Группа молекулярных инструментов для нейробиологии

Кто мы?

Формально группа была создана в 2014 году в рамках программы Президиума РАН «Молекулярная и клеточная биология», одновременно с образованием в ИБХ отдела молекулярной нейробиологии. Однако де факто группа существует с 2008 года. У нас молодой амбициозный коллектив, и мы всегда рады новым коллегам: аспиранты, студенты, приходите к нам!

Направления исследований

Чем мы занимаемся?

  1. Исследованием плохо изученных природных ядов: медуз, многоножек, некоторых видов пауков, жуков-листоедов, морских ежей и скатов-хвостоколов.
  2. Созданием молекулярных инструментов исследования нейрорецепторов.
  3. Созданием средств диагностики и терапии заболеваний.
Ф.И.О.ДолжностьЭл. почта
Беркут Антонина Анатольевнам.н.с.
Гиголаев Андрей Михайловичстуд.
Каневская Жанна Олеговналаб.
Коммер Антон Айгаровичасп.
Кузьменков Алексей Игоревичасп.
Кульдюшев Никита Александровичстуд.
Опарин Пётр Борисович, к. х. н.м.н.с.
Сачкова Мария Юрьевна, к. х. н.м.н.с.
Чудецкий Иван студ.

Избранные публикации

  1. Oparin P.B., Nadezhdin K.D., Berkut A.A., Arseniev A.S., Grishin E.V., Vassilevski A.A. (2016). Structure of purotoxin-2 from wolf spider: modular design and membrane-assisted mode of action in arachnid toxins. Biochem. J. 473 (19), 3113–26 [+]

    Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of β-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine knot (ICK, or knottin) β-structural domain and a C-terminal linear cationic domain. In aqueous solution, the C-terminal fragment is hyper-flexible, whereas the knottin domain is very rigid. In membrane-mimicking environment, the C-terminal domain assumes a stable amphipathic α-helix. This helix effectively tethers the toxin to membranes and serves as a membrane-access and membrane-anchoring device. Sequence analysis reveals that the knottin + α-helix architecture is quite widespread among arachnid toxins, and PT2 is therefore the founding member of a large family of polypeptides with similar structure motifs. Toxins from this family target different membrane receptors such as P2X in the case of PT2 and calcium channels, but their mechanism of action through membrane access may be strikingly similar.

    ID:1568
  2. Kuzmenkov A.I., Sachkova M.Y., Kovalchuk S.I., Grishin E.V., Vassilevski A.A. (2016). Lachesana tarabaevi, an expert in membrane-active toxins. Biochem. J. 473 (16), 2495–506 [+]

    In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18-79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present 'idealized' amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61-79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.

    ID:1557
  3. Kuzmenkov A.I., Krylov N.A., Chugunov A.O., Grishin E.V., Vassilevski A.A. (2016). Kalium: a database of potassium channel toxins from scorpion venom. Database (Oxford) 2016, [+]

    Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community.Database URL:http://kaliumdb.org/.

    ID:1504
  4. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561
  5. Dubovskii P.V., Vassilevski A.A., Kozlov S.A., Feofanov A.V., Grishin E.V., Efremov R.G. (2015). Latarcins: versatile spider venom peptides. Cell. Mol. Life Sci. 72 (23), 4501–22 [+]

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.

    ID:1395
  6. Kuzmenkov A.I., Grishin E.V., Vassilevski A.A. (2015). Diversity of Potassium Channel Ligands: Focus on Scorpion Toxins. Biochemistry Mosc. 80 (13), 1764–99 [+]

    Potassium (K+) channels are a widespread superfamily of integral membrane proteins that mediate selective transport of K+ ions through the cell membrane. They have been found in all living organisms from bacteria to higher multicellular animals, including humans. Not surprisingly, K+ channels bind ligands of different nature, such as metal ions, low molecular mass compounds, venom-derived peptides, and antibodies. Functionally these substances can be K+ channel pore blockers or modulators. Representatives of the first group occlude the channel pore, like a cork in a bottle, while the second group of ligands alters the operation of channels without physically blocking the ion current. A rich source of K+ channel ligands is venom of different animals: snakes, sea anemones, cone snails, bees, spiders, and scorpions. More than a half of the known K+ channel ligands of polypeptide nature are scorpion toxins (KTx), all of which are pore blockers. These compounds have become an indispensable molecular tool for the study of K+ channel structure and function. A recent special interest is the possibility of toxin application as drugs to treat diseases involving K+ channels or related to their dysfunction (channelopathies).

    ID:1503
  7. Kuzmenkov A.I., Vassilevski A.A., Kudryashova K.S., Nekrasova O.V., Peigneur S., Tytgat J., Feofanov A.V., Kirpichnikov M.P., Grishin E.V. (2015). Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY. J. Biol. Chem. 290 (19), 12195–209 [+]

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

    ID:1310
  8. Berkut A.A., Peigneur S., Myshkin M.Y., Paramonov A.S., Lyukmanova E.N., Arseniev A.S., Grishin E.V., Tytgat J., Shenkarev Z.O., Vassilevski A.A. (2015). Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae. J. Biol. Chem. 290 (1), 492–504 [+]

    We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

    ID:1138
  9. Pluzhnikov K.A., Kozlov S.A., Vassilevski A.A., Vorontsova O.V., Feofanov A.V., Grishin E.V. (2014). Linear antimicrobial peptides from Ectatomma quadridens ant venom. Biochimie 107 Pt B, 211–5 [+]

    Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105 μg/g.

    ID:1150
  10. Slavokhotova A.A., Naumann T.A., Price N.P., Rogozhin E.A., Andreev Y.A., Vassilevski A.A., Odintsova T.I. (2014). Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases. FEBS J. , [+]

    The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defense-related genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn-metalloproteinase from Fusarium verticillioides, named fungalysin Fv-cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein-like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP-1b or WAMP-2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.

    ID:1090
  11. Sachkova M.Y., Slavokhotova A.A., Grishin E.V., Vassilevski A.A. (2014). Structure of the yellow sac spider Cheiracanthium punctorium genes provides clues to evolution of insecticidal two-domain knottin toxins. Insect Mol. Biol. 23 (4), 527–38 [+]

    Yellow sac spiders (Cheiracanthium punctorium, family Miturgidae) are unique in terms of venom composition, because, as we show here, two-domain toxins have replaced the usual one-domain peptides as the major constituents. We report the structure of the two-domain Che. punctorium toxins (CpTx), along with the corresponding cDNA and genomic DNA sequences. At least three groups of insecticidal CpTx were identified, each consisting of several members. Unlike many cone snail and snake toxins, accelerated evolution is not typical of cptx genes, which instead appear to be under the pressure of purifying selection. Both CpTx modules present the inhibitor cystine knot (ICK), or knottin signature; however, the sequence similarity between the domains is low. Conversely, notable similarity was found between separate domains of CpTx and one-domain toxins from spiders of the Lycosidae family. The observed chimerism is a landmark of exon shuffling events, but in contrast to many families of multidomain protein genes no introns were found in the cptx genes. Considering the possible scenarios, we suggest that an early transcription-mediated fusion event between two related one-domain toxin genes led to the emergence of a primordial cptx-like sequence. We conclude that evolution of toxin variability in spiders appears to be quite different from other venomous animals.

    ID:1079
  12. Berkut A.A., Usmanova D.R., Peigneur S., Oparin P.B., Mineev K.S., Odintsova T.I., Tytgat J., Arseniev A.S., Grishin E.V., Vassilevski A.A. (2014). Structural similarity between defense peptide from wheat and scorpion neurotoxin permits rational functional design. J. Biol. Chem. 289 (20), 14331–40 [+]

    In this study, we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide was found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule, Tk-hefu, was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto the Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that although the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ∼ 35 μm) to κ-hefutoxin 1 (IC50 ∼ 40 μm). We conclude that α-hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design.

    ID:1103
  13. Sachkova M.Y., Slavokhotova A.A., Grishin E.V., Vassilevski A.A. (2014). Genes and evolution of two-domain toxins from lynx spider venom. FEBS Lett. 588 (5), 740–5 [+]

    Spiderines are comparatively long polypeptide toxins (∼110 residues) from lynx spiders (genus Oxyopes). They are built of an N-terminal linear cationic domain (∼40 residues) and a C-terminal knottin domain (∼60 residues). The linear domain empowers spiderines with strong cytolytic activity. In the present work we report 16 novel spiderine sequences from Oxyopes takobius and Oxyopes lineatus classified into two subfamilies. Strikingly, negative selection acts on both linear and knottin domains. Genes encoding Oxyopes two-domain toxins were sequenced and found to be intronless. We further discuss a possible scenario of lynx spider modular toxin evolution.

    ID:1048
  14. Slavokhotova A.A., Rogozhin E.A., Musolyamov A.K., Andreev Y.A., Oparin P.B., Berkut A.A., Vassilevski A.A., Egorov T.A., Grishin E.V., Odintsova T.I. (2014). Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution. Plant Mol. Biol. 84 (1-2), 189–202 [+]

    Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C(1)X3C(2)XnC(3)X3C(4)), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C(1)-C(4) and C(2)-C(3). Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal "tail" regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S-S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.

    ID:981
  15. Vassilevski A.A., Sachkova M.Y., Ignatova A.A., Kozlov S.A., Feofanov A.V., Grishin E.V. (2013). Spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules identified in the lynx spider Oxyopes takobius. FEBS J. 280 (23), 6247–61 [+]

    In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 μg·g(-1) ) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 μm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins.

    ID:980
  16. Чугунов А.О., Василевский А.А. (2013). Эволюционная «гонка вооружений»: нейротоксины против ионных каналов. Наука и Жизнь 11, 42–48 [+]

    Биологическая эволюция — общая форма существования живой материи. При детальном рассмотрении оказывается, что виды почти никогда не эволюционируют поодиночке. Обычно в этом принимают участие их экологические партнёры. Таким образом, изменение происходит в парах: паразит — хозяин, хищник — жертва. Эволюционное изменение одного вида неизбежно приводит к изменению другого. Более того, взаимную эволюцию, когда один вид совершенствует систему нападения, а другой вслед за ним — систему защиты (и наоборот), часто можно проследить на молекулярном уровне. И здесь наиболее действенной и элегантной оказалась система нападения с помощью ядов, содержащих в своём составе нейротоксины — вещества, воздействующие на нервную систему и мышцы жертвы.

    ID:976
  17. Utkina L.L., Andreev Y.A., Rogozhin E.A., Korostyleva T.V., Slavokhotova A.A., Oparin P.B., Vassilevski A.A., Grishin E.V., Egorov T.A., Odintsova T.I. (2013). Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence. FEBS J. 280 (15), 3594–608 [+]

    A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk-AMP-X1 and Tk-AMP-X2, were completely sequenced and shown to belong to the α-hairpinin structural family of plant peptides with a characteristic C1XXXC2-X(n)-C3XXXC4 motif. The peptides inhibit the spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk-AMP-X peptides. They code for precursor proteins of unusual multimodular structure, consisting of a signal peptide, several α-hairpinin (4-Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C-terminal prodomain. Three types of precursor proteins, with five, six or seven 4-Cys peptide modules, were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk-AMP-X precursors have no introns in the protein-coding regions and are upregulated by fungal pathogens and abiotic stress, providing conclusive evidence for their role in stress response. A combined PCR-based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically-related Triticum and Aegilops species, including polyploid wheat genome donors. Association of the Tk-AMP-X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk-AMP-X-related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize.

    ID:863
  18. Chugunov A.O., Koromyslova A.D., Berkut A.A., Peigneur S., Tytgat J., Polyansky A.A., Pentkovsky V.M., Vassilevski A.A., Grishin E.V., Efremov R.G. (2013). Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets, Sodium Channels. J. Biol. Chem. 288 (26), 19014–27 [+]

    To gain success in the evolutionary "arms race," venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Navs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Navs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid "core module" is supplemented with the "specificity module" (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Navs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Navs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Navs.

    ID:858
  19. Kuzmenkov A.I., Fedorova I.M., Vassilevski A.A., Grishin E.V. (2013). Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments. Biochim. Biophys. Acta 1828 (2), 724–31 [+]

    Venom of Lachesana tarabaevi (Zodariidae, "ant spiders") exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.

    ID:804
  20. Kudryashova K.S., Nekrasova O.V., Kuzmenkov A.I., Vassilevski A.A., Ignatova A.A., Korolkova Y.V., Grishin E.V., Kirpichnikov M.P., Feofanov A.V. (2013). Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study. Analytical and bioanalytical chemistry , [+]

    Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

    ID:782

Руководитель подразделения

Василевский Александр Александрович

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 51, комн. 365
  • Тел.: +7 (495) 336-65-40
  • Эл. почта: avas@ibch.ru

Биомолекулярные инструменты для визуализации ионных каналов (2016-11-19)

Мы создали высокоаффинные химерные молекулы на основе флуоресцентных белков и токсинов скорпионов, селективно воздействующих на потенциал-чувствительные калиевые каналы человека. Мы также продемонстрировали возможности использования таких химер в нейробиологии для визуализации ионных каналов: изучения их локализации, профиля экспрессии в клетках, тканях и органах. Кроме того, представляется возможным использование новых инструментов для скрининговых технологий и диагностики целого ряда заболеваний.

Публикации

  1. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561

Создана база данных пептидных токсинов из яда скорпионов – Kalium (2016-11-19)

Была создана исчерпывающая база данных токсинов скорпионов, действующих на калиевые каналы (KTx), получившая название Kalium.

Kalium имеет открытый доступ и соответствующие ссылки на другие базы данных, такие как UniProt, PDB и NCBI Taxonomy Browser. Основной особенностью Kalium является простота представленных данных в соответствии с актуальной классификацией KTx. Все данные показаны в виде одной таблицы с возможностью сортировки и фильтрации по разным параметрам. Для каждой молекулы в составе Kalium были учтены все посттрансляционные модификации и рассчитаны молекулярные массы. Было проанализировано порядка 300 публикаций, из которых были взяты экспериментальные данные по физиологической активности KTx. Актуальная информация сопровождается ссылкой на соответствующую статью.

Эта инициатива получила широкое одобрение сообщества, в качестве экспертов Kalium выступили ведущие мировые ученые, занимающиеся исследованием животных ядов. Подробнее читай в пресс-релизе на сайте ИБХ.

Публикации

  1. Kuzmenkov A.I., Krylov N.A., Chugunov A.O., Grishin E.V., Vassilevski A.A. (2016). Kalium: a database of potassium channel toxins from scorpion venom. Database (Oxford) 2016, [+]

    Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community.Database URL:http://kaliumdb.org/.

    ID:1504
  2. Kuzmenkov A.I., Grishin E.V., Vassilevski A.A. (2015). Diversity of Potassium Channel Ligands: Focus on Scorpion Toxins. Biochemistry Mosc. 80 (13), 1764–99 [+]

    Potassium (K+) channels are a widespread superfamily of integral membrane proteins that mediate selective transport of K+ ions through the cell membrane. They have been found in all living organisms from bacteria to higher multicellular animals, including humans. Not surprisingly, K+ channels bind ligands of different nature, such as metal ions, low molecular mass compounds, venom-derived peptides, and antibodies. Functionally these substances can be K+ channel pore blockers or modulators. Representatives of the first group occlude the channel pore, like a cork in a bottle, while the second group of ligands alters the operation of channels without physically blocking the ion current. A rich source of K+ channel ligands is venom of different animals: snakes, sea anemones, cone snails, bees, spiders, and scorpions. More than a half of the known K+ channel ligands of polypeptide nature are scorpion toxins (KTx), all of which are pore blockers. These compounds have become an indispensable molecular tool for the study of K+ channel structure and function. A recent special interest is the possibility of toxin application as drugs to treat diseases involving K+ channels or related to their dysfunction (channelopathies).

    ID:1503

Параллельная эволюция токсинов-модуляторов натриевых каналов у мигаломорфных и аранеоморфных пауков (2016-03-21)

Мы провели детальное структурное и функциональное исследование токсина Hm-3 из яда аранеоморфного паука Heriaeus melloteei. Структура токсина была исследована с помощью ЯМР-спектроскопии, а активность – с помощью электрофизиологии. Оказалось, что подобно многим токсинам мигаломорфных пауков, Hm-3 формирует мотив «цистинового узла», обладает амфифильной структурой, способен связываться с мембранами и ингибирует активацию потенциал-чувствительных натриевых каналов, по-видимому, по механизму «мембранного доступа». Гидрофобные «хребты» на поверхности молекул Hm-3 и токсинов мигаломорфных пауков однако расположены с противоположных сторон. Мы предполагаем параллельную эволюцию ингибиторов активации натриевых каналов у мигаломорфных и аранеоморфных пауков.

Публикации

  1. Berkut A.A., Peigneur S., Myshkin M.Y., Paramonov A.S., Lyukmanova E.N., Arseniev A.S., Grishin E.V., Tytgat J., Shenkarev Z.O., Vassilevski A.A. (2015). Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae. J. Biol. Chem. 290 (1), 492–504 [+]

    We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

    ID:1138

Разработка интегрального транскриптомного и протеомного подхода для поиска блокаторов калиевых каналов в яде животных (2016-03-21)

Разработан оригинальный подход поиска новых лигандов калиевых каналов, объединяющий биоинженерную клеточную тест-систему и транскриптомный и протеомный анализ яда животных. С применением этого подхода из яда скорпиона Mesobuthus eupeus были получены восемь высокоаффинных пептидных блокаторов потенциал-зависимого калиевого канала Kv1.1, включая пять новых пептидов. Предложенный подход является универсальным и эффективным инструментом для направленного поиска блокаторов калиевых каналов в природных ядах.

Публикации

  1. Kuzmenkov A.I., Vassilevski A.A., Kudryashova K.S., Nekrasova O.V., Peigneur S., Tytgat J., Feofanov A.V., Kirpichnikov M.P., Grishin E.V. (2015). Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY. J. Biol. Chem. 290 (19), 12195–209 [+]

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

    ID:1310

Предложен удобный «минималистичный» структурный каркас для «скаффолд-инженерии» – проектирования белков с заданной функцией (2015-01-21)

Белковые структуры (типы укладки) различаются по размерам и сложности и, следовательно, по удобству их использования в биоинженерии. Для открытого нами семейства защитных пептидов растений, называемых α-гарпининами, характерна простая укладка. Она состоит из двух коротких α-спиралей, скрепленных двумя дисульфидными мостиками. Подобная структура характерна для некоторых токсинов моллюсков-конусов и скорпионов – ингибиторов калиевых каналов. Мы пересадили функционально важные аминокислотные остатки из токсина в защитный пептид и получили химерную молекулу, полностью воспроизводящую функции токсина. α-Гарпининовая укладка представляется простым и удобным структурным мотивом для белковой инженерии и может быть использована для рационального дизайна лекарственных средств.

Публикации

  1. Berkut A.A., Usmanova D.R., Peigneur S., Oparin P.B., Mineev K.S., Odintsova T.I., Tytgat J., Arseniev A.S., Grishin E.V., Vassilevski A.A. (2014). Structural similarity between defense peptide from wheat and scorpion neurotoxin permits rational functional design. J. Biol. Chem. 289 (20), 14331–40 [+]

    In this study, we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide was found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule, Tk-hefu, was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto the Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that although the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ∼ 35 μm) to κ-hefutoxin 1 (IC50 ∼ 40 μm). We conclude that α-hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design.

    ID:1103