Biochemistry (Moscow) (v.75, #8)

Renalase is a recently discovered secretory enzyme responsible for selective degradation of blood catecholamines. The review summarizes literature data on expression of this enzyme and on its structure and functions. Special attention is paid to unsolved and questionable problems including: 1) prediction of the presence of FAD in the protein structure based on amino acid sequence similarity of renalase with known FAD-dependent enzymes; 2) identity of plasma and urinary renalase; 3) mechanism underlying conversion of inactive renalase into the active form.
Keywords: catecholamines; enzymatic degradation; renalase; structure; function; bioinformation analysis of amino acid sequence

DNA polymerases and carcinogenesis by V. M. Krutyakov; T. P. Kravetskaya (959-964).
There are many various chromosomal and gene mutations in human cancer cells. The total mutation rate in normal human cells is 2·10−7 mutations/gene/division. From 6 to 12 carcinogenic mutations can arise by the end of the life, and these can affect the structure of ∼150 protooncogenes and genes encoding suppressors of tumor growth. However, this does not explain the tens and hundreds of thousands of mutations detectable in cancer cells. Mutation is any change of nucleotide sequence in cellular DNA. Gene mutations are mainly consequences of errors of DNA polymerases, especially of their specialized fraction (inaccurate DNA polymerases β, ζ, η, θ, ι, κ, λ, μ, σ, ν, Rev1, and terminal deoxynucleotidyl transferase, and only polymerases θ and σ manifest a slight 3′-exonuclease activity) and also consequences of a decrease in the rate of repair of these errors. Inaccurate specialized human polymerases are able to synthesize DNA opposite lesions in the DNA template, but their accuracy is especially low during synthesis on undamaged DNA. In the present review fundamental features of such polymerases are considered. DNA synthesis stops in the area of its lesion, but this block is overcome due to activities of inaccurate specialized DNA polymerases. After the lesion is bypassed, DNA synthesis is switched to accurate polymerases α, δ, ɛ, or γ. Mechanisms of direct and reverse switches of DNA polymerases as well as their modifications during carcinogenesis are discussed.
Keywords: DNA polymerases and their lesions; carcinogenesis; mutagenesis; switch of DNA polymerases

RNA interference (RNAi) plays an important biological role in regulation of gene expression of eukaryotes. In addition, RNAi was shown to be an adaptive protective molecular immune mechanism against viral diseases. Antiviral RNAi initiates from generation of short interfering RNAs used in the subsequent recognition and degradation of the viral RNA molecules. As a response to protective reaction of plants, most of the viruses encode specific proteins able to counteract RNAi. This process is known as RNAi suppression. Viral suppressors act on various stages of RNAi and have biochemical properties that enable viruses to effectively counteract the protective system of plants. Modern molecular and biochemical investigations of a number of viral suppressors have significantly expanded our understanding of the complexity of the nature of RNAi suppression as well as mechanisms of interaction between viruses and plants.
Keywords: plant; RNA interference; suppression; suppressor; virus

Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na+/K+ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na+]i/[K+]i ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na+, K+, and H+ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.
Keywords: Na,K-ATPase; ouabain; ionophores; intracellular Na+ and K+ ; p38 MAPK; kidney; cell death

Fibrillarin is an evolutionarily-conserved and obligatory protein component of eukaryotic cell nucleoli involved in pre-rRNA processing and methylation. In vertebrates the fibrillarin molecule contains two cysteine residues (Cys99 and Cys268) whose sulfhydryl groups are able to establish intramolecular -S-S- bridges. However, the functional state of fibrillarin with reduced or oxidized thiol groups is still practically unstudied. Besides, there are no data in the literature concerning existence of the -S-S- fibrillarin form in human cells. To answer these questions, we used plasmids encoding native human fibrillarin and its mutant form devoid of cysteine residues (fibrillarinC99/268S) fused with EGFP for temporary transfection of HeLa cells. The mobile fraction localizing the enzymatically active protein molecules and the fluorescence half-recovery time characterizing the rate of enzymatic reactions were determined by the FRAP technique using a confocal laser scanning microscope. Measurements were carried out at 37 and 27°C. The results show that the fibrillarin pool in HeLa cells includes two protein forms, with reduced SH groups and with oxidized SH groups forming intramolecular -S-S- bridges between Cys99 and Cys268. However, the absence of Cys99 and Cys268 has no effect on intracellular localization of fibrillarin and its main dynamic parameters. The human fibrillarin form without disulfide bridges is included into the mobile protein fraction and is consistent with its functionally active state.
Keywords: fibrillarin; Cys99; Cys268; site-specific mutagenesis; FRAP; protein mobility; HeLa cells

Isolation and crystallization of a chimeric Qβ replicase containing Thermus thermophilus EF-Ts by N. N. Vasiliev; L. Jenner; M. M. Yusupov; A. B. Chetverin (989-994).
Qβ replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qβ. In addition to the phage-encoded catalytic β subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qβ replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the β subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qβ replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.
Keywords: RNA replication; RNA-dependent RNA polymerase; ribosomal protein S1; elongation factors EF-Tu and EF-Ts; two plasmid expression system; protein crystallization; structure stability

An important question that is addressed here is whether the modeling of protein folding can catch the difference between the folding of proteins with similar structures but with different folding mechanisms. In this work, the modeling of folding of four α-helical proteins from the homeodomain family, which are similar in size, was done using the Monte Carlo and dynamic programming methods. A frequently observed order of folding of α-helices for each protein was determined using the Monte Carlo method. A correlation between the experimental folding rate and the number of Monte Carlo steps was also demonstrated. Amino acid residues that are important for the folding were determined using the dynamic programming method. The defined regions correlate with the order of folding of secondary-structure elements in the proteins both in experiments and in modeling.
Keywords: protein folding; folding intermediates; folding nuclei; nucleation mechanism; diffusion-collision mechanism

A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides by K. V. Lavrov; I. A. Zalunin; E. K. Kotlova; A. S. Yanenko (1006-1013).
A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4′-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7–8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K m) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.
Keywords: Rhodococcus erythropolis ; amidase; N-substituted amides; acrylamide; N-acetylamino acids

Inhibition of transketolase by hexacyanoferrate(III) by V. A. Yurshev; I. A. Sevostyanova; O. N. Solovjeva; G. A. Kochetov (1014-1016).
The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.
Keywords: transketolase; nonequivalence of active sites; inhibitors; hexacyanoferrate

Comparison of models of thrombin-binding 15-mer DNA aptamer by molecular dynamics simulation by R. V. Reshetnikov; A. V. Golovin; A. M. Kopylov (1017-1024).
Two models of 15-mer thrombin-binding DNA aptamer (15TGT) were comparatively analyzed by molecular dynamics simulation using the GROMACS software package. The two original models of 15TGT were obtained by NMR and X-ray analyses. The models significantly differ in the topology of loops and the direction of oligodeoxyribonucleotide chain. The evolution of the two structures in parm99 force fields and parmbsc0 optimized for nucleic acids was analyzed in our adaptation of GROMACS architecture. It is shown that the best system for description of the 15TGT structure is the model obtained by X-ray analysis in the parmbsc0 force field.
Keywords: aptamer; molecular dynamics simulation; thrombin

Changes in erythrocyte shape during morphological response modified by benzalkonium chloride (BzA) were studied in sucrose solutions. Fixation of the cells with glutaraldehyde- and formaldehyde-containing fixatives at some time points is usually inadequate to maintain the current cell shape. Considering the reconstruction of erythrocyte shape, which takes into account the mode of fixative action, we showed that the echinocyte-forming activity of BzA depends on the concentration of this surfactant. It can induce a direct spherostomatocyte-spheroechinocyte transition without altering the near-spherical shape of the cells. On the other hand, the reverse spheroechinocyte-spherostomatocyte transition was always accompanied by some flattening of the cells, although in some instances discoidal shape was not achieved. The data point to asymmetric shape transitions of erythrocytes in sucrose solution, which contradicts the continuum and bilayer-couple models of shape regulation. It seems that the nonuniform structure of native erythrocyte membrane plays a more important role in morphological transitions of these cells than suggested earlier.
Keywords: erythrocyte; red blood cell; shape; morphological transition; morphological response; sucrose; benzalkonium chloride

Carboxypeptidase from Streptomyces bikiniensis: Primary structure, isolation, and properties by A. V. Serkina; I. A. Zalunin; E. I. Levitin; T. A. Voejkova; B. V. Tyaglov; L. M. Novikova; L. K. Emeljanova; G. E. Konstantinova; G. G. Chestukhina (1032-1038).
A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0–7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).
Keywords: Streptomyces bikiniensis ; metallocarboxypeptidase; substitution therapy

Using a specific fluorescent probe of mitochondrial membrane potential (tetramethylrhodamine ethyl ester), we have shown that glucose deprivation (GD) of cultured cerebellar granule neurons (CGN) for 3 h lowers mitochondrial membrane potential in these cells. Longer glucose starvation (24 h) causes CGN death that is not prevented by blockers of ionotropic glutamate receptors (MK-801 (10 μM) and NBQX (10 μM)). Glutamine or pyruvate (2 mM) maintain membrane potential of mitochondria and decrease CGN death under GD conditions. In the presence of glucose the mitochondrial respiratory chain blocker rotenone induces neuron death potentiated by glutamine. The potentiation effect is completely prevented by blockers of ionotropic glutamate receptors. These results show that glutamine under conditions of GD can be utilized by mitochondria as substrate, but at the same time, in the case of mitochondrial function deterioration, metabolism of this amino acid results in glutamate accumulation to toxic level.
Keywords: glutamine; cerebellar granule neurons; glutamate; mitochondria; glucose deprivation; chemical hypoxia

Staining by antibodies to rhodopsin (Rh) and fluorescence of N-retinylopsin (RO) have shown that digitonin (DIG)-, dodecyl-β-D-maltoside (DM)-, and sodium dodecyl sulfate (SDS)-solubilized frog Rh after BN- and HRCN-PAGE is situated in the gradient gel in the state of dimer with a slight content of higher oligomers (trimer, tetramer, etc.). With increasing detergent harshness (DIG < DM < SDS), the proportion of higher oligomers in extracts becomes more prominent. Formation of RO in rod outer segments (ROS) in the presence of 0.7 M NaBH3CN at pH 5.0 occurs only when Rh is simultaneously photolyzed during reduction. Dithiothreitol at the concentration of 0.005 M failed to induce RO production. Formation of a stable C-N bond between all-trans-retinal and opsin in RO is accompanied by decrease in the dimer share and increase in the share of the higher oligomers due to secondary dissociation-aggregation of solubilized opsin. The position of the Rh dimer in relation to the anode during both native electrophoreses is determined not only by its molecular mass, but probably also depends on unfolding degree (or form): the harsher the detergent, the closer to the anode the dimer is located. Treatment of ROS by agents modifying the cholesterol component of lipid membrane (MβCD, filipin III, nystatin, saponin) did not change the character of Rh oligomerization, thus showing that integrity of the cholesterol component of photoreceptor membrane is not a crucial factor for oligomerization of opsin. It is supposed that the dimer-oligomer “portrait” of frog Rh, which has been found by two methods of native electrophoresis in three detergents with different degree of harshness, corresponds to a physiological state of this protein in native photoreceptor membrane.
Keywords: frog rhodopsin; oligomerization; BN-PAGE; HRCN-PAGE; antibodies; fluorescence; retinylopsin; digitonin; dodecyl maltoside; SDS

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae with a mutation disturbing the function of vacuolar ATPase by A. A. Tomaschevsky; L. P. Ryasanova; T. V. Kulakovskaya; I. S. Kulaev (1052-1054).
A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.
Keywords: inorganic polyphosphates; Saccharomyces cerevisiae ; mutant; vma2 gene; ATPase; vacuole

Yeast Pma1 H+-ATPase is a key enzyme of cell metabolism generating electrochemical proton gradient across the plasma membrane, thus playing an important role in the maintenance of ion homeostasis in the cell. Using site-directed mutagenesis, we have previously replaced all 21 amino acid residues in the transmembrane segment M8 with Ala (Guerra et al. (2007) Biochim. Biophys. Acta, 1768, 2383–2392). In this work, we present new data on the role of these amino acid residues in the structure-function relationship in the enzyme and cell tolerance to heat shock. Mutations Q798A and I799A are lethal for cells regardless of expression of the enzyme in secretory vesicles or plasma membrane. The F796A mutation causes enzyme and cell sensitivity to heat shock when expressed in secretory vesicles. The I794A mutation increases temperature sensitivity of cells when the enzyme is expressed either in secretory vesicles or, to a lesser extent, in plasma membrane. The E803A mutation has no significant influence on the ATPase and cell sensitivity to heat shock; however, it causes a shift in the equilibrium between E1 and E2 conformations of the enzyme towards E1.
Keywords: yeast; plasma membrane; secretory vesicles; ATPase; transmembrane segment; heat shock; site-directed mutagenesis

Handbook of glycomics by G. Ya. Wiederschain (1064-1065).