Laboratory of molecular toxinology

Department of molecular bases of neurosignalization

Head: Yuri Utkin, D.Sc
utkin@ibch.ru+7(495)336-65-22

snake venoms, toxins, new proteins, isolation, properties, Nicotinic acetylcholine receptor

The Laboratory carries out the fundamental studies on the molecular structures of animal venoms' components as well as molecular mechanisms of action of animal venoms and their components. Toxins are used as the main instrument for investigation of mechanisms of the nervous system function and of the nerve impulse transduction, in particular. As a result of expansion of interests and tasks of the Laboratory, the studies of the venoms which affect the hemostasis have been initiated in recent years.

The Laboratory of molecular toxinology cooperates with several foreign groups and laboratories. Thus, the study of various receptors and search for new toxins are carried out jointly with Ruhr University Bochum (Germany, Ruhr-Universität Bochum). Besides, the toxins are studied in cooperation with Belarusian scientists (Institute of Physiology, National Academy of Sciences of Belarus), who studied the effect of neuroactive compounds from venoms on the development of tumors (Ehrlich carcinoma is used as a tumor model). Of particular interest is the cooperation with the specialists from Vietnam (The Institute of Applied Materials Science, Ho Chi Minh City), research on toxins of Vietnamese animals is carried out together with the Vietnamese scientists. Also the Laboratory of molecular toxinology has good relations with the Indian scientists.

In addition to the traditionally studied neurotoxins currently the Laboratory explores the peptides and proteins that affect blood clotting. Scientists are involved in the study of their structure and the determination of biological properties. In addition, the staff of the Laboratory, together with colleagues from the branch of Institute of Bioorganic Chemistry (Pushchino) investigates hypotensive peptides which have been found in the venom of a Burmese viper Azemiops feae. The Department of molecular bases of neurosignaling carries out preclinical studies on the peptide of this poisonous snake which interacts with the cholinergic receptors and is positioned as a local muscle relaxant. This is the reason why in the future it can be used for treatment of a number of diseases, where the local muscle relaxation is necessary.

The Laboratory has been organized in 2009 and together with the Laboratory of ligand-receptor interactions constitutes the Department of molecular bases of neurosignaling. The complementary methods of structural neurobiology allow the laboratories to carry out investigations in close cooperation. 

  • Conducting the fundamental studies on the molecular structures of animal venoms' components as well as molecular mechanisms of action of animal venoms and their components.
  • Exploring the peptides and proteins that affect blood clotting, studying their structure and the determination of biological properties.

As a result, more than three dozen of proteins with new structural and functional properties were isolated and characterized recently.

Thus, several new toxins were found during the study of Naja kaouthis cobra venom. In particular, for the first time a glycosylated three-fingered toxin – cytotoxin 3 from Naja kaouthia venom was found. So far it is the only glycosylated three-fingered toxin. It was demonstrated that glycosylation resulted in substantial decrease of toxin cytotoxicity. We have shown that so called weak toxins which were well know for many years are capable to interact with nicotinic acetylcholine receptors. Although acting only at micromolar concentrations, weak toxins have an advantage of being practically nontoxic while producing a long lasting effect. Moreover these toxins allosterically interact with muscarinic acetylcholine receptors. A disulfide bound dimers of three-fingered toxins were also found in the Naja kaouthia venom for the first time. It was shown that such a dimerization results in the change of biological activity of toxins forming dimer.

Crystal structure of alpha-cobratoxin dimer (Osipov et al., (2012) J. Biol. Chem. 287(9), 6725-6734.)

  • Several neurotoxic proteins were isolated from viper venoms. Thus two heterodimeric phospholipases A2 possessing presynaptic activity were isolated from Nikolski’s viper venom. From puff adder Bitis arietans venom, we isolated and characterized the novel protein bitanarin that reversibly blocks nAChRs. Bitanarin is the first described phospholipase A2 that contains 14 disulfide bonds within a single polypeptide chain, and thus it represents a new structural type of phospholipases A2 by both the length of polypeptide chain and the number of cysteine residues. Bitanarin possesses the neurotoxic properties unique for phospholipases A2: it binds to nicotinic acetylcholine receptors and blocks acetylcholine-elicited current. On the other hand, bitaranin is the first competitive acetylcholine receptor blocker possessing phospholipase A2 activity.
  • Several new proteins affecting hemostasis were also isolated from snake venoms and characterized. Thus, a new metalloproteinase oxiagin was isolated from Naja oxiana cobra venom. Oxiagin was found to inhibit the classical pathway of the complement system by preventing the formation of C3-convertase. To achieve it, oxiagin binds to IgG on the surface of sheep erythrocytes sensitized with rabbit antibodies, thus, preventing the interaction of component C2 (without its inactivation) with immobilized C4b. A new anticoagulant phospholipase A2 have been isolated from the venom of the Egyptian cobra Naja haje. The protein retards fibrin clot formation by inhibiting thrombin. This protein is the first thrombin inhibitor found in the venoms of the Elapidae family and a first example of new structural type of polypeptide thrombin inhibitors.
NamePositionContacts
Yuri Utkin, D.ScHead of lab.utkin@ibch.ru+7(495)336-65-22
Aleksej Osipov, Ph.D.s. r. f.osipov@mx.ibch.ru+7(495)336-65-22
Irina Shelukhina, Ph.D.s. r. f.shelukhina.iv@yandex.ru+7(495)330-73-74
Vladislav Starkovr. f.vladislavstarkov@mail.ru+7(495)336-65-22
Vladimir Kostj. r. f.goron.dekar@gmail.com+7(916)9726071
Andrei Siniavinj. r. f.andreysi93@ya.ru
Evgeniia GorbachevaPhD stud.eogorbacheva@gmail.com
Dmitrij Suhovt. q. - lab. as.
Tat'jana Andreevares. eng.+7(495)330-73-74
Vera Mozhaevaeng.
Nikita Timofeeveng.

Former members:

Anna Ramazanovaj. r. f.lamsi@mail.ru
Alexandra Garifulinat. q. - lab. as.
Ekaterina Spirovaeng.katya_spirova@mail.ru

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Yuri Utkin

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 31, office. 606
  • Phone: +7(495)336-65-22
  • E-mail: utkin@ibch.ru

The encapsulation in nanomaterials enhances the biological activity of the components of animal venoms

The strong anticoagulant activity of the dipeptide IleTrp (IW) and adenosine (Ado) found in scorpion venom was previously demonstrated. The results of further research showed that the inclusion of these compounds in nanomaterials significantly enhances their anticoagulant activity. Ado and IW were encapsulated in a heat-sensitive nanogel consisting of heparin, to which pluronic P123 polymer was covalently attached. Biological activity in vivo was determined by measuring the time of bleeding from the tail of the mouse and it found that encapsulation of Ado and IW in the nanomaterial significantly enhances their effect, which leads to an increase in bleeding time. Thus, the inclusion of low molecular weight anticoagulants Ado and IW in nanomaterials can be considered as a way to increase their biological activity.

Publications

  1. Nguyen TD, Nguyen TN, Nguyen TTT, Ivanov IA, Nguyen KC, Tran QN, Hoang AN, Utkin YN (2019). Nanoencapsulation Enhances Anticoagulant Activity of Adenosine and Dipeptide IleTrp. Nanomaterials (Basel) 9 (9),
  2. Nguyen TD, Nguyen TN, Nguyen KC, Tran QN, Hoang AN, Egorova NS, Starkov VG, Tsetlin VI, Utkin YN (2019). Encapsulation of Neurotoxins, Blockers of Nicotinic Acetylcholine Receptors, in Nanomaterials Based on Sulfated Polysaccharides. Dokl Biochem Biophys 487 (1), 251–255

The first three-finger snake neurotoxin, which distinguishes two binding sites in the muscle nicotinic receptor by affinity and dissociation kinetics

In collaboration with Laboratory of ligand-receptor interactions

A new three-finger α-neurotoxin - αδ-bungarotoxin - was isolated and characterized from the venom of Bungarus candidus krait. It differs from α-bungarotoxin, the well-known antagonist of muscle, α7 and α9α10 neuronal subtypes of nicotinic receptors, by 11 substitutions of 74 amino acid residues. The new toxin also effectively interacted with the α7 receptor with nanomolar affinity, but it demonstrated a unique (for three-finger toxins) difference in affinity towards two different binding sites on the muscle receptor (affinity to the α-δ site was higher than to the α-γ site by 20 times). In addition, the new toxin showed kinetics of dissociation from one of the sites of muscle-type receptor much faster than α-bungarotoxin with its almost irreversible binding. Thus, a new tool for the study of muscle nicotinic receptors with a unique set of properties has been discovered.

Publications

  1. Utkin YN, Kuch U, Kasheverov IE, Lebedev DS, Cederlund E, Molles BE, Polyak IL, Ivanov IA, Prokopev NA, Ziganshin RH, Jornvall H, Alvelius G, Chanhome L, Warrell DA, Mebs D, Bergman T, Tsetlin VI (2019). Novel Long-chain Neurotoxins from Distinguish the Two Binding Sites in Muscle-type Nicotinic Acetylcholine Receptors. Biochem J 476 (8), 1285–1302

A new class of nicotinic acetylcholine receptor inhibitors has been proposed and synthesized

In collaboration with Laboratory of ligand-receptor interactions

Based on our own and published data showing that positively charged amino acid residues play an important role in the activity of α-conotoxins, a series of peptides consisting exclusively of arginine (R) residues was synthesized, and oligoarginines (R = 6-18) were shown to be inhibitors of various subtypes of nicotinic acetylcholine receptors (nAChR), with R8 having pronounced neuronal selectivity against α9/α10 nAChR, and R16 possessing the highest affinity for α7 nAChR. Inhibitory activity was also found for a number of positively charged polymers, which, along with oligoarginines, are used for intracellular delivery of potential drug compounds, which should be taken into account as possible positive and negative effects.

Publications

  1. Lebedev D, Kryukova E, Ivanov I, Egorova N, Timofeev N, Spirova E, Tufanova E, Siniavin A, Kudryavtsev D, Kasheverov I, Zouridakis M, Katsarava R, Zavradashvili N, Iagorshvili I, Tzartos S, Tsetlin V (2019). Oligoarginine Peptides, a New Family of nAChR Inhibitors. Mol Pharmacol 96 (5), 664–673

The first recombinant viper three finger toxins - antagonists of the nicotinic acetylcholine receptors of muscle and neuronal types

In collaboration with Laboratory of ligand-receptor interactions,  Laboratory of Molecular Diagnostics,  Science-Educational center

One of the main components of the venom of the snakes from the Elapidae family are three finger toxins, which possess various types of biological activity, including inhibition of synaptic transmission by blocking the nicotinic acetylcholine receptors. So far, three finger toxins have not been found in the venom of the Viperidae snakes, although the mRNA encoding these toxins has been found in the venom glands of snakes from this family. Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. We have shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors both of neuronal and muscle type.

Ehrlich carcinoma growth is inhibited by nerve growth factor and cobra venom factor

When studying the effect of cobra venom components on mice inoculated with Ehrlich carcinoma, we have established for the first time that the nerve growth factor (NGF) from cobra venom and the cobra venom factor (CVF) suppressed tumor growth. However, the antitumor effect of NGF depended on the status of the immune system and disappeared when the complement system was depleted; the disturbance of the inflammatory response also abolished the antitumor effect of NGF.

Publications

  1. Osipov AV, Terpinskaya TI, Kuznetsova TE, Ryzhkovskaya EL, Lukashevich VS, Rudnichenko JA, Ulashchyk VS, Starkov VG, Utkin YN (2017). Cobra venom factor and ketoprofen abolish the antitumor effect of nerve growth factor from cobra venom. Toxins (Basel) 9 (9),
  2. Terpinskaya TI, Ulashchik VS, Osipov AV, Tsetlin VI, Utkin YN (2016). Suppression of Ehrlich carcinoma growth by cobra venom factor. Dokl Biol Sci 470 (1), 240–243
  3. Osipov AV, Terpinskaya TI, Kryukova EV, Ulaschik VS, Paulovets LV, Petrova EA, Blagun EV, Starkov VG, Utkin YN (2014). Nerve growth factor from cobra venom inhibits the growth of Ehrlich tumor in mice. Toxins (Basel) 6 (3), 784–795

Structural/dynamic mode of S-type cytotoxin interaction with detergent micelles and lipid membranes: high-resolution NMR spectroscopy and molecular dynamics.

In collaboration with Laboratory of biomolecular NMR-spectroscopy,  Laboratory of biomolecular modeling

Determination of the spatial structure of membrane peptides and proteins requires membrane-mimicking environments. Most often, detergent micelles are used in the experiments. However, it is not clear how to transfer these results to lipid bilayers. In the current work, the solution to this question is suggested for a beta sheet protein, S-type cytotoxin 1, purified from the venom of N. oxiana cobra. The spatial structure of this toxin was determined by NMR spectroscopy in aqueous solution and dodecylphosphocholine (DPC) micelles. Full-atom and coarse-grained molecular dynamics (MD) was used to investigate the toxin partitioning into DPC micelles (Figure, left panel) and palmitoyloleoylphosphatidylcholine bilayer (Figure, right panel). It was shown that the toxin partitioning either in micelles, or in lipid membrane is accompanied with adaptation of the toxin molecule to hydrophobic/hydrophilic milieu and conformational rearrangement within the tip of the loop-II (Figure, left panel). As a result, it was shown that the single toxin/micelle binding mode exists – with the tips of the all three protein loops. In the bilayer, averaging between the three binding modes takes place: with the tip of the loop I; with the tips of the loops I and II; with the tips of the all three loops (Figure, right panel, from top to bottom).

New fluorescent probes to research the structure and functions of membranes

In collaboration with Laboratory of lipid chemistry

The use of anthrylvinyl-perylenoyl FRET-pair of phospholipid probes revealed the existence of regulatory interaction site(s) on the surface of ceramide-1 phosphate transfer protein that are specific to the polar head groups of phosphoglycerides in the lipid membrane. This finding delineates new differences between Glycolipid Transfer Proteins superfamily members that are specific for C1P versus glycolipid [1]. By means of new BODIPY FRET-pair of phosphatidylcholine probes, it was shown that heterodimeric V. nikolskii phospholipases A2 induce aggregation and stacking of negatively charged lipid bilayers [2]; this may be one of the mechanisms of PLA2 biological activity. A novel combination of FRET between BODIPY-ganglioside probes and Monte Carlo simulations (MC-FRET) identified directly 10 nm large nanodomains (rafts) composed of sphingomyelin and cholesterol in liquid-disordered model membranes that mimic the cytoplasmic membrane; the nanodomains are also fluid and disordered [3].

Publications

  1. Alekseeva AS, Tretiakova DS, Chernikov VP, Utkin YN, Molotkovsky JG, Vodovozova EL, Boldyrev IA (2017). Heterodimeric V. nikolskii phospholipases A2 induce aggregation of the lipid bilayer. Toxicon 133, 169–179
  2. Zhai X, Gao YG, Mishra SK, Simanshu DK, Boldyrev IA, Benson LM, Bergen HR, Malinina L, Mundy J, Molotkovsky JG, Patel DJ, Brown RE (2017). Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins. J Biol Chem 292 (6), 2531–2541
  3. Koukalová A, Amaro M, Aydogan G, Gröbner G, Williamson PTF, Mikhalyov I, Hof M, Šachl R (2017). Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered. Sci Rep 7 (1), 5460

Development of a new technique based on calcium imaging and functional characterization of mutant α7/α9 nAChRs with the use of this technique.

In collaboration with Laboratory of ligand-receptor interactions

On the basis of the calcium imaging method, we developed a new technique that allows to effectively express functionally active "problematic" subtypes of nicotinic receptors (nAChRs) in cell lines. It involves co-expressing with the appropriate receptor subtype a chaperone and a fluorescent calcium sensor Case12. This technique allowed us to obtain 6 mutant forms of α7 nAChR with selected single substitutions of amino acid residues from α9 nAChR subtype. All the mutants together with the wild-type receptors were analyzed for affinity to acetylcholine and epibatidine using the developed technique. This helped to identify two key mutations - L119D and F187S which are responsible for selectivity of these nAChR subtypes to above-mentioned ligands. Computer simulations showed a significant change in the arrangement of ligands’ molecules in binding sites of these two mutant forms of the receptor, explaining the data obtained.

Publications

  1. Shelukhina I, Spirova E, Kudryavtsev D, Ojomoko L, Werner M, Methfessel C, Hollmann M, Tsetlin V (2017). Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants. PLoS One 12 (8), e0181936

SLURP-1 (81 amino-acid residues, 5 disulfides), identical in the amino-acid sequence to the endogenous human protein, has been synthesized and shown to differ from all known recombinant

In collaboration with Laboratory of ligand-receptor interactions

SLURP-1 (81 amino acid residues, 5 disulfides) has been synthesized with the amino acid sequence identical to that of the endogenous human toxin-like protein. 1H-NMR revealed the same structure as in the recombinant rSLURP-1 bearing additional N-terminal Met0. These proteins  have some differences in molecular dynamics, but differ greatly in their activity towards distinct subtypes of nicotinic receptors. Our work in general stresses the necessity of maximal approach to the structure of naturally-occurring proteins to solve the mechanisms of their endogenous activities and choosing appropriate medical applications.  

Publications

  1. Durek T, Shelukhina IV, Tae HS, Thongyoo P, Spirova EN, Kudryavtsev DS, Kasheverov IE, Faure G, Corringer PJ, Craik DJ, Adams DJ, Tsetlin VI (2018). Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors. Sci Rep 7 (1), 16606

Interaction of snake neurotoxins and alpha-conotoxin ImI with ionotropic receptors of the gamma-aminobutyric acid was discovered

For the first time it was found that snake venom neurotoxins and alpha-conotoxin inhibited currents induced by gamma-aminobutyric acid in GABA(A) receptors heterologously expressed in Xenopus oocytes. The degree of inhibition depended on the nature of the neurotoxin and the subunit composition of the receptor. The highest inhibitory activity had alpha-cobratoxin from  cobra Naja kaouthia. Inhibition is of a mixed competitive and noncompetitive type. Central polypeptide loop of alpha-cobratoxin played a major role in the interaction of the toxin with GABA(A) receptor.