Бочарова Ольга Владимировна


Период обученияСтрана, городУчебное заведениеДополнительная информация
1989–1995 Россия, Москва Российский Государственный Медицинский Университет, отделение биофизики Диплом биофизика
2002 Россия, Москва ГУ НИИ эпидемиологии и микробиологии имени Н.Ф. Гамалеи Присуждена учёная степень кандидата медицинских наук за диссертацию "Непрерывные В-эпитопы искусственного белка альбебетина и его биологически активных производных"

Избранные публикации

  1. Bocharova O.V., Urban A.S., Nadezhdin K.D., Bocharov E.V., Arseniev A.S. (2016). Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies. Protein Expr. Purif. 123, 105–11 [+]

    Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP686-726, containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the (13)C,(15)N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP671-726WT fragment.

  2. Bocharova O.V., Bragin P.E., Bocharov E.V., Mineev K.S., Goncharuk S.A., Arseniev A.S. (2016). Cell Free Expression and Purification of the Fragments of the Receptor Tyrosine Kynases of the EGFR Family, Containing the Transmembrane Domain with the Juxtamembrane Region, for Structural Studies. BIOLOGICHESKIE MEMBRANY 33 (2), 124–132 [+]

    The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the 13C,15N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.

  3. Bragin P.E., Mineev K.S., Bocharova O.V., Volynsky P.E., Bocharov E.V., Arseniev A.S. (2016). HER2 Transmembrane Domain Dimerization Coupled with Self-Association of Membrane-Embedded Cytoplasmic Juxtamembrane Regions. J. Mol. Biol. 428 (1), 52–61 [+]

    Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.

  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. Nadezhdin K.D., Bocharova O.V., Bocharov E.V., Arseniev A.S. (2012). Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment. FEBS Lett. 586 (12), 1687–92 [+]

    Some pathogenic mutations associated with Alzheimer's disease are thought to affect structural-dynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln(686)-Lys(726) was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning α-helix Lys(699)-Lys(724) self-associates in a left-handed parallel dimer through extended heptad repeat motif I(702)X(3)M(706)X(2)G(709)X(3)A(713)X(2)I(716)X(3)I(720)X(2)I(723), whereas the juxtamembrane region Gln(686)-Val(695) constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-β peptide.

  6. Mineev K.S., Bocharov E.V., Pustovalova Y.E., Bocharova O.V., Chupin V.V., Arseniev A.S. (2010). Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases. J. Mol. Biol. 400 (2), 231–243 [+]

    Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed alpha-helical bundle through their N-terminal double GG4-like motif T(648)G(649)X(2)G(652)A(653) and glycine zipper motif T(652)X(3)S(656)X(3)G(660), respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.

  7. Sun Y., Breydo L., Makarava N., Yang Q., Bocharova O.V., Baskakov I.V. (2007). Site-specific conformational studies of prion protein (PrP) amyloid fibrils revealed two cooperative folding domains within amyloid structure. J. Biol. Chem. 282 (12), 9090–7 [+]

    Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.

  8. Bocharova O.V., Makarava N., Breydo L., Anderson M., Salnikov V.V., Baskakov I.V. (2006). Annealing prion protein amyloid fibrils at high temperature results in extension of a proteinase K-resistant core. J. Biol. Chem. 281 (4), 2373–9 [+]

    Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.

  9. 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.

  10. Bocharova O.V., Breydo L., Salnikov V.V., Baskakov I.V. (2005). Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils. Biochemistry 44 (18), 6776–87 [+]

    In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu(2+) on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu(2+) inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for Cu(2+), and less so for Zn(2+), while Mn(2+) had no effect on the conversion. Cu(2+)-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu(2+)-binding capacity. Using rPrP 89-230, we found that Cu(2+)-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu(2+) inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu(2+) also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu(2+) induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu(2+) induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu(2+) in the pathogenesis of prion diseases is complex. Because Cu(2+) may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.

  11. Bocharova O.V., Breydo L., Salnikov V.V., Gill A.C., Baskakov I.V. (2005). Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease. Protein Sci. 14 (5), 1222–32 [+]

    In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP(Sc) produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.