Biochemistry (Moscow) (v.75, #2)

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants — the “Skulachev-ion” derivatives by D. S. Izyumov; L. V. Domnina; O. K. Nepryakhina; A. V. Avetisyan; S. A. Golyshev; O. Y. Ivanova; M. V. Korotetskaya; K. G. Lyamzaev; O. Y. Pletjushkina; E. N. Popova; B. V. Chernyak (123-129).
Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.
Keywords: oxidative stress; mitochondria-targeted antioxidants; SkQ; mitochondria; apoptosis

Lipofuscin granule dynamics during development of age-related macular degeneration by V. B. Saprunova; D. I. Pilipenko; A. V. Alexeevsky; A. Zh. Fursova; N. G. Kolosova; L. E. Bakeeva (130-138).
The pigment epithelium cell structure and therapeutic effect of antioxidant SkQ1, selectively penetrating into mitochondria from eye drops, were studied upon development in OXYS rats of age-related retinopathy as a model of macular degeneration. The characteristic dynamics and ultrastructural peculiarities of the layer of electron-dense cytoplasmic structures of the pigment epithelium apex part and incorporated lipofuscin granules were revealed. The therapy of OXYS animals for 68 days using 250 nM SkQ1 drops decreased the extent of development of age-related macular degeneration. Electron-microscopic investigation showed that SkQ1 prevented development of ultrastructural changes in the pigment epithelium characteristic of macular degeneration, the condition of which after therapy with SkQ1 drops corresponded to ultrastructure of pigment epithelium in Wistar rats of the same age having no symptoms of retinal damage. It is supposed that ultrastructural changes in the electron-dense layer upon development of age-related macular degeneration are indicative of disturbances in the optical cycle functioning, especially of disturbances in functioning of photoreceptor membranes.
Keywords: electron microscopy; age dependent macular degeneration; pigment epithelium; mitochondria; lipofuscin granules; oxidative stress; SkQ1

Interaction of yeast mitochondria with fatty acids and mitochondria-targeted lipophilic cations by E. I. Sukhanova; T. A. Trendeleva; R. A. Zvyagilskaya (139-144).
The effect of fatty acids and mitochondria-targeted lipophilic cations (SkQ1, SkQ3, MitoQ, and C12TPP) on tightly-coupled mitochondria from yeasts Dipodascus (Endomyces) magnusii and Yarrowia lipolytica was investigated. Micromolar concentrations of saturated and unsaturated fatty acids were found to decrease the membrane potential, which was recovered almost totally by ATP and BSA. At low, micromolar concentrations, mitochondria-targeted lipophilic cations are “relatively weak, mild uncouplers”, at higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their prooxidant and detergent action. At very low, not uncoupling concentrations, mitochondria-targeted lipophilic cations profoundly promote (potentiate) the uncoupling effect of fatty acids. It is conceivable that the observed uncoupling effect of lipophilic cations can be, at least partially, due to their interactions with the endogenous pool of fatty acids.
Keywords: yeast; Yarrowia lipolytica ; Dipodascus (Endomyces) magnusii ; mitochondria; fatty acids; SkQ1; SkQ3; MitoQ; C12TPP; respiration; transmembrane potential; swelling

New-generation Skulachev ions exhibiting nephroprotective and neuroprotective properties by E. Y. Plotnikov; D. N. Silachev; A. A. Chupyrkina; M. I. Danshina; S. S. Jankauskas; M. A. Morosanova; E. V. Stelmashook; A. K. Vasileva; E. S. Goryacheva; Y. A. Pirogov; N. K. Isaev; D. B. Zorov (145-150).
A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6′-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. An analog of SkQR1 not containing plastoquinone (C12R19) was not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.
Keywords: Skulachev ions; SkQ; SkQR1; kidney; oxidative stress; mitochondria-targeted antioxidants; mitochondria; rhabdomyolysis; pyelonephritis; brain ischemia; magnetic resonance tomography

Mechanisms of regulation of transcription factor HIF under hypoxia by E. B. Anokhina; L. B. Buravkova (151-158).
The importance of transcription factor HIF in cellular response to hypoxia and various regulatory levels determining its functioning within cells are considered. Special attention is paid to important links of the hypoxia-triggered HIFmediated mechanism. The review contains retrospective analysis of the search for cellular primary oxygen sensors and their interactions with HIF. It also deals with the interrelation of HIF-mediated and other hypoxia-activated mechanisms and outlines importance of HIF subunits in the cell.
Keywords: hypoxia; transcription factor HIF

Formation of plant cell wall supramolecular structure by T. A. Gorshkova; P. V. Mikshina; O. P. Gurjanov; S. B. Chemikosova (159-172).
Plant cell wall is an example of a widespread natural supramolecular structure: its components are considered to be the most abundant organic compounds renewable by living organisms. Plant cell wall includes numerous components, mainly polysaccharidic; its formation is largely based on carbohydrate-carbohydrate interactions. In contrast to the extracellular matrix of most other organisms, the plant cell compartment located outside the plasma membrane is so structured that has been named “wall”. The present review summarizes data on the mechanisms of formation of this supramolecular structure and considers major difficulties and results of research. Existing approaches to the study of interactions between polysaccharides during plant cell wall formation have been analyzed, including: (i) characterization of the structure of natural polysaccharide complexes obtained during cell wall fractionation; (ii) analysis of the interactions between polysaccharides “at mixing in a tube”; (iii) study of the interactions between isolated individual plant cell wall matrix polysaccharides and microfibrils formed by cellulose-synthesizing microorganisms; and (iv) investigation of cell wall formation and modification directly in plant objects. The key stages in formation of plant cell wall supramolecular structure are defined and characterized as follows: (i) formation of cellulose microfibrils; (ii) interactions between matrix polysaccharides within Golgi apparatus substructures; (iii) interaction between matrix polysaccharides, newly secreted outside the plasma membrane, and cellulose microfibrils during formation of the latter; (iv) packaging of the formed complexes and individual polysaccharides in cell wall layers; and (v) modification of deposited cell wall layers.
Keywords: cell wall; supramolecular structure; cellulose microfibrils; cross-linking glycans; pectin substances; carbohydrate-carbohydrate interactions

Interaction of murine Dnmt3a with DNA containing O6-methylguanine by D. V. Maltseva; E. S. Gromova (173-181).
O6-Methylguanine (O6meG) is one of the most toxic, mutagenic, and carcinogenic lesions caused by the interaction of DNA with several catabolism products as well as with environmental methylating agents. Carcinogenic impact of O6meG can be conditioned not only by its mutagenic properties but also by alteration in enzymatic methylation of the C5 carbon atom of cytosine residue in CpG sequences. In this study, the effect of O6meG on DNA methylation by the catalytic domain of murine DNA methyltransferase (MTase) Dnmt3a (Dnmt3a-CD) is assessed. Damaged DNA duplexes cooperatively bind with Dnmt3a-CD, and O6meG changes the stability of enzyme-substrate complexes. Kinetic analysis of the methylation reaction revealed that O6meG varies the ratio of productive and nonproductive enzyme-substrate complexes and, depending on localization in substrate, causes decrease or increase in DNA methylation. Dnmt3a-CD is less sensitive to the presence of O6meG in DNA substrate than procaryotic MTase SssI recognizing CpG.
Keywords: cooperative binding; murine DNA methyltransferase Dnmt3a; O6-methylguanine; nonproductive complex

Enhancer element potentially involved in human survivin gene promoter regulation in lung cancer cell lines by M. V. Mityaev; E. P. Kopantzev; A. A. Buzdin; T. V. Vinogradova; E. D. Sverdlov (182-191).
We have revealed evolutionarily conserved regions in a 4500-bp DNA sequence 5′-adjacent to the survivin (BIRC5) gene. In the transcribed region of the BIRC5 gene we have detected and characterized in detail a 3′-fragment of the CpG island that stimulated in enhancer-like way the gene promoter activity in normal cells and in a number of cancer, in particular lung cancer, cell lines. When added to the initial 1498-bp survivin promoter region, a transcribed DNA fragment of a CpG island approximately twofold enhanced the promoter activity in cancer cells and in normal lung fibroblasts. The observed effect did not depend upon the orientation of the fragment and distances between the fragment and the transcription initiation site. In the case of a heterologous SV40 virus promoter, the effect was less pronounced. In addition to earlier reports, the results provide new information on the BIRC5 gene expression regulation and also demonstrate that this gene exon sequences can play a significant role in BIRC5 gene expression regulation. The data provide another possibility to increase survivin promoter activity without changing its cancer specificity for application in cancer (in particular, lung cancer) gene therapy.
Keywords: regulation of promoter activity; survivin; BIRC5 gene; enhancer; cis-element; conserved sequence

Statistical analysis of unstructured amino acid residues in protein structures by M. Yu. Lobanov; S. O. Garbuzynskiy; O. V. Galzitskaya (192-200).
We have performed a statistical analysis of unstructured amino acid residues in protein structures available in the databank of protein structures. Data on the occurrence of disordered regions at the ends and in the middle part of protein chains have been obtained: in the regions near the ends (at distance less than 30 residues from the N- or C-terminus), there are 66% of unstructured residues (38% are near the N-terminus and 28% are near the C-terminus), although these terminal regions include only 23% of the amino acid residues. The frequencies of occurrence of unstructured residues have been calculated for each of 20 types in different positions in the protein chain. It has been shown that relative frequencies of occurrence of unstructured residues of 20 types at the termini of protein chains differ from the ones in the middle part of the protein chain; amino acid residues of the same type have different probabilities to be unstructured in the terminal regions and in the middle part of the protein chain. The obtained frequencies of occurrence of unstructured residues in the middle part of the protein chain have been used as a scale for predicting disordered regions from amino acid sequence using the method (FoldUnfold) previously developed by us. This scale of frequencies of occurrence of unstructured residues correlates with the contact scale (previously developed by us and used for the same purpose) at a level of 95%. Testing the new scale on a database of 427 unstructured proteins and 559 completely structured proteins has shown that this scale can be successfully used for the prediction of disordered regions in protein chains.
Keywords: unstructured regions; intrinsically disordered regions; natively unfolded proteins; globular proteins; stability

Examination of the antioxidative homeostasis in skeletal muscle cells in the presence or absence of selenoprotein W (SelW) is necessary to understand the importance of SelW in the antioxidative system. Depletion of SelW by RNA interference was achieved by introducing a synthetic small interfering RNA into the mouse skeletal muscle cell line C2C12 (C3H). Transfectant screening was performed by real-time reverse transcription-PCR, Western blotting, flow cytometry, fluorescence staining, cell viability, and glutathione assays. SelW expression and mRNA levels were downregulated by 62.1 and 72.4%, respectively. In addition, acute cytotoxicity and an apoptosis rate of ∼36% in SelW-depleted cells demonstrated that RNA interference was successful. As compared with non-SelW-depleted cells, the enzyme activities of glutathione peroxidase, superoxide dismutase, and catalase and total antioxidative capability and glutathione level increased by 47.6, 103.0, 31.0, 205.6, and 30.0%, respectively (P < 0.05). Thus, SelW is important for the antioxidative system of muscle cells. Depletion of SelW, however, could be compensated by other intracellular antioxidative enzymes because oxidative stress was not the causative factor for apoptosis in SelW-depleted cells. Thus, the main function of SelW in muscle cells is not in the antioxidative system.
Keywords: selenoprotein W; function; RNA interference antioxidation; muscle cell; in vitro

We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48°C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl BB can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl PA is the molecule of BChl tightly bound with the L-sub- unit in mutant RC as it was supposed earlier.
Keywords: bacterial photosynthesis; site-directed mutagenesis; coordination of magnesium atom; reaction center; bacteriochlorophyll; Rhodobacter sphaeroides ; stability of membrane-associated proteins

Antimicrobial and other oligopeptides of grapes by A. A. Zamyatnin; O. L. Voronina (214-223).
Structures and functions of about 700 oligopeptides of various plants are presently known. However, only one polypeptide has been isolated from grapes and characterized. At the same time, tens of thousands of uncharacterized amino acid sequences have been revealed in this plant, among which there can also be precursors of oligopeptide regulators. Due to the scientific and practical importance of innate immunity of agricultural plants, we have undertaken structural and functional investigation of these sequences to identify new regulatory oligopeptides including antimicrobial agents. For this purpose, we elaborated a special method of computer analysis enabling comparison of primary structures of putative precursors of grape oligopeptides with amino acid sequences of known oligopeptides of other plants. Structural similarity served as the basis for prediction of potential functional properties. As a result, over 20 new structures of antimicrobial and other grape oligopeptides have been found.
Keywords: oligopeptide; grape; precursor; antimicrobial peptide; bioinformatics

Characteristics of binding of zwitterionic liposomes to water-soluble proteins by A. S. Dudkina; A. A. Selischeva; N. I. Larionova (224-232).
The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to 31P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).
Keywords: phosphatidylcholine; phosphatidylethanolamine; liposomes; aprotinin; Bowman-Birk soybean proteinase inhibitor; differential scanning calorimetry

Thermodynamic analysis of protein kinase A Iα activation by O. N. Rogacheva; A. V. Popov; E. V. Savvateeva-Popova; V. E. Stefanov; B. F. Shchegolev (233-241).
Thermodynamic analysis of protein kinase A (PKA) Iα activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3′:5′-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (iso-density polarizable continuum model) computations (ɛ = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Iα activation is mediated by two processes. First, 3′:5′-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (ΔG R o = −23.9 ± 8.2 kJ/mol). Second, 3′:5′-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Iα activation is higher than that to inactive holoenzyme complex (ΔG 3′:5′−AMP o = −28.1 ± 9.7 kJ/mol). ATP is capable of docking into the 3′:5′-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3′:5′-AMP binding to PKA Iα holoenzyme were found to be 60 and 57 μM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 μM, are proved to be direct measures of 3′:5′-AMP-PKA Iα binding affinity. Their values are in a reasonable agreement with the changes in 3′:5′-AMP concentration in the cell (2-55 μM) and account for PKA Iα activation in response to adequate stimuli.
Keywords: protein kinase A Iα activation; 3′:5′-AMP (cyclic adenosine-3′,5′-monophosphate); ATP; standard Gibbs free energy change (ΔGo); stacking interaction

Purification and properties of alcohol oxidase from Pichia putida by A. R. Gvozdev; I. A. Tukhvatullin; R. I. Gvozdev (242-248).
Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8–9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2–3 cysteine residues. The pH optimum was within 8.5–9.0. Specific activity of the enzyme varied from 10 to 50 μmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30–50% (w/v) of sucrose. Different forms of 0.1–1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.
Keywords: alcohol oxidase; subunits; quaternary structure; crystallization

In the present study, a novel structural motif that can be represented as a combination of the known βαβ-unit and ψ-motif is described and analyzed. In theory, there are four possible combinations of the motifs since each of them can exist in two forms, left-handed and right-handed. For this study, we have selected 140 nonhomologous proteins in which 158 combinations of such types have been found. The combination of the right-handed ψ-motif and the right-handed βαβ-unit has been shown to occur most often (87 cases out of 158) and the combination of the left-handed βαβ-unit and the left-handed ψ-motif does not occur at all. Three novel structural trees in which the commonly occurring combinations are taken as the root structures have been constructed.
Keywords: βαβ-unit; modeling; ψ-motif; protein folding; structural classification

Programmed cell death in plants: Protective effect of phenolic compounds against chitosan and H2O2 by V. D. Samuilov; L. A. Vasil’ev; E. V. Dzyubinskaya; D. B. Kiselevsky; A. V. Nesov (257-263).
Addition of chitosan or H2O2 caused destruction of nuclei of epidermal cells (EC) in the epidermis isolated from pea leaves. Phenol, a substrate of the apoplastic peroxidase-oxidase, in concentrations of 10−10–10−6 M prevented the destructive effect of chitosan. Phenolic compounds 2,4-dichlorophenol, catechol, and salicylic acid, phenolic uncouplers of oxidative phosphorylation pentachlorophenol and 2,4-dinitrophenol, and a non-phenolic uncoupler carbonyl cyanide m-chlorophenylhydrazone, but not tyrosine or guaiacol, displayed similar protective effects. A further increase in concentrations of the phenolic compounds abolished their protective effects against chitosan. Malate, a substrate of the apoplastic malate dehydrogenase, replenished the pool of apoplastic NADH that is a substrate of peroxidase-oxidase, prevented the chitosan-induced destruction of the EC nuclei, and removed the deleterious effect of the increased concentration of phenol (0.1 mM). Methylene Blue, benzoquinone, and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) capable of supporting the optimal catalytic action of peroxidase-oxidase cancelled the destructive effect of chitosan on the EC nuclei. The NADH-oxidizing combination of TMPD with ferricyanide promoted the chitosan-induced destruction of the nuclei. The data suggest that the apoplastic peroxidase-oxidase is involved in the antioxidant protection of EC against chitosan and H2O2.
Keywords: apoplastic peroxidase; programmed cell death; chitosan; hydrogen peroxide; phenolic compounds; protective action; plants; epidermal cells