Шаронов Георгий Владимирович

Личная информация

Шаронов Г. В. является выпускником Московского физико-технического института 2003 г. и работает в Лаборатории оптической микроскопии и спектроскопии биомолекул ИБХ РАН с 2000 года.

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1997–2003 г. Долгопрудный, Московская обл. Московский физико-технический институт (МФТИ) Дипломы бакалавра и магистра прикладных физики и математики. Специализация: физико-химическая биология и биотехнология.

Научные интересы

Основной областью научных интересов Шаронова Г.В. является механизм действия рецепторов и передачи сигнала через плазматичекую мембрану. В этих исследованияю особое внимание уделяется роли липидов и цитоскелета. 

Основные научные результаты

Шароновым Г.В. впервые показано взаимодействие между трансмембранными доменами эфринового рецептора и роль этого взаимодействия в активации рецептора. На основании этих и других данных, полученных сотрудникаим отдела структурной биологии ИБХ РАН, предложен механизм активации рецепторных тирозиновых киназ за счет изменения их взаимодействия с липидными рафтами (Sharonov et. al. 2014, Bocharov et al. 2017). Работы по изучению ГФИ-заякоренных белков послужили основой для формулировки Шароновым Г.В. гипотезы, объясняющий их биологическую активность влиянием на примембранный цитоскелет (Шаронов и др.,  2016). Шароновым Г.В. были разработаны и использованы оригинальные подходы оптической микроскопии и сортировки клеток в потоке. В частности были оптимизированы подходы для сортировки малых количеств лимфоцитов с целью сиквенирования вариабильных участков генов антиген распознающих рецепторов. (Egorov et al.). С участием Шаронова Г.В. было обнаружено наличие нового типа взаимодействия между отдаленными участками внутри гена зеленого флуоресцентного белка, что было опубликовано в журнале Nature (Sarkisyan et al., 2016). 

Избранные публикации

  1. Serebrovskaya E.O., Yuzhakova D.V., Ryumina A.P., Druzhkova I.N., Sharonov G.V., Kotlobay A.A., Zagaynova E.V., Lukyanov S.A., Shirmanova M.V. (2016). Soluble OX40L favors tumor rejection in CT26 colon carcinoma model. Cytokine 84, 10–6 [+]

    OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice. We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.

    ID:1585
  2. Sharonov G.V., Balatskaya M.N., Tkachuk V.A. (2016). Glycosylphosphatidylinositol-Anchored Proteins as Regulators of Cortical Cytoskeleton. Biochemistry Mosc. 81 (6), 636–50 [+]

    Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are important players in reception and signal transduction, cell adhesion, guidance, formation of immune synapses, and endocytosis. At that, a particular GPI-AP can have different activities depending on a ligand. It is known that GPI-AP oligomer creates a lipid raft in its base on plasma membrane, which serves as a signaling platform for binding and activation of src-family kinases. Yet, this does not explain different activities of GPI-APs. Meanwhile, it has been shown that short-lived actomyosin complexes are bound to GPI-APs through lipid rafts. Here, we hypothesize that cell cortical cytoskeleton is the main target of GPI-AP signaling. Our hypothesis is based on the fact that the GPI-AP-induced lipid raft bound to actin filaments and anionic lipids of this raft is known to interact with and activate various actin-nucleating factors, such as formins and N-WASP. It is also known that these and other actin-regulating proteins are activated by src-family kinases directly or through their effectors, such as cortactin and abl-kinases. Regulation of cytoskeleton by GPI-APs may have impact on morphogenesis, cell guidance, and endocytosis, as well as on signaling of other receptors. To evaluate our hypothesis, we have comprehensively considered physiological activities of two GPI-APs - urokinase receptor and T-cadherin.

    ID:1584
  3. Sarkisyan K.S., Bolotin D.A., Meer M.V., Usmanova D.R., Mishin A.S., Sharonov G.V., Ivankov D.N., Bozhanova N.G., Baranov M.S., Soylemez O., Bogatyreva N.S., Vlasov P.K., Egorov E.S., Logacheva M.D., Kondrashov A.S., Chudakov D.M., Putintseva E.V., Mamedov I.Z., Tawfik D.S., Lukyanov K.A., Kondrashov F.A. (2016). Local fitness landscape of the green fluorescent protein. Nature 533 (7603), 397–401 [+]

    Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

    ID:1529
  4. Zlobovskaya O.A., Sergeeva T.F., Shirmanova M.V., Dudenkova V.V., Sharonov G.V., Zagaynova E.V., Lukyanov K.A. (2016). Genetically encoded far-red fluorescent sensors for caspase-3 activity. BioTechniques 60 (2), 62–8 [+]

    Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.

    ID:1373
  5. TyurinKuzmin P.A., Zhdanovskaya N.D., Sukhova A.A., Sagaradze G.D., Albert E.A., Ageeva L.V., Sharonov G.V., Vorotnikov A.V., Tkachuk V.A. (2016). Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF. PLoS ONE 11 (4), e0154157 [+]

    Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.

    ID:1586
  6. Sarkisyan K.S., Goryashchenko A.S., Lidsky P.V., Gorbachev D.A., Bozhanova N.G., Gorokhovatsky A.Y., Pereverzeva A.R., Ryumina A.P., Zherdeva V.V., Savitsky A.P., Solntsev K.M., Bommarius A.S., Sharonov G.V., Lindquist J.R., Drobizhev M., Hughes T.E., Rebane A., Lukyanov K.A., Mishin A.S. (2015). Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime. Biophys. J. 109 (2), 380–9 [+]

    Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

    ID:1305
  7. Egorov E.S., Merzlyak E.M., Shelenkov A.A., Britanova O.V., Sharonov G.V., Staroverov D.B., Bolotin D.A., Davydov A.N., Barsova E., Lebedev Y.B., Shugay M., Chudakov D.M. (2015). Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers. J. Immunol. 194 (12), 6155–63 [+]

    Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.

    ID:1313
  8. Sarkisyan K.S., Zlobovskaya O.A., Gorbachev D.A., Bozhanova N.G., Sharonov G.V., Staroverov D.B., Egorov E.S., Ryabova A.V., Solntsev K.M., Mishin A.S., Lukyanov K.A. (2015). KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS ONE 10 (12), e0145287 [+]

    Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.

    ID:1355
  9. 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.

    ID:1139
  10. Turchaninova M.A., Britanova O.V., Bolotin D.A., Shugay M., Putintseva E.V., Staroverov D.B., Sharonov G., Shcherbo D., Zvyagin I.V., Mamedov I.Z., Linnemann C., Schumacher T.N., Chudakov D.M. (2013). Pairing of T-cell receptor chains via emulsion PCR. European journal of immunology , [+]

    Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains - both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are co-expressed in the same living cells. This article is protected by copyright. All rights reserved.

    ID:849
  11. 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.

    ID:1479
  12. Mamedov I.Z., Britanova O.V., Bolotin D.A., Chkalina A.V., Staroverov D.B., Zvyagin I.V., Kotlobay A.A., Turchaninova M.A., Fedorenko D.A., Novik A.A., Sharonov G.V., Lukyanov S., Chudakov D.M., Lebedev Y.B. (2011). Quantitative tracking of T cell clones after haematopoietic stem cell transplantation. EMBO Mol Med 3 (4), 201–7 [+]

    Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.

    ID:453
  13. Serebrovskaya E.O., Gorodnicheva T.V., Ermakova G.V., Solovieva E.A., Sharonov G.V., Zagaynova E.V., Chudakov D.M., Lukyanov S., Zaraisky A.G., Lukyanov K.A. (2011). Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein. Biochem. J. 435 (1), 65–71 [+]

    Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

    ID:550
  14. Mamedov I.Z., Britanova O.V., Chkalina A.V., Staroverov D.B., Amosova A.L., Mishin A.S., Kurnikova M.A., Zvyagin I.V., Mutovina Z.Y., Gordeev A.V., Khaidukov S.V., Sharonov G.V., Shagin D.A., Chudakov D.M., Lebedev Y.B. (2009). Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients. Autoimmunity 42 (6), 525–36 [+]

    Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

    ID:276
  15. Chertkova R.V., Sharonov G.V., Feofanov A.V., Bocharova O.V., Latypov R.F., Chernyak B.V., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P. (2008). Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences. Mol. Cell. Biochem. 314 (1-2), 85–93 [+]

    Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) > h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

    ID:1685
  16. Sharonov G.V., Karmakova T.A., Kassies R., Pljutinskaya A.D., Grin M.A., Refregiers M., Yakubovskaya R.I., Mironov A.F., Maurizot J.C., Vigny P., Otto C., Feofanov A.V. (2006). Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution. Free Radic. Biol. Med. 40 (3), 407–19 [+]

    В работе проведено сравнительное исследование 11 новых фотосенсибилиаторов для фотодинамической терапии рака — циклоимидных производных бактериохлорина р с различными боковыми заместителями. Показано, что данные соединения обладают интенсивным поглощением в спектральном диапазоне прозрачности биологической ткани (780—830 нм), высоким квантовым выходом генерации синглетного кислорода (0,54—0,56) и способностью проникать и накапливаться в опухолевых клетках in vitro. Установлено, что с помощью боковых заместителей можно направлять данные соединения преимущественно в аппарат Гольджи, или липидные капли, или лизосомы и обеспечивать высокие коэффициенты накопления этих соединений в раковых клетках. На модели перевивной опухоли мышей выявлено усиленное накопление изучаемых соединений в опухоли и окружающей соединительной ткани, что с учетом перечисленных выше свойств позволяет рекомендовать циклоимидные производные бактериохлорина р в качестве перспективных фотосенсибилизаторов для фотодинамической терапии рака.

    ID:92
  17. 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.

    ID:93
  18. 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.

    ID:94
  19. Feofanov A.V., Sharonov G.V., Dubinnyi M.A., Astapova M.V., Kudelina I.A., Dubovskii P.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. (2004). Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje. Biochemistry Mosc. 69 (10), 1148–57 [+]

    Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

    ID:341
  20. Feofanov A., Sharonov G., Grichine A., Karmakova T., Pljutinskaya A., Lebedeva V., Ruziyev R., Yakubovskaya R., Mironov A., Refregier M., Maurizot J.C., Vigny P. (2004). Comparative study of photodynamic properties of 13,15-N-cycloimide derivatives of chlorin p6. Photochem. Photobiol. 79 (2), 172–88 [+]

    В работе проведено сравнительное исследование четырех новых фотосенсибилизаторов на основе циклоимидных производных хлорина p6 с различными боковыми заместителями. Установлено, что фотодинамическая активность данных соединений в отношении опухолевых клеток in vitro в несколько сот раз выше, чем активность используемого в клинике препарата Фотогем. Показано, что данные соединения эффективно образуют синглетный кислород (квантовый выход 0,35—0,66) и накапливаются в клетках в мономерной фотоактивной форме в связанном с липидными структурами состоянии. В работе продемонстрировано, что с помощью боковых заместителей можно направлять данные соединения в различные внутриклеточные компартменты: аппарат Гольджи, митохондрии, ядерную мембрану и липидные капли. Под действием света данные фотосенсибилизаторы вызывают гибель клеток преимущественно по механизму апоптоза при сублетальном режиме (гибель 50% клеток) и по механизму некроза — при летальном (гибель 100% клеток).

    ID:95