Laboratory of neuroreceptors and neuroregulators

Department of Molecular Neurobiology

Head: Sergey Kozlov, D.Sc
serg@ibch.ru+7(495)336 4022

Toxinology, neurotoxins, ionotropic receptors, protein structure, recombinant polypeptides, bioactive molecules for medicine

The laboratory conducts research of biologically active components from natural sources, interacting with a variety of targets in the cell membrane, as well as developing a common approach for the identification, isolation, structural and functional analysis and use as a thin tool which modulate the activity of the nerve cells membrane receptors. Laboratory research allows to be closer to an understanding the molecular mechanisms of the natural ligand specificity action and lays the foundation for the development of fundamentally new highly specific drugs and insecticides.

The laboratory cooperates with The Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, (Magazanik L.G.), Federal Scientific Clinical Center of Physical-Chemical Medicine (Govorun V.M.), Sadykov Institute of Bioorganic Chemistry of the Academy of Sciences of Uzbekistan (Salikhov S.I.), Vavilov Institute of General Genetics RAS (Odintsova T.I., PhD), All-Russian Research Institute of Agricultural Biotechnology (prof. Babakov A.V.), University of Leuven, Belgium (Prof. Tytgat J.).

The laboratory has also performed studies in cooperation with Monsanto, Bayer, Zeneka, DuPont and other companies.

Young people are always welcome and get first class practical courses. Since its foundation, 23 PhD dissertations have been successfully defended in the Laboratory. High quality equipment, use of state-of-the-art methods, ongoing staff practical skill training, open-mindedness and ambitious scientific goals – this is what best describes the Laboratory today.

The Laboratory was founded in 1987 by Professor Eugene V. Grishin, member of the Russian Academy of Sciences. The laboratory has evolved into a world-leading scientific team studying natural neurotoxins and their neuronal receptors. The focus is on toxic components of animal venoms acting on different targets in the cell membrane. General approaches have been elaborated to active venom components identification, isolation, structure-function analyses and use as high-precision tools modulating neural membrane receptor activity.

  • Search and investigation of polypeptide toxins and lower-modulated compounds that modulate activity of membrane receptors involved in processes of generation and pain signaling (Korolkova Y.V., PhD; Andreev Ya.A., PhD; Koshelev S.G., PhD)
  • Studies of antimicrobial peptides from arthropods and plant defense peptides (Rogozhin E.A., PhD)
  • Production of recombinant polypeptide toxins (Andreev Ya.A. PhD); study of DNA complex nanostructure (Danilevich V.N.).
  • Development of agricultural crop protection methods to increase crop (Rogozhin E.)
  • Development of methods for the production of recombinant polypeptide toxins in various heterologous expression systems (Yaroslav A. Andreev, Catherine E. Maleeva)
  • Omixed technologies implementation in search and structural identification of biologically active compounds of complex multi-component mixtures (Mikov A.)
  • Creation of cell lines for the analysis of ligand-receptor interactions (Julia Korol'kova, Yaroslav A. Andreev)
  • Studying the DNA nanostructure complexes (Vasiliy Danilevich)
  • a new class of toxins such as spiders argiope – polyamine blocker of glutamate receptors (Grishin et al., 1983)
  • a family of high-selective neurotoxins from the venom of the Black Widow ("black widow") – latrotoxin (Kiyatkin et al., 1990)
  • membrane-active peptides from the venom of ants and spiders (Kozlov et al., 2006)
  • polypeptide toxins specific for sodium, potassium, calcium, proton-sensitive, TRPV1, TRPA1-channels.
  • low-molecular weight biologically active compounds from medicinal plants with analgesic effect.
  • In the study of neuronal receptors isolated and characterized for the first time all of the individual components of the mammalian sodium channel electro-excitability membranes.
NamePositionContacts
Anna Slavokhotova
Sergey Kozlov, D.Scdepart. dir.serg@ibch.ru+7(495)336 4022
Yaroslav Andreev, Ph.D.s. r. f.ay@land.ru+7(495)336-40-20
Alexander Vassilevski, Ph.D.s. r. f.avas@ibch.ru+7(495)336-65-40, +7(495)336-40-22
Vasilij Danilevich, Ph.D.s. r. f.dan@ibch.ru+7(495)336-40-22
Elena Eljakova, Ph.D.s. r. f.elyakova@yandex.ru+7(495)429-87-20
Yulija Korol'kova, Ph.D.s. r. f.july@ibch.ru+7(495)3366540
Sergej Koshelev, Ph.D.s. r. f.sknew@yandex.ru+7(495)3306683
Evgenij Rogozhinr. f.rea21@list.ru+7(495)336-40-22
Dmitry Osmakovr. f.
Alexander Mikovj. r. f.mikov.alexander@gmail.com+7(495)3366540
Ekaterina Maleevaj. r. f.+7()3364022
Irina Mosharovaj. r. f.+7()3306683
Yulia Logashinaj. r. f.julek022@rambler.ru
Ljudmila Aleksandrovaeng.+7(495)336-40-22
Ksenia Lybovaeng.
Anna Emelianovares. eng.annaemelyan@gmail.com

Former members:

Tseziy Egorov, D.Scl. r. f.ego@ibch.ru
Tat'jana Valjakina, Ph.D.s. r. f.valyakina@ibch.ru
Kirill Pluzhnikov, Ph.D.s. r. f.
Maria Simonova, Ph.D.r. f.simonova@ibch.ru
Ravilja Komalevar. f.
Ol'ga Lahtinar. f.
Elena Petrova, Ph.D.r. f.petrova@ibch.ru
Aleksandr Musoljamovj. r. f.musolyamov@ibch.ru
Larisa Samohvalovaj. r. f.
A Astafevaj. r. f.a_sanya@mail.ru
Maria Sachkova, Ph.D.PhD stud.sachkovamasha@mail.ru
Alexander Arzamasovstud.Alex_arzamasov@hotmail.com
Zhanna Kanaevskayalab. as.zh-kanewskaya@yandex.ru
Evgeny Grishin, member of the academy of sciencesdept. head

Selected publications

  1. Babenko V.V., Mikov A.N., Manuvera V.A., Anikanov N.A., Kovalchuk S.I., Andreev Y.A., Logashina Y.A., Kornilov D.A., Manolov A.I., Sanamyan N.P., Sanamyan K.E., Kostryukova E.S., Kozlov S.A., Grishin E.V., Govorun V.M., Lazarev V.N. (2017). Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus. Sci Rep 7 (1), 14534 [+]

    Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.

    ID:1908
  2. Kozlov S. (2017). Animal toxins for channelopathy treatment. Neuropharmacology , [+]

    Ion channels are transmembrane proteins that allow passive flow of ions inside and/or outside of cells or cell organelles. Except mutations lead to nonfunctional protein production or abolished receptor entrance on the membrane surface an altered channel may have two principal conditions that can be corrected. The channel may conduct fewer ions through (loss-of-function mutations) or too many ions (gain-of-function mutations) compared to a normal channel. Toxins from animal venoms are specialised molecules that are generally oriented toward interactions with ion channels. This is a result of long coevolution between predators and their prey. On the molecular level, toxins activate or inhibit ion channels, so they are ideal molecules for restoring conductance in mutated channels. Another aspect of this long coevolution is that a broad variety of toxins have been fine tuned to recognize the channels of different species, keeping many amino acids substitution among sequences. Many peptide ligands with high selectivity to specific receptor subtypes have been isolated from animal venoms, some of which are absolutely non-toxic to humans and mammalians. It is expected that molecules that are selective to each known receptor can be found in animal venoms, but the pool of toxins currently does not override all receptors described as being involved in channelopathies. Modern investigating methods have enhanced the search process for selective ligands. One prominent method is a site-directed mutagenesis of existing toxins to change the selectivity or/and affinity to the selected receptor, which has shown positive results.

    ID:1927
  3. Logashina Y.A., Solstad R.G., Mineev K.S., Korolkova Y.V., Mosharova I.V., Dyachenko I.A., Palikov V.A., Palikova Y.A., Murashev A.N., Arseniev A.S., Kozlov S.A., Stensvåg K., Haug T., Andreev Y.A. (2017). New Disulfide-Stabilized Fold Provides Sea Anemone Peptide to Exhibit Both Antimicrobial and TRPA1 Potentiating Properties. Toxins (Basel) 9 (5), [+]

    A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.

    ID:1914
  4. Korolkova Y., Makarieva T., Tabakmakher K., Shubina L., Kudryashova E., Andreev Y., Mosharova I., Lee H.S., Lee Y.J., Kozlov S. (2017). Marine Cyclic Guanidine Alkaloids Monanchomycalin B and Urupocidin A Act as Inhibitors of TRPV1, TRPV2 and TRPV3, but not TRPA1 Receptors. Mar Drugs 15 (4), [+]

    Marine sponges contain a variety of low-molecular-weight compounds including guanidine alkaloids possessing different biological activities. Monanchomycalin B and urupocidin A were isolated from the marine sponge Monanchora pulchra. We found that they act as inhibitors of the TRPV1, TRPV2, and TRPV3 channels, but are inactive against the TRPA1 receptor. Monanchomycalin B is the most active among all published marine alkaloids (EC50 6.02, 2.84, and 3.25 μM for TRPV1, TRPV2, and TRPV3, correspondingly). Moreover, monanchomycalin B and urupocidin A are the first samples of marine alkaloids affecting the TRPV2 receptor. Two semi-synthetic urupocidin A derivatives were also obtained and tested against TRP (Transient Receptor Potential) receptors that allowed us to collect some data concerning the structure-activity relationship in this series of compounds. We showed that the removal of one of three side chains or double bonds in the other side chains in urupocidin A led to a decrease of the inhibitory activities. New ligands specific to the TRPV subfamily may be useful for the design of medicines as in the study of TRP channels biology.

    ID:1929
  5. Logashina Y.A., Mosharova I.V., Korolkova Y.V., Shelukhina I.V., Dyachenko I.A., Palikov V.A., Palikova Y.A., Murashev A.N., Kozlov S.A., Stensvåg K., Andreev Y.A. (2017). Peptide from Sea Anemone Metridium senile Affects Transient Receptor Potential Ankyrin-repeat 1 (TRPA1) Function and Produces Analgesic Effect. J. Biol. Chem. 292 (7), 2992–3004 [+]

    The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.

    ID:1928
  6. Nikolaev M.V., Dorofeeva N.A., Komarova M.S., Korolkova Y.V., Andreev Y.A., Mosharova I.V., Grishin E.V., Tikhonov D.B., Kozlov S.A. (2017). TRPV1 activation power can switch an action mode for its polypeptide ligands. PLoS ONE 12 (5), e0177077 [+]

    TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone Heteractis crispa (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca2+ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3-300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 μM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength-potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.

    ID:1930
  7. Osmakov D.I., Koshelev S.G., Andreev Y.A., Kozlov S.A. (2017). Endogenous Isoquinoline Alkaloids Agonists of Acid-Sensing Ion Channel Type 3. Front Mol Neurosci 10, 282 [+]

    Acid-sensing ion channels (ASICs) ASIC3 expressed mainly in peripheral sensory neurons play an important role in pain perception and inflammation development. In response to acidic stimuli, they can generate a unique biphasic current. At physiological pH 7.4, human ASIC3 isoform (hASIC3) is desensitized and able to generate only a sustained current. We found endogenous isoquinoline alkaloids (EIAs), which restore hASIC3 from desensitization and recover the transient component of the current. Similarly, rat ASIC3 isoform (rASIC3) can also be restored from desensitization (at pH < 7.0) by EIAs with the same potency. At physiological pH and above, EIAs at high concentrations were able to effectively activate hASIC3 and rASIC3. Thus, we found first endogenous agonists of ASIC3 channels that could both activate and prevent or reverse desensitization of the channel. The decrease of EIA levels could be suggested as a novel therapeutic strategy for treatment of pain and inflammation.

    ID:1932
  8. Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Y.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2016). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol , [+]

    Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K(+)-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

    ID:1611
  9. Osmakov D.I., Koshelev S.G., Andreev Y.A., Dyachenko I.A., Bondarenko D.A., Murashev A.N., Grishin E.V., Kozlov S.A. (2015). Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination. Toxicon , [+]

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins.

    ID:1472
  10. 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
  11. Astafieva A.A., Enyenihi A.A., Rogozhin E.A., Kozlov S.A., Grishin E.V., Odintsova T.I., Zubarev R.A., Egorov T.A. (2015). Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity. Plant Sci. 238, 323–9 [+]

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.

    ID:1474
  12. Mikov A.N., Fedorova I.M., Potapieva N.N., Maleeva E.E., Andreev Y.A., Zaitsev A.V., Kim K.K., Bocharov E.V., Bozin T.N., Altukhov D.A., Lipkin A.V., Kozlov S.A., Tikhonov D.B., Grishin E.V. (2015). ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom. Sci Rep 5, 17232 [+]

    Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making "scissors-like mutual motions".

    ID:1341
  13. 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
  14. Osmakov D.I., Kozlov S.A., Andreev Y.A., Koshelev S.G., Sanamyan N.P., Sanamyan K.E., Dyachenko I.A., Bondarenko D.A., Murashev A.N., Mineev K.S., Arseniev A.S., Grishin E.V. (2013). Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity. J. Biol. Chem. 288 (32), 23116–27 [+]

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.

    ID:864
  15. Kabanova N.V., Vassilevski A.A., Rogachevskaja O.A., Bystrova M.F., Korolkova Y.V., Pluzhnikov K.A., Romanov R.A., Grishin E.V., Kolesnikov S.S. (2012). Modulation of P2X3 receptors by spider toxins. Biochim. Biophys. Acta 1818 (11), 2868–2875 [+]

    Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.

    ID:730
  16. Andreev Y.A., Kozlov S.A., Koshelev S.G., Ivanova E.A., Monastyrnaya M.M., Kozlovskaya E.P., Grishin E.V. (2008). Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1). J. Biol. Chem. 283 (35), 23914–21 [+]

    The first inhibitor of thermoreceptors TRPV1 that play an important role in inflammation and pain sensation was isolated from nematocyst extract of the sea anemone Heteractis crispa. Recombinant analogue of the peptide named APHC1 was found to exert high analgesic activity comparable to that of opioids in vivo. Unlike opioids, however, the action of APHC1 does not lead to a narcotic effect. The newly described polypeptide has great practical value in terms of new analgesic and anti-inflammatory drug production.

    ID:87
  17. Kozlov S.A., Vassilevski A.A., Feofanov A.V., Surovoy A.Y., Karpunin D.V., Grishin E.V. (2006). Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J. Biol. Chem. 281 (30), 20983–92 [+]

    A family of short linear polypeptide molecules, latarcins, was found in the venom of the spider Lachesana tarabaevi. Some peptides were chemically synthesized and their antimicrobial properties were studied. The bactericidal activity of latarcins matched that of the most active known antimicrobial peptides from different animals. The high therapeutic potential of these structurally simple polypeptide molecules will be brought to clinic in the near future.

    ID:86
  18. Kozlov S., Malyavka A., McCutchen B., Lu A., Schepers E., Herrmann R., Grishin E. (2005). A novel strategy for the identification of toxinlike structures in spider venom. Proteins 59 (1), 131–40 [+]

    We compared two different approaches to sequence information analysis from the expressed sequence tag (EST) library constructed for the venom glands of the spider Agelena orientalis. Some results were more illustrative and reliable by the contig analysis technique, whereas our novel method, with specific structural markers introduced for protein structure detection, allowed us to overcome some limitations of the contig analysis. A novel technique was suggested for the identification in data banks of the spider's ion channel inhibitor toxins using primary structure features common to all spiders. Analysis of about 150 polypeptides made it possible to introduce 3 primary structure motifs for spider toxins: the Principal Structural Motif (PSM), which postulates the existence of 6 amino acid residues between the first and second cysteine residue and the Cys-Cys sequence at a distance of 5-10 amino acid residues from the second cysteine; the Extra Structural Motif (ESM), which postulates the existence of a pair of CXC fragments in the C-region; and the Processing Quadruplet Motif (PQM), which specifies the Arg residue at position -1 and Glu residues at positions -2, -3, and/or -4 in the precursor sequences just before the postprocessing site. In the processed data bank we found 48 toxinlike structures with ion channel inhibitor motifs. These include agelenin earlier isolated from Agelena opulenta and 25 more homologous sequences, 15 homologs of mu-agatoxin 2 from the spider Agelenopsis aperta, 3 structures with low homology to omega-agatoxin-IIIA, and 4 new structures. Also we showed that toxinlike structures exceed two thirds of the overall database sequences.

    ID:194
  19. Korolkova Y.V., Bocharov E.V., Angelo K., Maslennikov I.V., Grinenko O.V., Lipkin A.V., Nosyreva E.D., Pluzhnikov K.A., Olesen S.P., Arseniev A.S., Grishin E.V. (2002). New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1. J. Biol. Chem. 277 (45), 43104–9 [+]

    The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.

    ID:195
  20. Korolkova Y.V., Kozlov S.A., Lipkin A.V., Pluzhnikov K.A., Hadley J.K., Filippov A.K., Brown D.A., Angelo K., Strøbaek D., Jespersen T., Olesen S.P., Jensen B.S., Grishin E.V. (2001). An ERG channel inhibitor from the scorpion Buthus eupeus. J. Biol. Chem. 276 (13), 9868–76 [+]

    The first selective blocker of K+ channels of ERG type BeKm was isolated from the venom of the scorpion Buthus eupeus. The peptide along with its mutants was produced in a heterological expression system and their properties were assessed on M-currents in the NG108-15 cell line, which allowed establishing the peptide’s pharmacophore. BeKm toxin became the first published selective inhibitor of hERG channels. At present it may be found on the market of bioactive substances.

    ID:85

Sergey Kozlov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 51, office. 363
  • Phone: +7(495)336 4022
  • E-mail: serg@ibch.ru

Fax: +7 (495) 330 5892

First peptide ligands potentiating the TRPA1 response to agonists and producing the analgesic and anti-inflammatory effects. (2017-11-24)

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.

Publications

  1. Logashina Y.A., Solstad R.G., Mineev K.S., Korolkova Y.V., Mosharova I.V., Dyachenko I.A., Palikov V.A., Palikova Y.A., Murashev A.N., Arseniev A.S., Kozlov S.A., Stensvåg K., Haug T., Andreev Y.A. (2017). New Disulfide-Stabilized Fold Provides Sea Anemone Peptide to Exhibit Both Antimicrobial and TRPA1 Potentiating Properties. Toxins (Basel) 9 (5), [+]

    A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.

    ID:1914
  2. Logashina Y.A., Mosharova I.V., Korolkova Y.V., Shelukhina I.V., Dyachenko I.A., Palikov V.A., Palikova Y.A., Murashev A.N., Kozlov S.A., Stensvåg K., Andreev Y.A. (2017). Peptide from Sea Anemone Metridium senile Affects Transient Receptor Potential Ankyrin-repeat 1 (TRPA1) Function and Produces Analgesic Effect. J. Biol. Chem. 292 (7), 2992–3004 [+]

    The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.

    ID:1928

Novel endogenous ligands of ASIC3 receptor can activate the channel independent from protons, and release human ASIC3 from the steady state desensitization at physiological pH. (2017-11-24)

First activators of acid-sensing ion channels (ASIC) with unique mechanism of action were found. The milimolar concentration of isoquinoline alkaloids (THP, reticuline), which are synthesized by mammalian cells as intermediates in the cycle of endogenous morphine synthesis from tyrosine, can decrease ASIC3 proton-mediated desensitization and at the same time effectively activates hASIC3 and rASIC3 at physiological pH 7.4 and slightly alkaline conditions. To date, a very limited pool of ASICs activators is known. All these molecules shift the channels activation limit to the higher pH values, that making the receptor to be more responsible to a small pH spikes. But still the protons contribution in the process of the channels opening considered as primary. The isoquinoline alkaloids are the first described "non-proton" activators of ASIC3 receptors.

A pharmacological characterization of "non-proton" activators at human and rodents ASIC3 isoforms revealed a strong functional difference of the effect between the rat channels (minor effect) and human (strong potentiate response to acidification) that questioned the effectiveness of ligands proved in animal tests by their transfer to clinical study. In addition, we suggested a new approach for the new analgesics development. Bu our opinion, two systems of nociception (ASIC3) and anti-nociception (opioid receptors) shared common biosynthetic pathway of ligands and may be in mutually balanced state. The inhibition or activation of enzymes involved in the endogenous morphine biosynthesis cascade may shift the equilibrium in some direction that results in analgesia or hyperalgesia.

ASICs play an important role not only in nociception but in the signal transduction, synaptic plasticity, learning, and neuronal cells death. The discovery of endogenous "non-proton" activators proves the existence of several regulatory ways for these channels. It is not clear which of them can more be implemented in the CNS, it is possible that acidification only accompanied the ligand gated regulation. The answer to this question will be obtained in further study.

Publications

  1. Osmakov D.I., Koshelev S.G., Andreev Y.A., Kozlov S.A. (2017). Endogenous Isoquinoline Alkaloids Agonists of Acid-Sensing Ion Channel Type 3. Front Mol Neurosci 10, 282 [+]

    Acid-sensing ion channels (ASICs) ASIC3 expressed mainly in peripheral sensory neurons play an important role in pain perception and inflammation development. In response to acidic stimuli, they can generate a unique biphasic current. At physiological pH 7.4, human ASIC3 isoform (hASIC3) is desensitized and able to generate only a sustained current. We found endogenous isoquinoline alkaloids (EIAs), which restore hASIC3 from desensitization and recover the transient component of the current. Similarly, rat ASIC3 isoform (rASIC3) can also be restored from desensitization (at pH < 7.0) by EIAs with the same potency. At physiological pH and above, EIAs at high concentrations were able to effectively activate hASIC3 and rASIC3. Thus, we found first endogenous agonists of ASIC3 channels that could both activate and prevent or reverse desensitization of the channel. The decrease of EIA levels could be suggested as a novel therapeutic strategy for treatment of pain and inflammation.

    ID:1932

The multidirectional mechanism of action of peptide modulators of TRPV1 receptor in different conditions of receptor activation has been established. (2017-11-24)

The effect of polypeptides APHC1, APHC2 and APHC3 isolated from sea anemones Heteractis crispa was studied on rTRPV1 expressed in CHO cells. Method of electrophysiology, fluorescence spectroscopy and molecular modeling was utilized. It was established that APHCs potentiate the response of TRPV1 to low (3-300 nM) capsaicin concentrations and inhibit the response to high (>3.0 µM) concentrations of the activator. Also the dependence of peptides action onto TRPV1 was investigated for varying concentrations of activators, such as 2APB and protons, and for the activation by combined stimuli. As a result, the bimodal mechanism of APHCs action onto the receptor is depended to the stimulus power then peptides potentiate low signals but inhibit / neutral to high-amplitude responses. The proposed dual "Gating" model of TRPV1 activation suggests that APHC-polypeptides can stabilize an intermediate state during the receptor activation. By molecular modeling the expected binding site of peptides located between a pair of P-loops on TRPV1 top.

Modulators like APHCs with a dualistic effect have a definite advantage for practical medical applications, since such compounds do not inhibit the normal functioning of the receptor, but produce the desired therapeutic effect at pathologically strong activation.

Publications

  1. Nikolaev M.V., Dorofeeva N.A., Komarova M.S., Korolkova Y.V., Andreev Y.A., Mosharova I.V., Grishin E.V., Tikhonov D.B., Kozlov S.A. (2017). TRPV1 activation power can switch an action mode for its polypeptide ligands. PLoS ONE 12 (5), e0177077 [+]

    TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone Heteractis crispa (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca2+ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3-300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 μM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength-potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.

    ID:1930

ω-Tbo-IT1 — selective inhibitor of insect channels isolated from Tibellus oblongus spider venom (2016-03-28)

Novel disulfide-containing polypeptide toxin was found in venom Tibellus oblongus spider from Central Asia region. Here, we report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin called ω-Tbo-IT1 It has insect toxic effect with LD50 14.3 μg/g in experiments on Musca domestica larvae. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in Calliphora vicina neuromuscular junction while parameters of spontaneous ones were not affected. Inhibition was concentration dependent with IC50 value 40±10 nM and Hill coefficient 3.4±0.3. The toxin did not affect frog neuro-muscular junction, glutamatergic and GABAergic transmission in rat brain. Ca2+ currents in Calliphora vicina muscle were not inhibited, whereas in cockroach neurons at least one type of voltage gated Ca2+ currents were inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as selective inhibitor of presynaptic insect Ca2+ channels. Spatial structure analysis of recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed the set of 20 structures. The toxin comprises the conventional ICK (knottin) fold containing extended β-hairpin loop and γ-turn that are capable of doing — as we called it — “scissors-like mutual motions”. Alongside with alternative twisting of the β-sheet observed in the major β-hairpin loop such plasticity of the molecule (dynamic epitope) may play a crucial role for the receptor binding/recognition.

Publications

  1. Mikov A.N., Fedorova I.M., Potapieva N.N., Maleeva E.E., Andreev Y.A., Zaitsev A.V., Kim K.K., Bocharov E.V., Bozin T.N., Altukhov D.A., Lipkin A.V., Kozlov S.A., Tikhonov D.B., Grishin E.V. (2015). ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom. Sci Rep 5, 17232 [+]

    Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making "scissors-like mutual motions".

    ID:1341

A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers (2016-03-28)

A novel peptide named ToAMP 4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, an d RP-H PLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids , and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 doe s not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4 , it was success fully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4 -mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and function al diversity of AMPs in plants.

Publications

  1. Astafieva A.A., Enyenihi A.A., Rogozhin E.A., Kozlov S.A., Grishin E.V., Odintsova T.I., Zubarev R.A., Egorov T.A. (2015). Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity. Plant Sci. 238, 323–9 [+]

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.

    ID:1474