Biochemistry (Moscow) (v.83, #6)

A large body of experimental data have shown that aerobic exercise of different duration, intensity, and pattern affect molecular mechanisms regulating mitochondrial biogenesis in skeletal muscles. This review focuses on the effects of exercise duration and intensity on the molecular mechanisms of mitochondrial biogenesis regulation in skeletal muscles, namely PGC-1α-dependent signaling. Studies of the effects of acute exercise and exercise training showed that an increase in the duration of aerobic exercise from 30 to 90 min does not provide additional stimuli to activate signaling pathways regulating post-translational modification of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and expression of the PGC-1α gene (PPARGC1A). Conversely, exercise intensity substantially affects mitochondrial biogenesis due to the increase in the recruitment of type II muscle fibers with accompanying pronounced metabolic shift leading to the activation of signaling cascades and expression of genes regulating mitochondrial biogenesis. Therefore, intermittent exercise, which recruits type II muscle fibers, is more efficient in the activation of mitochondrial biogenesis than work-matched continuous exercise. In skeletal muscle adapted to aerobic training, intensity-dependent activation of mitochondrial biogenesis after acute exercise is associated primarily with the AMP-activated protein kinase/PGC-1α pathway, expression of PGC-1α-regulated genes, and expression of PPARGC1A from the alternative (distal) inducible promoter regulated by the cAMP response element-binding protein 1-related transcription factors and their coactivators. Elucidation of the effects of duration and intensity of aerobic exercise on the PGC-1α-dependent and -independent mechanisms of mitochondrial biogenesis is important for treatment of patients with various metabolic disorders, as well as for optimization of training in athletes.
Keywords: exercise duration; exercise intensity; PGC-1α; AMPK; CaMK; CREB

Practical Recommendations for Improving Efficiency and Accuracy of the CRISPR/Cas9 Genome Editing System by M. N. Karagyaur; Y. P. Rubtsov; P. A. Vasiliev; V. A. Tkachuk (629-642).
CRISPR/Cas9 genome-editing system is a powerful, fairly accurate, and efficient tool for modifying genomic DNA. Despite obvious advantages, it is not devoid of certain drawbacks, such as propensity for introduction of additional nonspecific DNA breaks, insufficient activity against aneuploid genomes, and relative difficulty in delivering its components to cells. In this review, we focus on the difficulties that can limit the use of CRISPR/Cas9 and suggest a number of practical recommendations and information sources that will make it easier for the beginners to work with this outstanding technological achievement of the XXI century.
Keywords: genome editing; CRISPR/Cas9; NHEJ; HDR; HITI; gRNA design; HDR/HITI template design

Import of Proteins and Nucleic Acids into Mitochondria by N. A. Verechshagina; Yu. M. Konstantinov; P. A. Kamenski; I. O. Mazunin (643-661).
Many mitochondrial genes have been transferred to the nucleus in course of evolution. The products of expression of these genes, being still necessary for organelle function, are imported there from the cytosol. Molecular mechanisms of protein import are studied much deeper than those of nucleic acids. The latter, it seems to us, retards the development of mitochondrial genome editing technologies. In this review, we describe mechanisms of DNA, RNA, and protein import into mitochondria of different eukaryotes. The description is given for the natural processes, as well as for artificial targeting of macromolecules into mitochondria for therapy. Also, we discuss different approaches to introduce changes into the mitochondrial DNA sequence.
Keywords: mitochondria; import; proteins; nucleic acids; mtDNA editing

Aureochromes – Blue Light Receptors by A. B. Matiiv; E. M. Chekunova (662-673).
A variety of living organisms including bacteria, fungi, animals, and plants use blue light (BL) to adapt to changing ambient light. Photosynthetic forms (plants and algae) require energy of light for photosynthesis, movements, development, and regulation of activity. Several complex light-sensitive systems evolved in eukaryotic cells to use the information of light efficiently with photoreceptors selectively absorbing various segments of the solar spectrum, being the first components in the light signal transduction chain. They are most diverse in algae. Photosynthetic stramenopiles, which received chloroplasts from red algae during secondary symbiosis, play an important role in ecosystems and aquaculture, being primary producers. These taxa acquired the ability to use BL for regulation of such processes as phototropism, chloroplast photo-relocation movement, and photomorphogenesis. A new type of BL receptor–aureochrome (AUREO)–was identified in Vaucheria frigida in 2007. AUREO consists of two domains: bZIP (basic-region leucine zipper) domain and LOV (light-oxygen-voltage-sensing) domain, and thus this photoreceptor is a BL-sensitive transcription factor. This review presents current data on the structure, mechanisms of action, and biochemical features of aureochromes.
Keywords: photoreceptors; aureochromes; Vaucheria frigida ; Phaeodactylum tricornutum

Calcineurin and Its Role in Synaptic Transmission by E. O. Tarasova; A. E. Gaydukov; O. P. Balezina (674-689).
Calcineurin (CaN) is a serine/threonine phosphatase widely expressed in different cell types and structures including neurons and synapses. The most studied role of CaN is its involvement in the functioning of postsynaptic structures of central synapses. The role of CaN in the presynaptic structures of central and peripheral synapses is less understood, although it has generated a considerable interest and is a subject of a growing number of studies. The regulatory role of CaN in synaptic vesicle endocytosis in the synapse terminals is actively studied. In recent years, new targets of CaN have been identified and its role in the regulation of enzymes and neurotransmitter secretion in peripheral neuromuscular junctions has been revealed. CaN is the only phosphatase that requires calcium and calmodulin for activation. In this review, we present details of CaN molecular structure and give a detailed description of possible mechanisms of CaN activation involving calcium, enzymes, and endogenous and exogenous inhibitors. Known and newly discovered CaN targets at pre-and post-synaptic levels are described. CaN activity in synaptic structures is discussed in terms of functional involvement of this phosphatase in synaptic transmission and neurotransmitter release.
Keywords: calcineurin; calcineurin inhibitors; endocytosis; L-type calcium channels; regulation of receptors/channels by phosphatases and kinases

Mobility of Nuclear Components and Genome Functioning by E. A. Arifulin; Y. R. Musinova; Y. S. Vassetzky; E. V. Sheval (690-700).
Cell nucleus is characterized by strong compartmentalization of structural components in its three-dimensional space. Certain genomic functions are accompanied by changes in the localization of chromatin loci and nuclear bodies. Here we review recent data on the mobility of nuclear components and the role of this mobility in genome functioning.
Keywords: nucleus; genome; chromatin; nuclear bodies; mobility

Structural Study of the Complex Formed by Ceruloplasmin and Macrophage Migration Inhibitory Factor by A. V. Sokolov; L. A. Dadinova; M. V. Petoukhov; G. Bourenkov; K. M. Dubova; S. V. Amarantov; V. V. Volkov; V. A. Kostevich; N. P. Gorbunov; N. A. Grudinina; V. B. Vasilyev; V. R. Samygina (701-707).
Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with p-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.
Keywords: X-ray analysis; small-angle X-ray scattering; protein–protein interactions; ceruloplasmin; macrophage migration inhibitory factor

Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display by L. N. Shingarova; L. E. Petrovskaya; A. V. Zlobinov; S. Sh. Gapizov; E. A. Kryukova; K. R. Birikh; E. F. Boldyreva; S. A. Yakimov; D. A. Dolgikh; M. P. Kirpichnikov (708-716).
Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.
Keywords: tumor necrosis factor; TNF-binding proteins; 10th human fibronectin type III domain; bacterial display; autotransporter from Psychrobacter cryohalolentis K5T

Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and “Rathayibacter tanaceti” VKM Ac-2596 by A. S. Shashkov; E. M. Tul’skaya; A. S. Dmitrenok; G. M. Streshinskaya; N. V. Potekhina; S. N. Senchenkova; N. F. Piskunkova; L. V. Dorofeeva; L. I. Evtushenko (717-726).
On p. 725 in section Acknowledgments instead of:Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers–a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2: 4: 1, respectively (the structure is presented in the text). The “Rathayibacter tanaceti” VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.
Keywords: Rathayibacter ; cell wall; rhamnomannan; neutral glycopolymers; D-rhamnose; NMR spectroscopy

Localization of Galectins within Glycocalyx by E. M. Rapoport; V. K. Matveeva; O. A. Vokhmyanina; I. M. Belyanchikov; H.-J. Gabius; N. V. Bovin (727-737).
Galectins are involved in various biological processes, e.g. cell–cell and cell–matrix adhesion and the transmission of cellular signals. Despite the diversity of functions, little is known about the nature of their physiological cognate ligands on the cell surface and the localization of galectins in the glycocalyx, although this information is important for understanding the functional activity of galectins. In this work, localization of endogenous and exogenously loaded galectins in the glycocalyx was studied. The following main conclusions are drawn: 1) galectins are not evenly distributed within the glycocalyx, they are accumulated in patches. Patching is not the result of a cross-linking of cellular glycans by galectins. Instead, patch-wise localization is the consequence of irregular distribution of glycans forming the glycocalyx; 2) galectins are accumulated in the inner zone of the glycocalyx rather than at its outer face or directly in vicinity of the cell membrane; 3) patches are not associated with cell rafts.
Keywords: galectins; glycans; glycocalyx; mammalian cell

Blocking the expression of integrin α2β1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2β1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2β1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the “reversed” (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2β1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2β1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.
Keywords: integrins; tumor growth; proliferation; invasion; Akt protein kinase; signaling

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide by O. V. Glushkova; S. B. Parfenyuk; T. V. Novoselova; M. O. Khrenov; S. M. Lunin; E. G. Novoselova (746-754).
The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.
Keywords: RAW 264.7; lipopolysaccharide; p38; casein kinase II; NF-κB and SAPK/JNK signaling pathways; cytokines; heat shock proteins; TLR4

Transgenic Arabidopsis Plants Expressing Grape Glutathione S-Transferase Gene (VvGSTF13) Show Enhanced Tolerance to Abiotic Stress by Jing Xu; Ai-Qing Zheng; Xiao-Juan Xing; Lei Chen; Xiao-Yan Fu; Ri-He Peng; Yong-Sheng Tian; Quan-Hong Yao (755-765).
Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.
Keywords: glutathione S-transferase; abiotic stress; stress tolerance; oxidative stress; grape

MicroRNAs (miRNA) play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. However, the miRNAs from skin of Andrias davidianus have not been reported. In this study, a small-RNA cDNA library was constructed and sequenced from skin of A. davidianus. A total of 513 conserved miRNAs belonging to 174 families were identified. The remaining 108 miRNAs we identified were novel and likely to be skin tissue-specific but were expressed at low levels. The presence of randomly selected 15 miRNAs identified and their expression in eight different tissues from A. davidianus were validated by stem-loop qRT-PCR. For better understanding the functions of miRNAs, 129,791 predicated target genes were analyzed by GO and their pathways illustrated by KEGG pathway analyses. The results show that these identified miRNAs from A. davidianus skin are involved in a broad range of physiological functions including metabolism, growth, development, and immune responses. This study exhaustively identifies miRNAs and their target genes, which will ultimately pave the way for understanding their role in skin of A. davidianus and other amphibians. Further studies are necessary to better understand miRNA-mediated gene regulation.
Keywords: Andrias davidianus ; microRNA; skin; deep sequencing; target gene