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Yuri N Utkin,
Christoph Weise,
Igor E Kasheverov,
Tatyana V Andreeva,
Elena V Kryukova, Maxim N Zhmak,
Vladislav G Starkov,
Ngoc Anh Hoang,
Daniel Bertrand,
Joachim Ramerstorfer,
Werner Sieghart,
Andrew J Thompson,
Sarah C R Lummis,
Victor I Tsetlin
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ABSTRACT: Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.
Journal of Biological Chemistry 05/2012; 287(32):27079-86. · 4.77 Impact Factor
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ABSTRACT: Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.
Analytical and Bioanalytical Chemistry 02/2011; 399(10):3547-54. · 3.78 Impact Factor
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ABSTRACT: A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog, single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.
Marine Drugs 01/2011; 9(10):1698-714. · 3.85 Impact Factor
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ABSTRACT: Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.
Biochemical and Biophysical Research Communications 09/2007; 360(1):156-62. · 2.48 Impact Factor
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ABSTRACT: Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.
FEBS Journal 11/2006; 273(19):4470-81. · 3.79 Impact Factor
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ABSTRACT: alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.
FEBS Journal 05/2006; 273(7):1373-88. · 3.79 Impact Factor
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ABSTRACT: α-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in α-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of α-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their 1H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced 125I-labeled GI from the Torpedo californica nAChR. 125I-labeled GI(Bpa12) bound to two sites on the receptor (Kd 13 and 1800 nm), and on UV irradiation specifically photolabeled the α, γ and δ subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the α subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at αSer173 and containing the agonist-binding site segment C. In the γ subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at γAla167 and γAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.
FEBS Journal 03/2006; 273(7):1373 - 1388. · 3.79 Impact Factor
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ABSTRACT: Alpha-conotoxins, neurotoxic peptides from poisonous Conus marine snails, can be subdivided into several groups targeting distinct subtypes of nicotinic acetylcholine receptors (nAChRs). Such alpha-conotoxins as, for example, GI, MI, or SIA potently block muscle-type nAChRs from muscles and from the electric organ of Torpedo ray, whereas others target distinct neuronal nAChRs: alpha-conotoxins ImI and PnIB block pentaoligomeric alpha7 nAChRs, and alpha-conotoxins MII or PnIA inhibit heteromeric nAChRs made of combinations of alpha3 or alpha6 subunits with beta2 subunit. alpha-Conotoxins interact with N-terminal extracellular ligand-binding domains of nAChRs and are indispensable tools for distinguishing various subtypes of AChRs at normal and pathological states. Although many alpha-conotoxins have been isolated from Conus venoms, there is still a great need in more potent and selective tools, which in principle can be obtained by design and synthesis of novel alpha-conotoxin analogs.
Journal of Molecular Neuroscience 02/2006; 30(1-2):77-8. · 2.50 Impact Factor
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Patrick H N Celie,
Igor E Kasheverov,
Dmitry Y Mordvintsev,
Ronald C Hogg,
Pim van Nierop,
René van Elk,
Sarah E van Rossum-Fikkert, Maxim N Zhmak,
Daniel Bertrand,
Victor Tsetlin,
Titia K Sixma,
August B Smit
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ABSTRACT: Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.
Nature Structural & Molecular Biology 08/2005; 12(7):582-8. · 12.71 Impact Factor
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ABSTRACT: The antibodies against synthetic (183-192) fragments of beta2- and beta4-subunits of rat neuronal nicotinic acetylcholine receptor were used to study a beta-subunit composition of nicotinic receptors in the inferior mesenteric ganglion of the guinea pig by both immunocytochemical staining and blocking of excitatory postsynaptic potentials induced by electric stimulation of the pre-ganglionic nerve (intermesenteric trunk). The beta4-specific antibody stained 59.8 +/- 7.5% of neurons and inhibited the synaptic responses in all (n = 10) neurons studied by 25.5 +/- 1.8%. The beta2-specific antibody did not stain ganglionic neurons and did not affect the synaptic transmission. Taking into account the previously obtained data on the alpha-subunits found in this ganglion, it is concluded that the neurons of inferior mesenteric ganglion contain nicotinic receptors of alpha3(alpha5)beta4 subtypes involved in synaptic transmission through the intermesenteric tract.
Neuroscience Letters 08/2004; 365(2):143-6. · 2.11 Impact Factor
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ABSTRACT: Comparative structure-function studies have been carried out for alpha-conotoxin GI acting on nicotinic acetylcholine receptors (AChR) from mammalian muscles and from the electric organ of the Torpedo californica ray and for alpha-conotoxin ImI, which targets the neuronal alpha7 AChR. A series of analogs has been prepared for this purpose: chemically modified derivatives, including a covalently linked dimer of GI, as well as analogs wherein one or several amino acid residues have been changed using solid-phase peptide synthesis. The activity of all compounds was assessed in competition with radioiodinated and/or tritiated alpha-conotoxin GI for binding to the membrane-bound AChR of Torpedo californica. Binding of radioiodinated alpha-conotoxin GI dimer was also monitored directly, revealing the largest, as compared to all other analogues, difference in the affinity between the two binding sites in the receptor (KD approximately 11 and 1200 nM). Comparison of binding data with the results of CD measurements point to important role of the spatial organization of the alpha-conotoxin second loop in manifestation of their "muscle" or "neuronal" specificity.
Neurochemical Research 05/2003; 28(3-4):599-606. · 2.24 Impact Factor
