Лаборатория молекулярной токсинологии

Отдел молекулярных основ нейросигнализации

Руководитель: Уткин Юрий Николаевич, д. х. н.
utkin@ibch.ru+7(495)336-65-22

яды змей, токсины, выделение и свойства новых белков, Никотиновый холинорецептор

Лаборатория проводит фундаментальные исследования по изучению молекулярной структуры компонентов животных ядов и механизмов их действия. В качестве основного инструмента для исследований механизмов функционирования нервной системы и проведения нервных импульсов используются токсины. В результате расширения круга интересов и задач Лаборатории ее сотрудники изучают яды, которые не только воздействуют на нейрорецепторы, но и влияют на гемостаз.

Лаборатория молекулярной токсинологии сотрудничает с рядом зарубежных лабораторий и групп. Так, изучением различных рецепторов и поиском новых токсинов она занимается совместно с Рурским университетом (Ruhr-Universität Bochum, Германия). Кроме того, исследование токсинов проводится совместно с белорусскими учеными («Институт физиологии НАН Беларуси»), которые изучают влияние нейроактивных соединений ядов на развитие опухолей (в качестве модельной опухоли используется карцинома Эрлиха). Особый интерес представляет сотрудничество Лаборатории со специалистами из Вьетнама (Institute of Applied materials science, Хошимин), вместе с которыми изучаются яды вьетнамских животных. Также Лаборатория молекулярной токсинологии  налаживает связи с учеными из Индии.

Помимо традиционно изучаемых нейротоксинов Лаборатория исследует пептиды и белки, влияющие на свертываемость крови. Ученые занимаются установлением их структуры и определением биологических свойств. Кроме того, сотрудники Лаборатории вместе с коллегами из филиала ИБХ (г. Пущино) изучают снижающие кровяное давление пептиды, которые были найдены в яде бирманской гадюки Azemiops feae.  Отдел молекулярных основ нейросигнализации занимается доклиническими исследованиями одного из пептидов этой ядовитой змеи, который взаимодействует с холинорецепторами и позиционируется как местный миорелаксант. В будущем это позволит использовать его для лечения  ряда заболеваний, в которых существует необходимость в местном расслаблении мышц.

Лаборатория молекулярной токсинологии была создана в 2009 году. Вместе с Лабораторией лиганд-рецепторных взаимодействий она входит в состав Отдела молекулярных основ нейросигнализации, который, в свою очередь, возник вследствие  расширения круга задач существовавшей до этого Лаборатории рецепции нейропептидов. Имеющиеся у обеих Лабораторий взаимодополняемые методы структурной нейробиологии помогают сотрудникам проводить совместные исследования и наиболее эффективно выполнять главные задачи отдела.

  • Проведение фундаментальнх исследований по изучению молекулярной структуры компонентов животных ядов и механизмов их действия.
  • Исследование пептидов и белков, влияющих на свертываемость крови, установление их структуры и определение биологических свойств. 

В результате работы выделено и охарактеризовано более трех десятков новых белков, обладающих уникальными структурными и функциональными характеристиками. 

  • При изучении яда кобры Naja kaouthia впервые обнаружено несколько новых токсинов. Так, выделен и охарактеризован первый и пока единственный гликозилированный трехпетельный токсин - цитотоксин 3. Установлено, что гликозилирование этого токсина приводит к существенному снижению его цитотоксичности. Также впервые показано, что давно известные «слабые» токсины способны взаимодействовать с никотиновыми холинорецепторами. При этом, обладая слабым сродством к рецептору, такие токсины связываются практически необратимо. Кроме того, эти токсины способны также аллостерически связываться с мускариновыми холинорецепторами. В яде этой же кобры впервые обнаружены ковалентные димеры трех-петельных токсинов. Показано, что такая димеризация вызывает изменение биологической активности токсинов, образующих димер.

Кристаллическая структура димера альфа-кобратоксина (Osipov et al., (2012) J. Biol. Chem. 287(9), 6725-6734.)

  • Несколько нейротоксичных белков выделено из ядов гадюк. Так, из яда гадюки Никольского выделена гетеродимерная фосфолипаза А2, проявляющая пресинаптическую нейротоксичность. Из яда африканской шумящей гадюки Bitis arietans выделен блокирующий никотиновый холинорецептор (нХР) белок с молекулярной массой 27,4 кДа. N-концевая последовательность этого белка, названного битанарином, гомологична N-концевым аминокислотным последовательностям фосфолипаз А2 из ядов змей, имеющих молекулярную массу около 14 кДа и не обладающих способностью блокировать холинорецепторы. С использованием метода радиолигандного анализа установлено взаимодействие битанарина с нХР мышечного и альфа7 типа, а также с ацетилхолин-связывающими белками моллюсков L. stagnalis и A. californica. По своей активности битанарин близок так называемым необычным или слабым трехпетельным токсинам из ядов кобр. Этот белок обладает также фосфолипазной активностью, сопоставимой с активностью фософлипаз А2 яда кобр. Битанарин представляет собой фосфолипазу А2 нового типа и является первой фософлипазой, для которой показано взаимодействие с нХР.
  • При исследовании компонентов ядов, воздействующих на гемостаз, также обнаружен ряд новых белков. Так, из яда кобры Naja oxiana выделена металлопротеиназа оксиагин, для которой впервые показан новый механизм ингибирования системы комплемента. В яде кобры Naja haje обнаружена фософлипаза А2, обладающая уникальной способностью ингибировать тромбин. Это – первый представитель нового структурного типа белковых ингибиторов тромбина.

Избранные публикации

  1. Osipov A.V., Terpinskaya T.I., Kuznetsova T.E., Ryzhkovskaya E.L., Lukashevich V.S., Rudnichenko J.A., Ulashchyk V.S., Starkov V.G., Utkin Y.N. (2017). Cobra Venom Factor and Ketoprofen Abolish the Antitumor Effect of Nerve Growth Factor from Cobra Venom. Toxins (Basel) 9 (9), [+]

    We showed recently that nerve growth factor (NGF) from cobra venom inhibited the growth of Ehrlich ascites carcinoma (EAC) inoculated subcutaneously in mice. Here, we studied the influence of anti-complementary cobra venom factor (CVF) and the non-steroidal anti-inflammatory drug ketoprofen on the antitumor NGF effect, as well as on NGF-induced changes in EAC histological patterns, the activity of lactate and succinate dehydrogenases in tumor cells and the serum level of some cytokines. NGF, CVF and ketoprofen reduced the tumor volume by approximately 72%, 68% and 30%, respectively. The antitumor effect of NGF was accompanied by an increase in the lymphocytic infiltration of the tumor tissue, the level of interleukin 1β and tumor necrosis factor α in the serum, as well as the activity of lactate and succinate dehydrogenases in tumor cells. Simultaneous administration of NGF with either CVF or ketoprofen abolished the antitumor effect and reduced all other effects of NGF, whereas NGF itself significantly decreased the antitumor action of both CVF and ketoprofen. Thus, the antitumor effect of NGF critically depended on the status of the immune system and was abolished by the disturbance of the complement system; the disturbance of the inflammatory response canceled the antitumor effect as well.

    ID:1843
  2. Utkin Y.N. (2017). Modern trends in animal venom research - omics and nanomaterials. World J Biol Chem 8 (1), 4–12 [+]

    Animal venom research is a specialized investigation field, in which a number of different methods are used and this array is constantly expanding. Thus, recently emerged omics and nanotechnologies have already been successfully applied to venom research. Animal venoms have been studied for quite a long time. The traditional reductionist approach has been to isolate individual toxins and then study their structure and function. Unfortunately, the characterization of the venom as a whole system and its multiple effects on an entire organism were not possible until recent times. The development of new methods in mass spectrometry and sequencing have allowed such characterizations of venom, encompassing the identification of new toxins present in venoms at extremely low concentrations to changes in metabolism of prey organisms after envenomation. In particular, this type of comprehensive research has become possible due to the development of the various omics technologies: Proteomics, peptidomics, transcriptomics, genomics and metabolomics. As in other research fields, these omics technologies ushered in a revolution for venom studies, which is now entering the era of big data. Nanotechnology is a very new branch of technology and developing at an extremely rapid pace. It has found application in many spheres and has not bypassed the venom studies. Nanomaterials are quite promising in medicine, and most studies combining venoms and nanomaterials are dedicated to medical applications. Conjugates of nanoparticles with venom components have been proposed for use as drugs or diagnostics. For example, nanoparticles conjugated with chlorotoxin - a toxin in scorpion venom, which has been shown to bind specifically to glioma cells - are considered as potential glioma-targeted drugs, and conjugates of neurotoxins with fluorescent semiconductor nanoparticles or quantum dots may be used to detect endogenous targets expressed in live cells. The data on application of omics and nanotechnologies in venom research are systematized concisely in this paper.

    ID:1842
  3. Vulfius C.A., Kasheverov I.E., Kryukova E.V., Spirova E.N., Shelukhina I.V., Starkov V.G., Andreeva T.V., Faure G., Zouridakis M., Tsetlin V.I., Utkin Y.N. (2017). Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors. PLoS ONE 12 (10), e0186206 [+]

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

    ID:1873
  4. Kovalchuk S.I., Ziganshin R.H., Starkov V.G., Tsetlin V.I., Utkin Y.N. (2016). Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A₂ are the Main Venom Components. Toxins (Basel) 8 (4), 105 [+]

    С использованием количественного протеомного анализа изучены яды четырех видов гадюк (V. kaznakovi, V. nikolskii, V. orlovi и V. renardi), которые обитают в различных регионах России. Установлено, что в отличие от ядов ранее изученных видов гадюк, в которых доминирующим являются металлоротеиназы, основными компонентами исследованных в данной работе ядов являются фософлипазы А2, содержание которых колеблется от 24 до 65%.

    ID:1603
  5. Ziganshin R.H., Kovalchuk S.I., Arapidi G.P., Starkov V.G., Hoang A.N., ThiNguyen T.T., Nguyen K.C., Shoibonov B.B., Tsetlin V.I., Utkin Y.N. (2015). Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. Toxicon 107 (Pt B), 197–209 [+]

    Kraits are venomous snakes of genus Bungarus from family Elapidae. Krait venoms are generally neurotoxic, but toxicity strongly depends on the particular species and regional origin of snakes. We analyzed the proteomes of Vietnamese Bungarus multicinctus and Bungarus fasciatus venoms both qualitatively and quantitatively. It should be noted that no proteomic data for B. multicinctus venom existed so far. We have found that in this venom, almost half (45%) of the proteins by weight was represented by β-bungarotoxins, followed by three finger toxins (28%) and phospholipases A2 (16%), other proteins being present at the level of 1-3%. In B. fasciatus venom, phospholipase A2 was the main component (71%), followed by oxidase of l-amino acids (8%), acetylcholinesterase (5%) and metalloproteinases (4%). Unexpectedly, extremely low amount of three finger toxins (1%) was found in this venom. Interestingly, the presence of complement depleting factor was observed in both venoms. Although our data showed the presence of the same toxin families in Vietnamese krait venoms as those found earlier in the venoms of kraits from other geographic regions, their relative ratio is completely different. This concerns especially B. fasciatus venom with predominant content of phospholipases A2 and very low amount of three finger toxins.

    ID:1466
  6. MalcaGarcia G.R., Hennig L., Shelukhina I.V., Kudryavtsev D.S., Bussmann R.W., Tsetlin V.I., Giannis A. (2015). Curare Alkaloids: Constituents of a Matis Dart Poison. J. Nat. Prod. , [+]

    A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of d-(-)-quinic acid, l-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. d-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

    ID:1317
  7. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Paramonov A.S., Chugunov A.O., Janickova H., Dolejsi E., Dolezal V., Utkin Y.N., Tsetlin V.I., Arseniev A.S., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2015). Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290 (39), 23616–30 [+]

    Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

    ID:1394
  8. Kudryavtsev D.S., Shelukhina I.V., Son L.V., Ojomoko L.O., Kryukova E.V., Lyukmanova E.N., Zhmak M.N., Dolgikh D.A., Ivanov I.A., Kasheverov I.E., Starkov V.G., Ramerstorfer J., Sieghart W., Tsetlin V.I., Utkin Y.N. (2015). Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic GABA Receptors. J. Biol. Chem. , [+]

    Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins (TFTs) from snake venoms, specifically stained the α1β3γ2 receptor; at 10 μM α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nM) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other TFTs: long α-neurotoxin Ls III and non-conventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and non-competitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accomodating under loop C of the receptors.

    ID:1307
  9. Utkin Y.N. (2015). Animal venom studies: Current benefits and future developments. World J Biol Chem 6 (2), 28–33 [+]

    Poisonous organisms are represented in many taxa, including kingdom Animalia. During evolution, animals have developed special organs for production and injection of venoms. Animal venoms are complex mixtures, compositions of which depend on species producing venom. The most known and studied poisonous terrestrial animals are snakes, scorpions and spiders. Among marine animals, these are jellyfishes, anemones and cone snails. The toxic substances in the venom of these animals are mainly of protein and peptide origin. Recent studies have indicated that the single venom may contain up to several hundred different components producing diverse physiological effects. Bites or stings by certain poisonous species result in severe envenomations leading in some cases to death. This raises the problem of bite treatment. The most effective treatment so far is the application of antivenoms. To enhance the effectiveness of such treatments, the knowledge of venom composition is needed. On the other hand, venoms contain substances with unique biological properties, which can be used both in basic science and in clinical applications. The best example of toxin application in basic science is α-bungarotoxin the discovery of which made a big impact on the studies of nicotinic acetylcholine receptor. Today compositions of venom from many species have already been examined. Based on these data, one can conclude that venoms contain a large number of individual components belonging to a limited number of structural types. Often minor changes in the amino acid sequence give rise to new biological properties. Change in the living conditions of poisonous animals lead to alterations in the composition of venoms resulting in appearance of new toxins. At the same time introduction of new methods of proteomics and genomics lead to discoveries of new compounds, which may serve as research tools or as templates for the development of novel drugs. The application of these sensitive and comprehensive methods allows studying either of venoms available in tiny amounts or of low abundant components in already known venoms.

    ID:1468
  10. Kudryavtsev D., Shelukhina I., Vulfius C., Makarieva T., Stonik V., Zhmak M., Ivanov I., Kasheverov I., Utkin Y., Tsetlin V. (2015). Natural compounds interacting with nicotinic acetylcholine receptors: from low-molecular weight ones to peptides and proteins. Toxins (Basel) 7 (5), 1683–701 [+]

    Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.

    ID:1306
  11. Ghazaryan N.A., Ghulikyan L., Kishmiryan A., Andreeva T.V., Utkin Y.N., Tsetlin V.I., Lomonte B., Ayvazyan N.M. (2015). Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms. Biochim. Biophys. Acta 1848 (2), 463–8 [+]

    We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

    ID:1161
  12. Dubovskii V., Vorontsova V., Utkin N., Arseniev S., Efremov G., Feofanov V. (2014). Cobra cytotoxins: determinants of antibacterial activity. Mendeleev Communications 25 (1), 70–71 [+]

    The investigation of antibacterial activity of three-finger cobra cytotoxins towards Gram-negative and Gram-positive bacteria showed no activity against the former species, whereas M. luteus was found most susceptible to cytotoxins. A correlation was revealed between this activity and hydrophobicity of the toxins (HTL scores), total charge and its distribution over the toxin molecule: the absence of Glu-16 residue and the presence of positively charged residues (Lys30/His31) in the tip of the loop 2.

    ID:1398
  13. Дубовский П.В., Уткин Ю.Н. (2014). Цитотоксины кобр: структурная организация и антибактериальная активность. Acta Naturae 6 (3), 12–19 ID:1125
  14. Osipov A.V., Terpinskaya T.I., Kryukova E.V., Ulaschik V.S., Paulovets L.V., Petrova E.A., Blagun E.V., Starkov V.G., Utkin Y.N. (2014). Nerve growth factor from cobra venom inhibits the growth of Ehrlich tumor in mice. Toxins (Basel) 6 (3), 784–95 [+]

    The effects of nerve growth factor (NGF) from cobra venom (cvNGF) on growth of Ehrlich ascites carcinoma (EAC) cells inoculated subcutaneously in mice have been studied. The carcinoma growth slows down, but does not stop, during a course of cvNGF injections and restores after the course has been discontinued. The maximal anti-tumor effect has been observed at a dose of 8 nmoles cvNGF/kg body weight. cvNGF does not impact on lifespan of mice with grafted EAC cells. K252a, a tyrosine kinase inhibitor, attenuates the anti-tumor effect of cvNGF indicating the involvement of TrkA receptors in the process. cvNGF has induced also increase in body weight of the experimental animals. In overall, cvNGF shows the anti-tumor and weight-increasing effects which are opposite to those described for mammalian NGF (mNGF). However in experiments on breast cancer cell line MCF-7 cvNGF showed the same proliferative effects as mNGF and had no cytotoxic action on tumor cells in vitro. These data suggest that cvNGF slows down EAC growth via an indirect mechanism in which TrkA receptors are involved.

    ID:1467
  15. Hoang A.N., Vo H.D., Vo N.P., Kudryashova K.S., Nekrasova O.V., Feofanov A.V., Kirpichnikov M.P., Andreeva T.V., Serebryakova M.V., Tsetlin V.I., Utkin Y.N. (2014). Vietnamese Heterometrus laoticus scorpion venom: evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel. Toxicon 77, 40–8 [+]

    The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel.

    ID:1082
  16. Vulfius C.A., Kasheverov I.E., Starkov V.G., Osipov A.V., Andreeva T.V., Filkin S.Y., Gorbacheva E.V., Astashev M.E., Tsetlin V.I., Utkin Y.N. (2014). Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2. PLoS ONE 9 (12), e115428 [+]

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

    ID:1162
  17. Utkin Y.N. (2013). Three-finger toxins, a deadly weapon of elapid venom--milestones of discovery. Toxicon 62, 50–5 [+]

    Three-finger toxins (TFTs) are the main venom components of snakes from Elapidae family. Amino acid sequences of more than five hundreds TFTs are determined; these toxins form one of the largest protein families present in snake venoms. The first TFT α-bungarotoxin was isolated almost half a century ago and so far it remains a valuable tool in the study of nicotinic acetylcholine receptors. TFTs possess diverse biological activities; for example, α-neurotoxins bind specifically with high affinity to nicotinic acetylcholine receptors, while cytotoxins induce non-specific lysis in great variety of cells. These toxins are widely used as instruments in different branches of life sciences. In this review the main landmarks in TFT study are considered. These are the discovery and isolation of TFTs, determination of their structure and mode of action as well as evolution and relationship within the family.

    ID:1469
  18. Dyachenko I.A., Murashev A.N., Andreeva T.V., Tsetlin V.I., Utkin Y.N. (2013). Analysis of nociceptive effects of neurotoxic phospholipase A2 from Vipera nikolskii venom in mice. J Venom Res 4, 1–4 [+]

    Phospholipases A2 are represented in snake venoms by several types and possess diverse biological activities including neurotoxicity. Previously, we isolated and characterized two neurotoxic phospholipases A2 (HDP-1 and HDP-2) from the venom of Nikolski's viper (Vipera nikolskii), which were heterodimers composed of two non-covalently bound subunits. Each heterodimer consisted of an enzymatically active basic subunit and an inactive acidic subunit. In this work, we studied the in vivo biological activity of HDP-2 in mice. The acute toxicity (LD50 = 0.38 μg/gm) and maximal tolerated dose (0.1 μg/gm) were determined. In the hot plate test, HDP-2 at the maximal tolerated dose, reliably prolonged the time of the mouse staying on the plate. However, taking into account the neurotoxicity of HDP-2, we believe that this effect may be explained by a general intoxication rather than specific decrease of pain sensitivity. In this respect HDP-2 differs from other heterodimeric phospholipases A2 like crotoxin, which possess analgesic activity. This difference can be explained by the dissimilarity in the structure of the acidic subunits, suggesting an important role of this subunit in analgesic activity.

    ID:1470
  19. Utkin Y.N., Weise C., Kasheverov I.E., Andreeva T.V., Kryukova E.V., Zhmak M.N., Starkov V.G., Hoang N.A., Bertrand D., Ramerstorfer J., Sieghart W., Thompson A.J., Lummis S.C., Tsetlin V.I. (2012). Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor. J. Biol. Chem. 287 (32), 27079–86 [+]

    Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

    ID:1083
  20. Osipov A.V., Rucktooa P., Kasheverov I.E., Filkin S.Y., Starkov V.G., Andreeva T.V., Sixma T.K., Bertrand D., Utkin Y.N., Tsetlin V.I. (2012). Dimeric α-Cobratoxin X-ray Structure: LOCALIZATION OF INTERMOLECULAR DISULFIDES AND POSSIBLE MODE OF BINDING TO NICOTINIC ACETYLCHOLINE RECEPTORS. J. Biol. Chem. 287 (9), 6725–34 [+]

    Установлена кристаллическая структура и положение дисульфидных связей в димере α-кобратоксина (αСТ-αСТ). Внутримолекулярные дисульфиды в центральных петлях II были восстановлены и исследовано взаимодействие полученных производных с различными типами никотиновых холинорецепторов. Обнаружено, что дисульфиды петили II необходимы для взаимодействия с α7 типом рецептора, но не с α3β2.

    ID:651
  21. Vulfius C.A., Gorbacheva E.V., Starkov V.G., Osipov A.V., Kasheverov I.E., Andreeva T.V., Astashev M.E., Tsetlin V.I., Utkin Y.N. (2011). An unusual phospholipase A₂ from puff adder Bitis arietans venom--a novel blocker of nicotinic acetylcholine receptors. Toxicon 57 (5), 787–93 [+]

    The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity.

    ID:537
  22. Osipov A., Utkin Y.u. (2011). Phospholipase A2 and Signaling Pathways in Pheochromocytoma PC12 Cells. Pheochromocytoma - A New View of the Old Problem, Jose Fernando Martin (Ed.), ISBN: 978-953-307-822-9, InTech, Available from: http://www.intechopen.com/articles/show/title/phospholipase-a2-and-signaling-pathways-in-pheochromocytoma-pc12-cells , [+]

    В данной главе рассматривается роль фосфолипаз А2 в сигнальных процессах, протекающих в клетках линии РС12 феохромоцитомы крысы. Обсуждаются возможные механизмы участия фосфолипаз А2 в этих процессах и их взаимосвязь с процессами дифференцировки и гибели клеток.

    ID:632
  23. Mordvintsev D.Y., Polyak Y.L., Rodionov D.I., Jakubik J., Dolezal V., Karlsson E., Tsetlin V.I., Utkin Y.N. (2009). Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors. FEBS J. 276 (18), 5065–75 [+]

    В этой работе показано, что слабый нейротоксин WTX может взаимодействовать как с никотиновыми, так и с мускариновыми холинорецепторами. При этом в никотиновых холинорецепторах взаимодействует с ортостерическим участком связывания, а в мускариновых холинорецепторам – с аллостерическим участком.

    ID:193
  24. Osipov A.V., Filkin S.Y., Makarova Y.V., Tsetlin V.I., Utkin Y.N. (2009). A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom. Toxicon 55 (2-3), 186–94 [+]

    Из яда кобры Naja haje выделен новый антикоагулянтный белок, обладающий способностью ингибировать тромбин. Это – первый ингибитор тромбина, обнаруженный в яде змеи, принадлежащей семейству Elapidae. Белок представляет собой фософлипазу А2 и является первым примером ферментов этой группы, обладающим способностью ингибировать тромбин.

    ID:364
  25. Osipov A.V., Kasheverov I.E., Makarova Y.V., Starkov V.G., Vorontsova O.V., Ziganshin R.K., Andreeva T.V., Serebryakova M.V., Benoit A., Hogg R.C., Bertrand D., Tsetlin V.I., Utkin Y.N. (2008). Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification. J. Biol. Chem. 283 (21), 14571–80 [+]

    Впервые обнаружены трех-петельные токсины нового структурного типа: соединенные дисульфидами димеры кобратоксина с цитотоксинами, а также гомодимер кобратоксина. Показано, что в результате димеризации кобратоксин приобретает способность взаимодействовать с еще одним типом никотинового холинорецептора. Такая пост-трансляционная модификация может рассматриваться как еще один путь диверсификации биологической активности трех-петельных токсинов.

    ID:105
  26. Ramazanova A.S., Zavada L.L., Starkov V.G., Kovyazina I.V., Subbotina T.F., Kostyukhina E.E., Dementieva I.N., Ovchinnikova T.V., Utkin Y.N. (2008). Heterodimeric neurotoxic phospholipases A2--the first proteins from venom of recently established species Vipera nikolskii: implication of venom composition in viper systematics. Toxicon 51 (4), 524–37 [+]

    For the first time the venom of recently established viper species Vipera nikolskii was fractionated and two heterodimeric phospholipases A(2) (HDP-1 and HDP-2) were isolated. Isolation of HDP-1 and HDP-2 is the first indication of the presence of two heterodimeric phospholipases A(2) in the venom of one viper species. When tested on the frog neuromuscular junction, isolated proteins affected neuromuscular transmission acting presynaptically. Using RP-HPLC, each heterodimer was separated into two monomeric subunits: basic phospholipase A(2) (HDP-1P and HDP-2P) and acidic component without enzymatic activity (HDP-In). The complete primary structures of subunits were deduced from corresponding sequences of cDNAs. The determined amino acid sequences were homologous to those of vipoxin from Vipera ammodytes and vaspin from Vipera aspis. Similar proteins were not found earlier in the well-studied venom of Vipera berus, the species from which V. nikolskii was recently separated. Our finding supports at the biochemical level the correctness of the establishment of V. nikolskii as an independent species. The finding of similar proteins (HDPs and vipoxin) in geographically remote species (V. nikolskii and V. ammodytes) corroborates the hypothesis about the pre-existence of genes encoding these proteins in all true viper species and their expression under certain conditions.

    ID:413
  27. Utkin Y.N., Osipov A.V. (2007). Non-lethal polypeptide components in cobra venom. Curr. Pharm. Des. 13 (28), 2906–15 [+]

    Яды кобр содержат целый ряд низкотоксичных компонентов, которые принадлежвт к различным структурным ифункциональным типам. Их роль в поражающем действии яда не всегда ясна. Представленный обзор является первой попыткой систематизировать данные о низкотоксичных компонентах ядов кобр.

    ID:367
  28. 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

    ID:192
  29. Utkin Y.N., Kukhtina V.V., Kryukova E.V., Chiodini F., Bertrand D., Methfessel C., Tsetlin V.I. (2001). "Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors. J. Biol. Chem. 276 (19), 15810–5 [+]

    Эта работа показала, что давно известные «слабые» токсины способны взаимодействовать с никотиновыми холинорецепторами. При этом, обладая слабым сродством к рецептору, такие токсины связываются практически необратимо.

    ID:103
  30. Kreienkamp H.J., Utkin Y.N., Weise C., Machold J., Tsetlin V.I., Hucho F. (1992). Investigation of ligand-binding sites of the acetylcholine receptor using photoactivatable derivatives of neurotoxin II from Naja naja oxiana. Biochemistry 31 (35), 8239–44 [+]

    В данной работе впервые с использованием фотоактивируемых производных нейротоксина установлено, какие субъединицы холинорецептора мышечного типа участвуют в формировании участка связывания агонистов/конкурентных антагонистов.

    ID:101

Уткин Юрий Николаевич

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 31, комн. 606
  • Тел.: +7(495)336-65-22
  • Эл. почта: utkin@ibch.ru

Проведен первый количественный протеомный анализ ядов змей двух различных семейств: гадюковых из рода Vipera (настоящие гадюки), обитающих на территории России, и аспидовых из рода Bungarus ( крайты), обитающих на территории Вьетнама. (2016-11-18)

Проведен первый количественный протеомный анализ ядов змей двух различных семейств: гадюковых из рода Vipera (настоящие гадюки), обитающих на территории России, и аспидовых из рода Bungarus ( крайты), обитающих на территории Вьетнама. При этом установлено, что яды разных видов змей сильно различаются по составу, а основными компонентами ядов гадюк являются фосфолипазы А2, содержание которых варьирует от 24 до 65%. Наибольшее количество фосфолипаз А2 (65%) содержится в яде гадюки Никольского. Очень большие различия в составе ядов обнаружены между двумя видами крайтов. Так, в яде многополосного крайта Bungarus multicinctus представлены в основном нейротоксины, составляющие более 70% яда. В то время как в состав яда ленточного крайта Bungarus fasciatus входят в основном фосфолипазы А2, составляющие более 70% яда. Выявленные различия следует учитывать при лечении укусов разными видами змей.

Публикации

  1. Kovalchuk S.I., Ziganshin R.H., Starkov V.G., Tsetlin V.I., Utkin Y.N. (2016). Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A₂ are the Main Venom Components. Toxins (Basel) 8 (4), 105 [+]

    С использованием количественного протеомного анализа изучены яды четырех видов гадюк (V. kaznakovi, V. nikolskii, V. orlovi и V. renardi), которые обитают в различных регионах России. Установлено, что в отличие от ядов ранее изученных видов гадюк, в которых доминирующим являются металлоротеиназы, основными компонентами исследованных в данной работе ядов являются фософлипазы А2, содержание которых колеблется от 24 до 65%.

    ID:1603
  2. Ziganshin R.H., Kovalchuk S.I., Arapidi G.P., Starkov V.G., Hoang A.N., ThiNguyen T.T., Nguyen K.C., Shoibonov B.B., Tsetlin V.I., Utkin Y.N. (2015). Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. Toxicon 107 (Pt B), 197–209 [+]

    Kraits are venomous snakes of genus Bungarus from family Elapidae. Krait venoms are generally neurotoxic, but toxicity strongly depends on the particular species and regional origin of snakes. We analyzed the proteomes of Vietnamese Bungarus multicinctus and Bungarus fasciatus venoms both qualitatively and quantitatively. It should be noted that no proteomic data for B. multicinctus venom existed so far. We have found that in this venom, almost half (45%) of the proteins by weight was represented by β-bungarotoxins, followed by three finger toxins (28%) and phospholipases A2 (16%), other proteins being present at the level of 1-3%. In B. fasciatus venom, phospholipase A2 was the main component (71%), followed by oxidase of l-amino acids (8%), acetylcholinesterase (5%) and metalloproteinases (4%). Unexpectedly, extremely low amount of three finger toxins (1%) was found in this venom. Interestingly, the presence of complement depleting factor was observed in both venoms. Although our data showed the presence of the same toxin families in Vietnamese krait venoms as those found earlier in the venoms of kraits from other geographic regions, their relative ratio is completely different. This concerns especially B. fasciatus venom with predominant content of phospholipases A2 and very low amount of three finger toxins.

    ID:1466

Открыто взаимодействие нейротоксинов змей и альфа-конотоксина с ионотропными рецепторами гамма-аминомасляной кислоты (2016-03-25)

Впервые установлено, что нейротоксины ядов змей и альфа-конотоксин ингибируют токи, индуцируемые гамма-аминомасляной кислотой в GABA(A) рецепторах, гетерологически экспрессированных в ооцитах Xenopus. Степень ингибирования зависит как от природы нейротоксина, так и от субъединичного состава рецептора. При этом наибольшей ингибирующей активностью обладает a-кобратоксин Naja kaouthia. Ингибирование имеет смешанный конкурентный и неконкурентный характер. Центральная полипептидная петля a-кобратоксина играет главную роль во взаимодействии токсина с GABA(A) рецептором.

Публикации

  1. Kudryavtsev D.S., Shelukhina I.V., Son L.V., Ojomoko L.O., Kryukova E.V., Lyukmanova E.N., Zhmak M.N., Dolgikh D.A., Ivanov I.A., Kasheverov I.E., Starkov V.G., Ramerstorfer J., Sieghart W., Tsetlin V.I., Utkin Y.N. (2015). Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic GABA Receptors. J. Biol. Chem. , [+]

    Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins (TFTs) from snake venoms, specifically stained the α1β3γ2 receptor; at 10 μM α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nM) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other TFTs: long α-neurotoxin Ls III and non-conventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and non-competitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accomodating under loop C of the receptors.

    ID:1307