[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Three species of the ant subfamily Ponerinae (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.
[Show abstract][Hide abstract] ABSTRACT: The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defense-related genes that results in the reinforcement of plant cell walls and 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, plant defense proteins that bind and degrade chitin of fungal cell walls. In this work, we studied plant response to such pathogen invasion and discovered novel inhibitors of fungalysin. We produced several recombinant hevein-like antimicrobial peptides named WAMPs containing different amino acids (A, K, E, and N) at the non-conservative position 34. An additional serine residue in the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, equal molar ratio of WAMP-1b or WAMP-2 to chitinase was sufficient to block the fungalysin activity keeping the chitinase intact. Thus, WAMPs represent a novel type of protease inhibitors being active against fungal metalloproteases. According to in vitro antifungal assays WAMPs demonstrated direct inhibition of 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.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Scorpion venom is a complex multicomponent mixture of biologically active substances, some of which possess very interesting properties and are used in quite unexpected areas. The family of chlorotoxin (CTX)-like peptides is a good example. These toxins exhibit insecticidal activity, however, the molecular mechanism of their action on the insect organism has not been established yet. Nevertheless, CTX-like peptides attracted considerable research effort due to their ability to interact specifically with cells of malignant brain tumors—gliomas. In the future these compounds may significantly simplify treatment of tumor diseases. The review summarizes the results accumulated over a forty years period of CTX-like peptides study. Aspects of their natural functions are considered, as well as the application area associated with gliomas.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In this paper we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide is 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 and was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto 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 while 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 structural templates, which may be used for functional engineering and drug design.
[Show abstract][Hide abstract] ABSTRACT: 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 O. takobius and O. 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Apart from the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines OtTx 1a, 1b, 2a and 2b. These toxins show both insecticidal activity (median lethal dose against flesh fly larvae of 75 µg/g) and potent antimicrobial effects (minimal inhibitory concentrations in the range of 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 membrane-mimicking environment, and shows potent cytolytic effects on cells of different origin. The C-terminal part is disulfide-rich (~60 residues; 5 S-S bonds) and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is much alike linear cytotoxic peptides found in different organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx 1a and compared their activity to that of the full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part that in both insecticidal and antimicrobial assays retained full-length toxin activity. The unique structure of spiderines fills up the missing part of the two-domain spider toxin ensemble. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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 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. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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 (Nav's) and thereby modify the excitability of muscle and nerve cells. Although more than a hundred α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Nav's 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 in spite of 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 vs. 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 Nav's extracellular loops 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 Nav's. 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 Nav's.
[Show abstract][Hide abstract] ABSTRACT: Antichlamydial activity of cyto-insectotoxin 1a (CIT 1a), representative of a unique class of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi, was studied. A plasmid vector expressing the cit 1a gene controlled by a human cytomegalovirus tetracycline-dependent promoter was constructed. Impressive inhibition of Chlamydia trachomatis infection in HEK 293 cells transfected by the cit 1a-harboring vector was achieved. With the use of various schemes of cell infection and gene expression induction, it was shown for the first time that an antimicrobial peptide exerts its potent antichlamydial action at an early stage of the pathogen life cycle.
Archives of Microbiology 03/2013; 195(3). DOI:10.1007/s00203-012-0863-5 · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Membrane-active peptides (MAPs) represent a broad variety of molecules, and biological functions of most are directly associated with their ability to interact with membranes. Taking into account the effect of MAPs on living cells they can be nominally divided into three major groups - fusion (FPs), antimicrobial/cytolytic (AMPs/CPs) and cellpenetrating (CPPs) peptides. Although spatial structure of different MAPs varies to a great extent, linear α-helical peptides represent the most studied class. These peptides possess relatively simple structural organization and share a set of similar molecular features, which make them very attractive to both experimental and computational studies. Here, we review different molecular modeling methods in prospective of their applications to study of α-helical MAPs. The most sophisticated of them, such as molecular dynamics simulations, give atomistic information about molecular interactions driving peptide binding to the water-lipid interface, cooperative mechanisms of membrane destabilization and thermodynamics of these processes. Significant progress has been achieved in this field during the last few years, resulting in a possibility to observe computationally MAPs action in realistic peptide-to-lipid ratios and over the microsecond timescale. Other relatively simple but powerful approaches allow assessment of important characteristics of MAPs such as α-helical propensity, amphiphilicity, total hydrophobicity, and spatial distribution of charge and hydrophobic/hydrophilic properties, etc. Altogether, computational methods provide efficient basis for rational design of MAPs with predefined properties and a spectrum of biological activities.
Current Protein and Peptide Science 11/2012; 13(7):644-57. DOI:10.2174/138920312804142147 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. Among them, a special place belongs to cyto-insectotoxins, a class of cytolytic molecules showing equally potent antimicrobial and insecticidal effects. We tested one of them, CIT 1a, for ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the cit 1a gene. Controlled expression of the transgene led to a significant decrease in C. trachomatis viability inside the infected cells. Using proteomic and transcriptomic approaches, we found alterations in protein expression patterns and identified differentially expressed genes in transfected cells.
Probiotics and Antimicrobial Proteins 09/2012; 4(3). DOI:10.1007/s12602-012-9108-9
[Show abstract][Hide abstract] ABSTRACT: 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.