Department of molecular bases of neurosignalization

All publications (show selected)

Victor Tsetlin

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 33, office. 237
  • Phone: +7(495)335-57-33
  • E-mail: vits@ibch.ru

Endovaniloids acyldopamines - regulators of cholesterol biosynthesis and inhibitors of cell migration in breast cancer

Laboratory of oxylipins

It was established for the first time that the level of cholesterol in cancer cells is an important parameter that determines their sensitivity to the cytotoxic action of endovanilloids acyldopamines. A decrease in the content of cholesterol in cells and, first of all, in the cytoplasmic membrane, leads to an increase in the cytotoxicity of acyldopamines, presumably as a result of the destruction of rafts. It was also shown for the first time that acyldopamines in breast cancer cells inhibit their migration, epithelial-mesynchymal transition, and stemness. In addition, acyl dopamines inhibit key genes for cholesterol biosynthesis in cancer cells. This result is important for the development of new therapeutic strategies for combating cancer, based on the use of endogenous anticancer compounds and small molecules synthesized on the template of their structures.

Publications

  1. Akimov MG, Dudina PV, Gamisonia AM, Gretskaya NM, Zinchenko GN, Mandal CC, Bezuglov VV (2020). The Influence of the Cholesterol Level in Cells on Endovanilloid Cytotoxicity. Dokl Biochem Biophys 493 (1), 167–170

Three-finger neurotoxins and new analogues of bradykinin-potentiating peptides were found in the venom of vipers for the first time, using transcriptomics and quantitative proteomics

Laboratory of ligand-receptor interactions,  Laboratory of Biological Testing,  Laboratory of molecular toxinology

From Azemiops feae viper venom, we have isolated previously azemiopsin, a new neurotoxin, that inhibited the nicotinic acetylcholine receptor. To characterize other A. feae toxins, quantitative proteomics was used and 120 unique proteins were identified. In total, identified toxins represented 14 families, among which bradykinin-potentiating peptides with unique amino acid sequences were found and manifested biological activity in vivo. Proteomic analysis also revealed non-conventional three-finger toxins belonging to the group of toxins with neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venoms. In parallel, transcriptome analysis of the venom gland was carried out by the next generation sequencing, and additionally identified 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

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

Laboratory of molecular toxinology,  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

Targeted drug delivery to dopaminergic neurons by the functionalized derivative of the dopamine transporter inhibitor GBR12909: a proof of concept.

Laboratory of oxylipins

A structure has been created that is capable of selectively delivering active molecules to dopaminergic neurons. The relevance of creating such drugs is due to the need to protect dopaminergic neurons, which progressively die in Parkinson's disease. The dopamine transporter inhibitor (DAT) GBR12909, which differs from other known inhibitors by its high selectivity and affinity for DAT, is selected as the address part of the prototype drug design being created. To test the applicability of this design for targeted delivery of pharmacologically active substances to dopaminergic neurons, a fluorescent analog of functionalized GBR12909 with BODIPY-FL-fluorophore was synthesized. By experiments on cells containing active DAT, it was shown that the fluorescent analog penetrates the PC12 pheochromocytoma cells and rat brain dopaminergic neurons through selective transport using DAT. Thus, it was found that the fluorescent analog of GB12909 is a ligand of the dopamine transporter and the DAT system can be used for targeted delivery of active compounds to dopaminergic neurons.

A.V. Lavrova, N.M. Gretskaya, M.G. Akimov and V.V. Bezuglov A Novel Fluorescent Analog of the Dopamine Reuptake Inhibitor GBR12909 // Russian J. Bioorgan. Chem. —2019. — Vol. 45. — No 5. — P. 416–424.

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

Laboratory of molecular toxinology

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

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

Laboratory of molecular toxinology,  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 molecular target for mammalian orphan neuropeptide - nocystatin associated with pain sensing.

Laboratory of ligand-receptor interactions,  Laboratory of neuroreceptors and neuroregulators

Nocystatin is an endogenous neuropeptide produced by neurons of the central and peripheral nervous system. It was belonged so far to the class of orphan proteins for which the molecular target has not been defined. In the Laboratory of Neuroreceptors and Neuroregulators of the IBCh RAS was discovered the peptides’ ability to evoked currents in X. laevis oocytes expressing rat ASIC1a, ASIC1b, ASIC2a, and ASIC3 that were very similar in kinetic parameters to the proton-gated response. The peptide was able to activate all subtypes of acid-sensitive channels, and its effect was concentration-dependent. General ASIC antagonists inhibited the current induced by the nocystatin application. Moreover, the channels activated by nocystatin desensitized and lose their ability to further activation by acid, therefore, low concentration of the peptide inhibits while a higher concentration stimulates the activity of ASIC channels. This can to explain its dual effect on pain sensitivity, where it acts both as a generator of pain signals and as an analgesic compound. No previously known ASICs’ ligands shown such biological effect. Thus, nocistatin is the first endogenous direct agonist of ASICs except protons which should be associated with a media property rather as the molecule.

Novel data could give a key to understanding ASICs activation regulation in the nervous system and also could be used to develop new drugs to treat pathological processes associated with ASICs activation, such as neurodegeneration, inflammation, and pain.

An article with the results of the first experiments was published in the journal Biomolecules.

The researchers from Yampolsky lab have successfully characterized three key low-molecular-weight components of Odontosyllis undecimdonta bioluminescence system: luciferin,  oxyluciferin (Green) and a nonspecific luciferin oxidation product (Pink). These compounds were revealed to be highly unusual tricyclic heterocycles containing three sulfur atoms in different electronic states. Together the structures of these low-molecular-weight components of Odontosyllis bioluminescent system have enabled us to propose chemical transformation pathways for the enzymatic (luminescent) and non-enzymatic (dark) oxidation of luciferin. Moreover Odontosyllis oxyluciferin was established to be the only green primary emitter described for any known bioluminescent marine organism.

Publications

  1. Kotlobay AA, Dubinnyi MA, Purtov KV, Guglya EB, Rodionova NS, Petushkov VN, Bolt YV, Kublitski VS, Kaskova ZM, Ziganshin RH, Nelyubina YV, Dorovatovskii PV, Eliseev IE, Branchini BR, Bourenkov G, Ivanov IA, Oba Y, Yampolsky IV, Tsarkova AS (2019). Bioluminescence chemistry of fireworm Odontosyllis. Proc Natl Acad Sci U S A 116 (38), 18911–18916

Characterization of the "analgesic" conotoxins' binding sites on the nicotinic receptor and its models

Laboratory of ligand-receptor interactions

A set of Cys-Cys isomers of "analgesic" conotoxins RgIA and GeXIVA acting through ortho- or allosteric binding sites of α9/α10 of nicotinic acetylcholine receptor, respectively, was synthesized. The study of them by direct or competitive radioligand analysis with water-soluble models of this receptor - acetylcholine-binding protein and recombinant extracellular domain of the α9 receptor subunit, showed that they effectively interact with the micromolar affinity only with the orthosteric binding site of these models.

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

Laboratory of ligand-receptor interactions,  Laboratory of Molecular Diagnostics,  Science-Educational center,  Laboratory of molecular toxinology

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.

Engineering antimicrobial peptide with a low hemolytic activity via combination of motifs of spider venom peptides. Using coarse-grained Molecular Dynamics, the depth of penetration of parent spider venom peptides (Ltc1, Oxt 4a) in model erythrocyte membrane was estimated. The artificial peptide (P5) is formed of fragments with the low depth of penetration (encircled - see Fig.), or penetrating deeply, and thus hemolytic (enclosed in ellipse). Hydrophobicity of the latter peptide was decreased via L/K mutation.

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

Laboratory of molecular toxinology

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

New fluorescent analogues of acyldopamines with the preserved the address moiety

Laboratory of oxylipins

New fluorescent analogues of natural acyldopamines mimicking residues of palmitic and oleic acids carrying the reporter BODIPY group at the distal end of molecule were synthesized. The specific uptake of mentioned compounds in rat pheochromocytoma PC12 cells was demonstrated and characteristics of this process were measured. Uptake rate of oleic analogue was more than three times as palmitic one. Synthesized compounds are ready to be used for investigation of acyldopamine transport in various cells of human or animal origin.

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

Laboratory of molecular toxinology,  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

Laboratory of molecular toxinology,  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

New fluorescent probes to research the structure and functions of membranes

Laboratory of molecular toxinology,  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

First peptide ligands potentiating the TRPA1 response to agonists and producing the analgesic and anti-inflammatory effects.

Laboratory of biomolecular NMR-spectroscopy,  Laboratory of ligand-receptor interactions,  Laboratory of neuroreceptors and neuroregulators

Two analgesic peptides Мs9а-1 and Ueq 12-1 were isolated from sea anemones Metridium senile and Urticina eques and characterized. Peptide Мs9а-1 contains 35 amino acid residues, and its spatial structure is stabilized by two disulfide bridges. The spatial structure of Мs9а-1 is similar to the sea anemones peptides structures described previously. Ueq 12-1 consists from 45 amino acid residues including 10 cysteine residues with an unusual distribution among sea anemone peptides. Its uncommon spatial structure resolved by NMR is partially similar to the structure of mammal’s alpha defensins. This similarity can explain a weak antimicrobial activity of Ueq 12-1 against gram-positive bacteria. Structurally different peptides Мs9а-1 and Ueq 12-1 have a similar mechanism of action onto the same biological target. Experiments in vitro on TRPA1 receptor expressed in oocytes of Xenopus laevis or in mammalian cells shown an increase of receptors’ respond to direct agonists, such as AITC and diclofenac. The intravenously peptides application in tests on mice in vivo resulted in significant analgesic and anti-inflammatory effects, while peptides’ administration did not cause pain or thermal hypersensitivity. We assume that observed effects are connected with the fact that peptides make the receptor more sensitive to their agonists (potentiating effect). So a release of endogenous inflammatory mediators leads to the desensitization of TRPA1-expressing neurons and a nociception decrease. Such enhance of the TRPA1 activity by peptides give novel opportunity for basic research and analgesic drug development.

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

Laboratory of biomolecular NMR-spectroscopy,  Laboratory of molecular toxinology,  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).

Modified peptides based on proglyprol structure with increased effectiveness

Laboratory of oxylipins

Hybrid compounds based on proglyprol (PGP) peptide carrying residues of docosahexaenoic acid and (or) dopamine were synthesized for the first time. Prepared compounds possess advanced neuroprotectory  and anti-inflammatory activity and may consider as prototypes of novel pharmaceuticals.

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

Laboratory of molecular toxinology

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.