Vol 11, No 1 (2019)


Ligands of Acid-Sensing Ion Channel 1a: Mechanisms of Action and Binding Sites

Tikhonov D.B., Magazanik L.G., Nagaeva E.I.


The proton-gated cationic channels belonging to the ASIC family are widely distributed in the central nervous system of vertebrates and play an important role in several physiological and pathological processes. ASIC1a are most sensitive to acidification of the external medium, which is the reason for the current interest in their function and pharmacology. Recently, the list of ASIC1a ligands has been rapidly expanding. It includes inorganic cations, a large number of synthetic and endogenous small molecules, and peptide toxins. The information on the mechanisms of action and the binding sites of the ligands comes from electrophysiological, mutational and structural studies. In the present review, we attempt to present a systematic view of the complex pattern of interactions between ligands and ASIC1a.

Acta Naturae. 2019;11(1):4-13
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Research Articles

Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1

Agapkina Y.Y., Pustovarova M.A., Korolev S.P., Zyryanova D.P., Ivlev V.V., Totmenin A.V., Gashnikova N.M., Gottikh M.B.


The high genetic variability of the human immunodeficiency virus (HIV-1) leads to a constant emergence of new genetic variants, including the recombinant virus CRF63_02A1, which is widespread in the Siberian Federal District of Russia. We studied HIV-1 CRF63_02A1 integrase (IN_CRF) catalyzing the incorporation of viral DNA into the genome of an infected cell. The consensus sequence was designed, recombinant integrase was obtained, and its DNA-binding and catalytic activities were characterized. The stability of the IN_CRF complex with the DNA substrate did not differ from the complex stability for subtype A and B integrases; however, the rate of complex formation was significantly higher. The rates and efficiencies of 3’-processing and strand transfer reactions catalyzed by IN_CRF were found to be higher, too. Apparently, all these distinctive features of IN_CRF may result from specific amino acid substitutions in its N-terminal domain, which plays an important role in enzyme multimerization and binding to the DNA substrate. It was also found that the drug resistance mutations Q148K/G140S and G118R/E138K significantly reduce the catalytic activity of IN_CRF and its sensitivity to the strand transfer inhibitor raltegravir. Reduction in sensitivity to raltegravir was found to be much stronger in the case of double-mutation Q148K/G140S.

Acta Naturae. 2019;11(1):14-22
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Isolation, Purification and Characterization of L,D-transpeptidase 2 from Mycobacterium tuberculosis

Baldin S.M., Shcherbakova T.A., Švedas V.K.


L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of nonclassical 3-3 peptidoglycan cross-links in a pathogen’s cell wall making it resistant to a broad range of penicillin antibiotics. The conditions of cultivation, isolation, and purification of recombinant L,D-transpeptidase 2 from M. tuberculosis have been optimized in this study. Oxidation of the free SH groups of catalytic cysteine Cys354 is an important factor causing the inactivation of the enzyme, which occurs during both the expression and storage of enzyme preparations. The biochemical characteristics of purified L,D-transpeptidase 2 and L,D-transpeptidase 2 lacking domain A were determined; the kinetic constants of enzyme-catalyzed nitrocefin transformation were evaluated.

Acta Naturae. 2019;11(1):23-28
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Thermodynamics of the DNA Repair Process by Endonuclease VIII

Kladova O.A., Kuznetsov N.A., Fedorova O.S.


In the present work, a thermodynamic analysis of the interaction between endonuclease VIII (Endo VIII) and model DNA substrates containing damaged nucleotides, such as 5,6-dihydrouridine and 2-hydroxymethyl-3-hydroxytetrahydrofuran (F-site), was performed. The changes in the fluorescence intensity of the 1,3-diaza-2-oxophenoxazine (tCO) residue located in the complementary chain opposite to the specific site were recorded in the course of the enzyme-substrate interaction. The kinetics was analyzed by the stopped-flow method at different temperatures. The changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage, were calculated. The comparison of the kinetic and thermodynamic data characterizing the conformational transitions of enzyme and DNA in the course of their interaction made it possible to specify the nature of the molecular processes occurring at the stages of substrate binding, recognition of the damaged base, and its removal from DNA.

Acta Naturae. 2019;11(1):29-37
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Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein

Ozharovskaia T.A., Zubkova O.V., Dolzhikova I.V., Gromova A.S., Grousova D.M., Tukhvatulin A.I., Popova O., Shcheblyakov D.V., Scherbinin D.N., Dzharullaeva A.S., Erokhova A.S., Shmarov M.M., Loginova S.Y., Borisevich S.V., Naroditsky B.S., Logunov D.Y., Gintsburg A.L.


The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 during the first Middle East respiratory syndrome (MERS) outbreaks. MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality rate of ~35.5%. Currently, there are no registered vaccines or means of therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein plays the most important role in the viral life cycle (virus internalization). The S protein is an immunodominant antigen and the main target for neutralizing antibodies. In the present study, the immunogenicities of five different forms of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein, the full-length S glycoprotein with the transmembrane domain of the G glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5) were used as delivery vehicles. Vaccination with all of the developed rAd5 vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral immune response was induced after the animal had been vaccinated with the membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing antibodies among all vaccinated animals. The most significant cellular immune response was induced after vaccination of the animals with the full-length S (rAd5-S). These investigations suggest that the full-length S and the membrane form of the RBD (RBD-G) are the most promising vaccine candidates among all the studied forms of S glycoprotein.

Acta Naturae. 2019;11(1):38-47
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A Novel Dipeptide NGF Mimetic GK-2 Selectively Activating the PI3K/AKT Signaling Pathway Promotes the Survival of Pancreatic β-Cells in a Rat Model of Diabetes

Ostrovskaya R.U., Ivanov S.V., Gudasheva T.A., Seredenin S.B.


We investigated the cytoprotective effect of a novel low-molecular-weight NGF mimetic, GK-2 (hexamethylenediamide bis-N-monosuccinyl-L-glutamyl-L-lysine), on pancreatic β-cells. The neuroprotective effect of GK-2 had been previously shown to be associated with selective activation of the PI3K/Akt signaling pathway. In this study, rats with streptozotocin (STZ)-induced type 2 diabetes mellitus were used. Metformin was used as a reference drug. STZ was immunohistochemically demonstrated to reduce the number of β-cells and affect their morphological structure. Treatment of diabetic animals with GK-2 (at a dose of 0.5 mg/kg intraperitoneally or 5 mg/kg orally) or metformin (300 mg/kg orally) for 28 days reduced the damaging effect of STZ. The effect of GK-2 on manifestations of STZ-induced diabetes, such as hyperglycemia, weight loss, polyphagia, and polydipsia, was comparable to that of metformin, while the cytoprotective activity of GK-2 was slightly stronger than that of metformin. A strong positive correlation between morphometric parameters and the blood glucose level was revealed. The GK-2 cytoprotective effect on β-cells is supposed to manifest through the PI3K/Akt signaling pathway.

Acta Naturae. 2019;11(1):48-57
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Structure of the Anti-C60 Fullerene Antibody Fab Fragment: Structural Determinants of Fullerene Binding

Osipov E.M., Hendrickson O.D., Tikhonova T.V., Zherdev A.V., Solopova O.N., Sveshnikov P.G., Dzantiev B.B., Popov V.O.


The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by π-π stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.

Acta Naturae. 2019;11(1):58-65
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Adaptation of the Newcastle Disease Virus to Cell Cultures for Enhancing Its Oncolytic Properties

Yurchenko K.S., Jing Y., Shestopalov A.M.


This study focuses on the adaptation of natural Newcastle disease virus (NDV) strains isolated from wild birds to human tumor cells. Many candidates for virotherapy are viruses pathogenic for human. During recombination of genetic material, there always exists a risk of getting a virus with an unstable genome. This problem can be solved by using natural apathogenic viruses as oncolytic agents. The Newcastle disease virus is the causative agent of contagious avian diseases. Its natural strains exhibit an antitumor effect and are considered safe for humans. As shown in earlier studies, the oncolytic properties of natural strains can be enhanced during adaptation to cell cultures, without interference in the virus genome. This study demonstrates that serial passaging increases the viral infectious titer in cancer cells. Moreover, the viability of tumor cells decreases post-infection when Newcastle disease virus strains are adapted to these cell cultures. The findings of this study complement the well-known data on the adaptation of the Newcastle disease virus to human cancer cells. Hence, it is possible to obtain a NDV strain with a more pronounced oncolytic potential during adaptation. This should be taken into account when choosing a strategy for designing anticancer drugs based on this virus.

Acta Naturae. 2019;11(1):66-73
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CASBench: A Benchmarking Set of Proteins with Annotated Catalytic and Allosteric Sites in Their Structures

Zlobin A.S., Suplatov D.А., Kopylov K.Е., Švedas V.К.


In recent years, the phenomenon of allostery has witnessed growing attention driven by a fundamental interest in new ways to regulate the functional properties of proteins, as well as the prospects of using allosteric sites as targets to design novel drugs with lower toxicity due to a higher selectivity of binding and specificity of the mechanism of action. The currently available bioinformatic methods can sometimes correctly detect previously unknown ligand binding sites in protein structures. However, the development of universal and more efficient approaches requires a deeper understanding of the common and distinctive features of the structural organization of both functional (catalytic) and allosteric sites, the evolution of their amino acid sequences in respective protein families, and allosteric communication pathways. The CASBench benchmark set contains 91 entries related to enzymes with both catalytic and allosteric sites within their structures annotated based on the experimental information from the Allosteric Database, Catalytic Site Atlas, and Protein Data Bank. The obtained dataset can be used to benchmark the performance of existing computational approaches and develop/train perspective algorithms to search for new catalytic and regulatory sites, as well as to study the mechanisms of protein regulation on a large collection of allosteric enzymes. Establishing a relationship between the structure, function, and regulation is expected to improve our understanding of the mechanisms of action of enzymes and open up new prospects for discovering new drugs and designing more efficient biocatalysts. The CASBench can be operated offline on a local computer or online using built-in interactive tools at https://biokinet.belozersky.msu.ru/casbench.

Acta Naturae. 2019;11(1):74-80
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Mauritian Endemic Medicinal Plant Extracts Induce G2/M Phase Cell Cycle Arrest and Growth Inhibition of Oesophageal Squamous Cell Carcinoma in Vitro

Rummun N., Hughes R.E., Beesoo R., Li W.W., Aldulaimi O., Macleod K.G., Bahorun T., Carragher N.O., Kagansky A., Neergheen-Bhujun V.S.


Terrestrial plants have contributed massively to the development of modern oncologic drugs. Despite the wide acceptance of Mauritian endemic flowering plants in traditional medicine, scientific evidence of their chemotherapeutic potential is lacking. This study aimed to evaluate the in vitro tumor cytotoxicity of leaf extracts from five Mauritian endemic medicinal plants, namely Acalypha integrifolia Willd (Euphorbiaceae), Labourdonnaisia glauca Bojer (Sapotaceae), Dombeya acutangula Cav. subsp. rosea Friedmann (Malvaceae), Gaertnera psychotrioides (DC.) Baker (Rubiaceae), and Eugenia tinifolia Lam (Myrtaceae).rolex oyster perpetual day date real or fake The cytotoxicities of the extracts were determined against six human cancer cell lines, including cervical adenocarcinoma, colorectal carcinoma, oesophageal adenocarcinoma, and oesophageal squamous cell carcinoma. The potent extracts were further investigated using cell cycle analysis and reverse phase protein array (RPPA) analysis. The antioxidant properties and polyphenolic profile of the potent extracts were also evaluated. Gas chromatography mass spectrometry (GC-MS) analyses revealed the presence of (+)-catechin and gallocatechin in E. tinifolia and L. glauca, while gallic acid was detected in A. integrifolia. L. glauca, A. integrifolia, and E. tinifolia were highly selective towards human oesophageal squamous cell carcinoma (KYSE-30) cells. L. glauca and E. tinifolia arrested KYSE- 30 cells in the G2/M phase, in a concentration-dependent manner. RPPA analysis indicated rolex fake that the extracts may partly exert their tumor growth-inhibitory activity by upregulating the intracellular level of 5′AMP-activated kinase (AMPK). The findings highlight the potent antiproliferative activity of three Mauritian endemic leaf extracts against oesophageal squamous ccheap replica luxury watches ell carcinoma and calls for further investigation into their chemotherapeutic application.

Acta Naturae. 2019;11(1):81-90
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