Астапова Мария Вячеславовна

Кандидат биологических наук

Эл. почта: ast-mariya@yandex.ru

Избранные публикации

  1. Shulepko M.A., Lyukmanova E.N., Shenkarev Z.O., Dubovskii P.V., Astapova M.V., Feofanov A.V., Arseniev A.S., Utkin Y.N., Kirpichnikov M.P., Dolgikh D.A. (2016). Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expr. Purif. 130, 13–20 [+]

    Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

  2. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

  3. Sharonov G.V., Bocharov E.V., Kolosov P.M., Astapova M.V., Arseniev A.S., Feofanov A.V. (2014). Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase. J. Biol. Chem. 289 (21), 14955–64 [+]

    The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.

  4. Nekrasova O.V., Sharonov G.V., Tikhonov R.V., Kolosov P.M., Astapova M.V., Yakimov S.A., Tagvey A.I., Korchagina A.A., Bocharova O.V., Wulfson A.N., Feofanov A.V., Kirpichnikov M.P. (2012). Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity. Biochemistry Mosc. 77 (12), 1387–94 [+]

    Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

  5. Feofanov A.V., Sharonov G.V., Astapova M.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. (2005). Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage. Biochem. J. 390 (Pt 1), 11–8 [+]

    Статья посвящена исследованию механизмов цитотоксического действия цитотоксинов (ЦТ) из яда кобр. В данной работе показано, что ЦТ 1 и 2 из яда кобры Naja oxiana, ЦТ 1 из Naja haje и ЦТ 3 из Naja kaouthia способны накапливаться в лизосомах клеток промиелоцитарной лейкемии человека HL60 и аденокарциномы легкого человека A549. Кинетика и концентрационная зависимость накопления ЦТ в лизосомах согласуется с кинетикой и концентрационной зависимостью гибели клеток, что свидетельствует о том, что лизосомы могут быть одной из мишеней ЦТ, а воздействие на эту мишень заключается в концентрационно-зависимой пермеабилизации мембраны лизосом.

  6. Sharonov G.V., Feofanov A.V., Bocharova O.V., Astapova M.V., Dedukhova V.I., Chernyak B.V., Dolgikh D.A., Arseniev A.S., Skulachev V.P., Kirpichnikov M.P. (2005). Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells. Apoptosis 10 (4), 797–808 [+]

    В работе докладывается о разработке метода измерения проапоптотическй активности экзогенного цитохрома с в живых клетках. Метод основан на введении белка в цитоплазму клетки с помощью электропорации и выявлении с помощью флуоресцентной микроскопии признаков развития апоптоза в клетках. С помощью данного метода были измерены относительные про-апоптозные активности лошадиного цитохрома с и четырех мутантных вариантов данного белка. Обнаружено, что аминокислотная замена К72W полностью блокирует про-апоптозную активность цитохрома с, но не влияет на его «дыхательную» функцию. Методом КОМИРСИ была впервые оценена минимальная цитоплазматическая концентрация лошадиного цитохрома с, необходимая для индукции апоптоза в клетках WEHI-3b.

  7. Osipov A.V., Astapova M.V., Tsetlin V.I., Utkin Y.N. (2004). The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom. Eur. J. Biochem. 271 (10), 2018–27 [+]

    В этой работе впервые был описан гликозилированный цитотоксин, выделенный из яда кобры Naja kaouthia. Это первый представитель обширного семейства трех-петельных токсинов, для которого обнаружена пост-трансляционная модификация

  8. Kostanyan I.A., Astapova M.V., Starovoytova E.V., Dranitsina S.M., Lipkin V.M. (1994). A new human leukemia cell 8.2 kDa differentiation factor: isolation and primary structure. FEBS Lett. 356 (2-3), 327–9 [+]

    A new 8.2 kDa differentiation factor has been purified to homogeneity from the cultural media of human myelogenous HL-60 leukemia cells induced by retinoic acid. cDNA clones encoding this factor were isolated from a cDNA library prepared from HL-60 differentiated cells and their nucleotide sequence has been determined. The deduced amino acid sequence of the differentiation factor molecule consists of 54 amino acid residues. The protein is shown to be glycosylated. It was shown by Northern blot experiments that the level of poly(A)+ RNA with a length of 450 nucleotides was higher in differentiated cells than in non-differentiated cells.