniche” integrates an activation of vascular endothelium of the microcirculatory vessels of target organs in the site of a future metastasis under conditions of chronic persistent productive inflammation that can be induced by cytokines from the primary tumor and independently of it. The endothelium activation is responsible for adhesion and clustering of the recruited myeloid progenitor cells and also for the retention of cells of malignant tumors. The preniche easily arises in organs enriched with organspecific macrophages (lungs, liver, brain, etc.) where the endothelium is predisposed for intensive recruiting of myeloid progenitor cells of macrophages, especially under conditions of inflammation. Introduction of the “preniche” concept allows us to avoid difficulties associated with the development of the metastatic niche concept, especially concerning the problem of organ-preferential localization of metastases, and to make some predictions for experimental verification and potential approaches for preventing metastasizing in some oncologic patients.
Keywords: preniche; organ-preferential metastasizing; metastatic niches