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1.
Toxicon ; 235: 107308, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37797725

ABSTRACT

Animal venoms are a promising source of potential bioinsecticides. To find hits with pronounced oral insect toxicity, we screened 82 venoms using Achroia grisella (Lepidoptera) and Tenebrio molitor (Coleoptera) larvae, and adult Drosophila melanogaster (Diptera). We also injected the most potent venoms in adult D. melanogaster to compare their efficiency in different routes of administration. 18 venoms from spiders and snakes show high oral toxicity and can be further exploited to isolate new insecticides.


Subject(s)
Insecticides , Spider Venoms , Animals , Venoms , Drosophila melanogaster , Spider Venoms/toxicity , Insecta , Insecticides/toxicity , Larva
2.
J Biol Chem ; 294(48): 18349-18359, 2019 11 29.
Article in English | MEDLINE | ID: mdl-31533989

ABSTRACT

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels Kv1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the Kv1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains Kv1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.


Subject(s)
Kv1.3 Potassium Channel/chemistry , Peptides/chemistry , Potassium Channel Blockers/chemistry , Proteins/chemistry , Amino Acid Sequence , Animals , Humans , Kv1.3 Potassium Channel/metabolism , Ligands , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Dynamics Simulation , Mutation , Peptides/genetics , Peptides/metabolism , Potassium Channel Blockers/metabolism , Protein Binding , Protein Conformation , Proteins/metabolism , Surface Properties
3.
FEBS Lett ; 593(19): 2779-2789, 2019 10.
Article in English | MEDLINE | ID: mdl-31276191

ABSTRACT

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage-gated ion channels, while most snake neurotoxins target ligand-gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α-bungarotoxin binding to both muscle-type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK-1 (α-KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ-KTx family) from Heterometrus laoticus, both being blockers of voltage-gated potassium channels. With an IC50 of 1.6 µm, OSK1 inhibits acetylcholine-induced current through mouse muscle-type nAChR heterologously expressed in Xenopus oocytes. Other well-characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.


Subject(s)
Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Scorpion Venoms/pharmacology , Animals , Mice , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/classification , Protein Binding , Receptors, Nicotinic/chemistry , Scorpion Venoms/chemistry , Scorpion Venoms/classification , Xenopus
4.
Protein Sci ; 26(3): 611-616, 2017 03.
Article in English | MEDLINE | ID: mdl-27997708

ABSTRACT

We have recently demonstrated that a common phenomenon in evolution of spider venom composition is the emergence of so-called modular toxins consisting of two domains, each corresponding to a "usual" single-domain toxin. In this article, we describe the structure of two domains that build up a modular toxin named spiderine or OtTx1a from the venom of Oxyopes takobius. Both domains were investigated by solution NMR in water and detergent micelles used to mimic membrane environment. The N-terminal spiderine domain OtTx1a-AMP (41 amino acid residues) contains no cysteines. It is disordered in aqueous solution but in micelles, it assumes a stable amphiphilic structure consisting of two α-helices separated by a flexible linker. On the contrary, the C-terminal domain OtTx1a-ICK (59 residues) is a disulfide-rich polypeptide reticulated by five S-S bridges. It presents a stable structure in water and its core is the inhibitor cystine knot (ICK) or knottin motif that is common among single-domain neurotoxins. OtTx1a-ICK structure is the first knottin with five disulfide bridges and it represents a good reference for the whole oxytoxin family. The affinity of both domains to membranes was measured with NMR using titration by liposome suspensions. In agreement with biological tests, OtTx1a-AMP was found to show high membrane affinity explaining its potent antimicrobial properties.


Subject(s)
Arthropod Proteins/chemistry , Membranes, Artificial , Spider Venoms/chemistry , Spiders/chemistry , Animals , Nuclear Magnetic Resonance, Biomolecular , Protein Domains
5.
Biochem J ; 473(19): 3113-26, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27412961

ABSTRACT

Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of ß-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) ß-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.


Subject(s)
Spider Venoms/chemistry , Amino Acid Sequence , Cell Membrane/drug effects , Circular Dichroism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microbial Sensitivity Tests , Protein Structure, Secondary , Sequence Homology, Amino Acid , Spider Venoms/pharmacology
6.
J Biol Chem ; 289(20): 14331-40, 2014 May 16.
Article in English | MEDLINE | ID: mdl-24671422

ABSTRACT

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.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/genetics , Neurotoxins/chemistry , Protein Engineering , Scorpions/chemistry , Triticum/chemistry , Animals , Antimicrobial Cationic Peptides/pharmacology , Disulfides/chemistry , Electrophysiological Phenomena/drug effects , Models, Molecular , Neurotoxins/genetics , Nuclear Magnetic Resonance, Biomolecular , Potassium Channel Blockers/chemistry , Potassium Channel Blockers/pharmacology , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology
7.
Plant Mol Biol ; 84(1-2): 189-202, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24081691

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.


Subject(s)
Antifungal Agents/metabolism , Plant Proteins/biosynthesis , Plant Proteins/metabolism , Seeds/metabolism , Stellaria/chemistry , Amino Acid Sequence , Antifungal Agents/chemistry , Cloning, Molecular , Evolution, Molecular , Fungi/drug effects , Gene Expression Regulation, Plant/physiology , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/genetics , Seeds/chemistry , Seeds/genetics , Stellaria/metabolism
8.
FEBS J ; 280(15): 3594-608, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23702306

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 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.


Subject(s)
Antimicrobial Cationic Peptides/genetics , Disease Resistance/genetics , Plant Proteins/genetics , Seedlings/genetics , Triticum/genetics , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Conserved Sequence , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Protein Processing, Post-Translational , Protein Structure, Secondary , Proteolysis , Sequence Homology, Amino Acid , Species Specificity , Stress, Physiological
9.
Biochem J ; 446(1): 69-77, 2012 Aug 15.
Article in English | MEDLINE | ID: mdl-22612157

ABSTRACT

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.


Subject(s)
Fagopyrum/chemistry , Peptides/chemistry , Plant Proteins/chemistry , Trypsin Inhibitors/chemistry , Amino Acid Sequence , Chromatography, Reverse-Phase , Fagopyrum/physiology , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptides/genetics , Peptides/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Conformation , Protein Folding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seeds/chemistry , Trypsin Inhibitors/isolation & purification
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