Tsetlin VI


Full name : Tsetlin Victor I

First name : Victor I

Mail : Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, UI. Miklukho-Maklaya, 161120 117871, GSP-7, Moscow V-437

Zip Code :

City :

Country : Russia

Email : victortsetlin3f@gmail.com

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Fax : 7 095 335 5733

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References (42)

Title : Three-finger proteins from snakes and humans acting on nicotinic receptors: Old and new - Tsetlin_2021_J.Neurochem_158_1223
Author(s) : Tsetlin VI , Kasheverov IE , Utkin YN
Ref : Journal of Neurochemistry , 158 :1223 , 2021
Abstract : The first toxin to give rise to the three-finger protein (TFP) family was alpha-bungarotoxin (alpha-Bgt) from Bungarus multicinctus krait venom. alpha-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, alphadelta-bungarotoxins differ from alpha-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia alpha-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas alpha-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as alpha-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits alpha7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its (1) H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same microM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.
ESTHER : Tsetlin_2021_J.Neurochem_158_1223
PubMedSearch : Tsetlin_2021_J.Neurochem_158_1223
PubMedID: 32648941

Title : Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System - Akimov_2020_Biomolecules_10_
Author(s) : Akimov MG , Kudryavtsev DS , Kryukova EV , Fomina-Ageeva EV , Zakharov SS , Gretskaya NM , Zinchenko GN , Serkov IV , Makhaeva GF , Boltneva NP , Kovaleva NV , Serebryakova OG , Lushchekina SV , Palikov VA , Palikova Y , Dyachenko IA , Kasheverov IE , Tsetlin VI , Bezuglov VV
Ref : Biomolecules , 10 : , 2020
Abstract : Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both alpha7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing alpha7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca(2+) rise, but inhibited the acetylcholine-evoked Ca(2+) rise with IC50 9 to 12 muM. In the A549 lung cancer cells, where alpha7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 +/- 1.5 muM and alphaKi = 51.4 +/- 4.1 muM for AChE and Ki = 70.5 +/- 6.3 muM and alphaKi = 214 +/- 17 muM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.
ESTHER : Akimov_2020_Biomolecules_10_
PubMedSearch : Akimov_2020_Biomolecules_10_
PubMedID: 32059521

Title : Human Secreted Ly-6\/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of alpha7 Nicotinic Acetylcholine Receptor - Lyukmanova_2016_PLoS.One_11_e0149733
Author(s) : Lyukmanova EN , Shulepko MA , Kudryavtsev D , Bychkov ML , Kulbatskii DS , Kasheverov IE , Astapova MV , Feofanov AV , Thomsen MS , Mikkelsen JD , Shenkarev ZO , Tsetlin VI , Dolgikh DA , Kirpichnikov MP
Ref : PLoS ONE , 11 :e0149733 , 2016
Abstract : SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-alpha7-nAChRs antibodies revealed alpha7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the alpha7-nAChRs. Exposure of Xenopus oocytes expressing alpha7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 muM). It was shown that rSLURP-1 binds to alpha7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-alpha-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with alpha7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of alpha7-nAChRs (mecamylamine, alpha-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of alpha-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through alpha7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.
ESTHER : Lyukmanova_2016_PLoS.One_11_e0149733
PubMedSearch : Lyukmanova_2016_PLoS.One_11_e0149733
PubMedID: 26905431

Title : Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors - Lyukmanova_2016_Toxicon_119_274
Author(s) : Lyukmanova EN , Shulepko MA , Shenkarev ZO , Kasheverov IE , Chugunov AO , Kulbatskii DS , Myshkin MY , Utkin YN , Efremov RG , Tsetlin VI , Arseniev AS , Kirpichnikov MP , Dolgikh DA
Ref : Toxicon , 119 :274 , 2016
Abstract : 'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-alpha-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human alpha7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at alpha7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.
ESTHER : Lyukmanova_2016_Toxicon_119_274
PubMedSearch : Lyukmanova_2016_Toxicon_119_274
PubMedID: 27343701

Title : Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors - Lyukmanova_2015_J.Biol.Chem_290_23616
Author(s) : Lyukmanova EN , Shenkarev ZO , Shulepko MA , Paramonov AS , Chugunov AO , Janickova H , Dolejsi E , Dolezal V , Utkin YN , Tsetlin VI , Arseniev AS , Efremov RG , Dolgikh DA , Kirpichnikov MP
Ref : Journal of Biological Chemistry , 290 :23616 , 2015
Abstract : Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
ESTHER : Lyukmanova_2015_J.Biol.Chem_290_23616
PubMedSearch : Lyukmanova_2015_J.Biol.Chem_290_23616
PubMedID: 26242733

Title : Cloning, synthesis, and characterization of alphaO-conotoxin GeXIVA, a potent alpha9alpha10 nicotinic acetylcholine receptor antagonist - Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
Author(s) : Luo S , Zhangsun D , Harvey PJ , Kaas Q , Wu Y , Zhu X , Hu Y , Li X , Tsetlin VI , Christensen S , Romero HK , McIntyre M , Dowell C , Baxter JC , Elmslie KS , Craik DJ , McIntosh JM
Ref : Proc Natl Acad Sci U S A , 112 :E4026 , 2015
Abstract : We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, alphaO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the alpha9alpha10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist alpha-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most alpha-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.
ESTHER : Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
PubMedSearch : Luo_2015_Proc.Natl.Acad.Sci.U.S.A_112_E4026
PubMedID: 26170295

Title : Quantitative proteomic analysis of Vietnamese krait venoms: Neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus - Ziganshin_2015_Toxicon_107_197
Author(s) : Ziganshin RH , Kovalchuk SI , Arapidi GP , Starkov VG , Hoang AN , Thi Nguyen TT , Nguyen KC , Shoibonov BB , Tsetlin VI , Utkin YN
Ref : Toxicon , 107 :197 , 2015
Abstract : Kraits are venomous snakes of genus Bungarus from family Elapidae. Krait venoms are generally neurotoxic, but toxicity strongly depends on the particular species and regional origin of snakes. We analyzed the proteomes of Vietnamese Bungarus multicinctus and Bungarus fasciatus venoms both qualitatively and quantitatively. It should be noted that no proteomic data for B. multicinctus venom existed so far. We have found that in this venom, almost half (45%) of the proteins by weight was represented by beta-bungarotoxins, followed by three finger toxins (28%) and phospholipases A2 (16%), other proteins being present at the level of 1-3%. In B. fasciatus venom, phospholipase A2 was the main component (71%), followed by oxidase of l-amino acids (8%), acetylcholinesterase (5%) and metalloproteinases (4%). Unexpectedly, extremely low amount of three finger toxins (1%) was found in this venom. Interestingly, the presence of complement depleting factor was observed in both venoms. Although our data showed the presence of the same toxin families in Vietnamese krait venoms as those found earlier in the venoms of kraits from other geographic regions, their relative ratio is completely different. This concerns especially B. fasciatus venom with predominant content of phospholipases A2 and very low amount of three finger toxins.
ESTHER : Ziganshin_2015_Toxicon_107_197
PubMedSearch : Ziganshin_2015_Toxicon_107_197
PubMedID: 26341420

Title : Human neuromodulator SLURP-1: bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution - Shulepko_2013_Biochemistry.(Mosc)_78_204
Author(s) : Shulepko MA , Lyukmanova EN , Paramonov AS , Lobas AA , Shenkarev ZO , Kasheverov IE , Dolgikh DA , Tsetlin VI , Arseniev AS , Kirpichnikov MP
Ref : Biochemistry (Mosc) , 78 :204 , 2013
Abstract : Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E. coli. The bacterial systems engineered for SLURP-1 expression as fused with thioredoxin and secretion with leader peptide STII failed in the production of milligram quantities of the protein. The SLURP-1 was produced with high-yield in the form of inclusion bodies, and different methods of the protein refolding were tested. Milligram quantities of recombinant SLURP-1 and its (15)N-labeled analog were obtained. The recombinant SLURP-1 competed with (125)I-alpha-bungarotoxin for binding to muscle-type Torpedo californica nAChR at micromolar concentrations, indicating a partial overlap in the binding sites for SLURP-1 and alpha-neurotoxins on the receptor surface. NMR study revealed conformational heterogeneity of SLURP-1 in aqueous solution, which was associated with cis-trans isomerization of the Tyr39-Pro40 peptide bond. The two structural forms of the protein have almost equal population in aqueous solution, and exchange process between them takes place with characteristic time of about 4 ms. Almost complete (1)H and (15)N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel beta-sheets formed from five beta-strands and closely resembles those of three-finger snake neurotoxins.
ESTHER : Shulepko_2013_Biochemistry.(Mosc)_78_204
PubMedSearch : Shulepko_2013_Biochemistry.(Mosc)_78_204
PubMedID: 23581991

Title : Water-soluble LYNX1 residues important for interaction with muscle-type and\/or neuronal nicotinic receptors - Lyukmanova_2013_J.Biol.Chem_288_15888
Author(s) : Lyukmanova EN , Shulepko MA , Buldakova SL , Kasheverov IE , Shenkarev ZO , Reshetnikov RV , Filkin SY , Kudryavtsev DS , Ojomoko LO , Kryukova EV , Dolgikh DA , Kirpichnikov MP , Bregestovski PD , Tsetlin VI
Ref : Journal of Biological Chemistry , 288 :15888 , 2013
Abstract : Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the alpha7 nAChR extracellular ligand-binding domain and the transmembrane domain of alpha1 glycine receptor (alpha7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at alpha7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on alpha7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for alpha-bungarotoxin and similar snake alpha-neurotoxins also targeting alpha7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.
ESTHER : Lyukmanova_2013_J.Biol.Chem_288_15888
PubMedSearch : Lyukmanova_2013_J.Biol.Chem_288_15888
PubMedID: 23585571

Title : The new peptide from the Fea's viper Azemiops feae venom interacts with nicotinic acetylcholine receptors -
Author(s) : Utkin YN , Weise C , Hoang NA , Kasheverov IE , Starkov VG , Tsetlin VI
Ref : Dokl Biochem Biophys , 442 :33 , 2012
PubMedID: 22419091

Title : Dimeric alpha-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors - Osipov_2012_J.Biol.Chem_287_6725
Author(s) : Osipov AV , Rucktooa P , Kasheverov IE , Filkin SY , Starkov VG , Andreeva TV , Sixma TK , Bertrand D , Utkin YN , Tsetlin VI
Ref : Journal of Biological Chemistry , 287 :6725 , 2012
Abstract : In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of alpha-cobratoxin (alphaCT-alphaCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the alphaCT-alphaCT crystal structure at 1.94 A where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric alpha-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the alphaCT-alphaCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the kappa-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the alpha7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents alpha7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in alphaCT-alphaCT considerably potentiates inhibition of the alpha3beta2 nAChR by the toxin. The heteromeric dimer of alpha-cobratoxin and cytotoxin has an activity similar to that of alphaCT-alphaCT against the alpha7 nAChR and is more active against alpha3beta2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by alpha7 nAChR, although it is less important for interaction of covalent TFT dimers with the alpha3beta2 nAChR.
ESTHER : Osipov_2012_J.Biol.Chem_287_6725
PubMedSearch : Osipov_2012_J.Biol.Chem_287_6725
PubMedID: 22223648

Title : Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor - Utkin_2012_J.Biol.Chem_287_27079
Author(s) : Utkin YN , Weise C , Kasheverov IE , Andreeva TV , Kryukova EV , Zhmak MN , Starkov VG , Hoang NA , Bertrand D , Ramerstorfer J , Sieghart W , Thompson AJ , Lummis SC , Tsetlin VI
Ref : Journal of Biological Chemistry , 287 :27079 , 2012
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 beta-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 alpha-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 +/- 0.03 mum) and with lower efficiency to human alpha7 nAChR (IC(50) 22 +/- 2 mum). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (alpha1beta1epsilondelta) than the fetal form (alpha1beta1gammadelta), EC(50) being 0.44 +/- 0.1 mum and 1.56 +/- 0.37 mum, respectively. The peptide had no effect on GABA(A) (alpha1beta3gamma2 or alpha2beta3gamma2) receptors at a concentration up to 100 mum or on 5-HT(3) receptors at a concentration up to 10 mum. 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.
ESTHER : Utkin_2012_J.Biol.Chem_287_27079
PubMedSearch : Utkin_2012_J.Biol.Chem_287_27079
PubMedID: 22613724

Title : [Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y] - Surin_2012_Bioorg.Khim_38_214
Author(s) : Surin AM , Kriukova EV , Strukov AS , Zhmak MN , Talka R , Tuominen R , Salminen O , Khiroug L , Kasheverov IE , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 38 :214 , 2012
Abstract : Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.
ESTHER : Surin_2012_Bioorg.Khim_38_214
PubMedSearch : Surin_2012_Bioorg.Khim_38_214
PubMedID: 22792725

Title : Design of new alpha-conotoxins: from computer modeling to synthesis of potent cholinergic compounds - Kasheverov_2011_Mar.Drugs_9_1698
Author(s) : Kasheverov IE , Zhmak MN , Khruschov AY , Tsetlin VI
Ref : Mar Drugs , 9 :1698 , 2011
Abstract : A series of 14 new analogs of alpha-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human alpha7 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 alpha-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 alpha7 nAChR, as found in competition with radioiodinated alpha-bungarotoxin. That is why we prepared radioiodinated derivatives of these alpha-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive alpha-conotoxins as compared to competition with radioactive alpha-bungarotoxin. Thus, radioiodinated alpha-conotoxins are a more sensitive tool for checking the activity of novel alpha-conotoxins and other compounds quickly dissociating from the receptor complexes.
ESTHER : Kasheverov_2011_Mar.Drugs_9_1698
PubMedSearch : Kasheverov_2011_Mar.Drugs_9_1698
PubMedID: 22072993

Title : An unusual phospholipase A(2) from puff adder Bitis arietans venom--a novel blocker of nicotinic acetylcholine receptors - Vulfius_2011_Toxicon_57_787
Author(s) : Vulfius CA , Gorbacheva EV , Starkov VG , Osipov AV , Kasheverov IE , Andreeva TV , Astashev ME , Tsetlin VI , Utkin YN
Ref : Toxicon , 57 :787 , 2011
Abstract : The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A(2) (PLA(2)s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca(2)+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [(1)(2)(5)I]iodinated alpha-bungarotoxin for binding to human alpha7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC(5)(0) being 20 +/- 1.5, 4.3 +/- 0.2, and 10.6 +/- 0.6 muM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC(5)(0) of 11.4 muM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA(2)s from snake venoms. Indeed, the protein possessed high PLA(2) activity, which was 1.95 mmol/min/mumol. Bitanarin is the first described PLA(2) that contains 14 disulphide bonds and the first nAChR blocker possessing PLA(2) activity.
ESTHER : Vulfius_2011_Toxicon_57_787
PubMedSearch : Vulfius_2011_Toxicon_57_787
PubMedID: 21333664

Title : [Bacterial expression of water-soluble domain of Lynx1, endogenic neuromodulator of human nicotinic acetylcholine receptors] - Shulepko_2011_Bioorg.Khim_37_609
Author(s) : Shulepko MA , Liukmanova EN , Kasheverov IE , Dolgikh DA , Tsetlin VI , Kirpichnikov MP
Ref : Bioorganicheskaia Khimiia , 37 :609 , 2011
Abstract : Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.
ESTHER : Shulepko_2011_Bioorg.Khim_37_609
PubMedSearch : Shulepko_2011_Bioorg.Khim_37_609
PubMedID: 22332355

Title : NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1 - Lyukmanova_2011_J.Biol.Chem_286_10618
Author(s) : Lyukmanova EN , Shenkarev ZO , Shulepko MA , Mineev KS , D'Hoedt D , Kasheverov IE , Filkin SY , Krivolapova AP , Janickova H , Dolezal V , Dolgikh DA , Arseniev AS , Bertrand D , Tsetlin VI , Kirpichnikov MP
Ref : Journal of Biological Chemistry , 286 :10618 , 2011
Abstract : Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake alpha-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 muM, ws-LYNX1 competed with (125)I-alpha-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing alpha7 nAChRs to 1 muM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on alpha4beta2 and alpha3beta2 nAChRs. Increasing ws-LYNX1 concentration to 10 muM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.
ESTHER : Lyukmanova_2011_J.Biol.Chem_286_10618
PubMedSearch : Lyukmanova_2011_J.Biol.Chem_286_10618
PubMedID: 21252236

Title : Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors - Mordvintsev_2009_FEBS.J_276_5065
Author(s) : Mordvintsev DY , Polyak YL , Rodionov DI , Jakubik J , Dolezal V , Karlsson E , Tsetlin VI , Utkin YN
Ref : Febs J , 276 :5065 , 2009
Abstract : Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain alpha-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.
ESTHER : Mordvintsev_2009_FEBS.J_276_5065
PubMedSearch : Mordvintsev_2009_FEBS.J_276_5065
PubMedID: 19682302

Title : Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors - Kasheverov_2009_Curr.Pharm.Des_15_2430
Author(s) : Kasheverov IE , Utkin YN , Tsetlin VI
Ref : Curr Pharm Des , 15 :2430 , 2009
Abstract : Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.
ESTHER : Kasheverov_2009_Curr.Pharm.Des_15_2430
PubMedSearch : Kasheverov_2009_Curr.Pharm.Des_15_2430
PubMedID: 19601841

Title : Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor - Kasheverov_2009_J.Neurochem_111_934
Author(s) : Kasheverov IE , Zhmak MN , Fish A , Rucktooa P , Khruschov AY , Osipov AV , Ziganshin RH , D'Hoedt D , Bertrand D , Sixma TK , Smit AB , Tsetlin VI
Ref : Journal of Neurochemistry , 111 :934 , 2009
Abstract : alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 microM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 microM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 microM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 microM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 microM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)> or = 90 microM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.
ESTHER : Kasheverov_2009_J.Neurochem_111_934
PubMedSearch : Kasheverov_2009_J.Neurochem_111_934
PubMedID: 19712060

Title : Bacterial production and refolding from inclusion bodies of a weak toxin, a disulfide rich protein - Lyukmanova_2009_Biochemistry.(Mosc)_74_1142
Author(s) : Lyukmanova EN , Shulepko MA , Tikhonov RV , Shenkarev ZO , Paramonov AS , Wulfson AN , Kasheverov IE , Ustich TL , Utkin YN , Arseniev AS , Tsetlin VI , Dolgikh DA , Kirpichnikov MP
Ref : Biochemistry (Mosc) , 74 :1142 , 2009
Abstract : The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.
ESTHER : Lyukmanova_2009_Biochemistry.(Mosc)_74_1142
PubMedSearch : Lyukmanova_2009_Biochemistry.(Mosc)_74_1142
PubMedID: 19916927

Title : Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification - Osipov_2008_J.Biol.Chem_283_14571
Author(s) : Osipov AV , Kasheverov IE , Makarova YV , Starkov VG , Vorontsova OV , Ziganshin R , Andreeva TV , Serebryakova MV , Benoit A , Hogg RC , Bertrand D , Tsetlin VI , Utkin YN
Ref : Journal of Biological Chemistry , 283 :14571 , 2008
Abstract : Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.
ESTHER : Osipov_2008_J.Biol.Chem_283_14571
PubMedSearch : Osipov_2008_J.Biol.Chem_283_14571
PubMedID: 18381281

Title : Bacterial expression, NMR, and electrophysiology analysis of chimeric short\/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors - Lyukmanova_2007_J.Biol.Chem_282_24784
Author(s) : Lyukmanova EN , Shenkarev ZO , Schulga AA , Ermolyuk YS , Mordvintsev DY , Utkin YN , Shoulepko MA , Hogg RC , Bertrand D , Dolgikh DA , Tsetlin VI , Kirpichnikov MP
Ref : Journal of Biological Chemistry , 282 :24784 , 2007
Abstract : Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.
ESTHER : Lyukmanova_2007_J.Biol.Chem_282_24784
PubMedSearch : Lyukmanova_2007_J.Biol.Chem_282_24784
PubMedID: 17576769

Title : Alpha-conotoxin analogs with enhanced affinity for nicotinic receptors and acetylcholine-binding proteins - Tsetlin_2006_J.Mol.Neurosci_30_77
Author(s) : Tsetlin VI , Kasheverov IE , Zhmak MN , Utkin YN , Vulfius CA , Smit AB , Bertrand D
Ref : Journal of Molecular Neuroscience , 30 :77 , 2006
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.
ESTHER : Tsetlin_2006_J.Mol.Neurosci_30_77
PubMedSearch : Tsetlin_2006_J.Mol.Neurosci_30_77
PubMedID: 17192636

Title : alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor - Kasheverov_2006_FEBS.J_273_1373
Author(s) : Kasheverov IE , Chiara DC , Zhmak MN , Maslennikov IV , Pashkov VS , Arseniev AS , Utkin YN , Cohen JB , Tsetlin VI
Ref : Febs J , 273 :1373 , 2006
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.
ESTHER : Kasheverov_2006_FEBS.J_273_1373
PubMedSearch : Kasheverov_2006_FEBS.J_273_1373
PubMedID: 16689926

Title : [Natural alpha-conotoxins and their synthetic analogues in studies of nicotinic acetylcholine receptors] - Kasheverov_2006_Bioorg.Khim_32_115
Author(s) : Kasheverov IE , Utkin Iu N , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 32 :115 , 2006
Abstract : alpha-Conotoxins, peptide neurotoxins from poisonous marine snails of the genus Conus that highly specifically block nicotinic acetylcholine receptors (AChRs) of various types, are reviewed. Preliminarily, the structural organization of AChRs of the muscular and neuronal types, their involvement in physiological processes, and their role in various diseases are briefly discussed. In this connection, the necessity of quantitative determination of AChR subtypes using neurotoxins and other approaches is substantiated. The chemical structure, spatial organization, and specificity of alpha-conotoxins are mainly discussed, taking into consideration the recent results on the ability of alpha-conotoxins to interact with muscular or neuronal hetero- and homooligomeric AChRs exhibiting a high species specificity. Particular emphasis is placed upon a thorough characterization of the surfaces of interaction of alpha-conotoxins with AChRs using synthetic analogues of alpha-conotoxins, mutations in AChRs, and pairwise mutations in both alpha-conotoxins and AChRs. The discovery in 2001 of the acetylcholine-binding protein from the pond snail Lymnaea stagnalis and the determination of its crystalline structure led to rapid progress in understanding the structural organization of ligand-binding domains of AChRs with which alpha-conotoxins also interact. We discuss the interaction of various alpha-conotoxins with acetylcholine-binding proteins, the recently reported X-ray structure of the complex of the acetylcholine-binding protein from Aplysia californica with the alpha-conotoxin analogue PnIA, and the application of this structure to the modeling of complexes of alpha-conotoxins with various AChRs.
ESTHER : Kasheverov_2006_Bioorg.Khim_32_115
PubMedSearch : Kasheverov_2006_Bioorg.Khim_32_115
PubMedID: 16637282

Title : Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors - Kasheverov_2006_FEBS.J_273_4470
Author(s) : Kasheverov IE , Zhmak MN , Vulfius CA , Gorbacheva EV , Mordvintsev DY , Utkin YN , van Elk R , Smit AB , Tsetlin VI
Ref : Febs J , 273 :4470 , 2006
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.
ESTHER : Kasheverov_2006_FEBS.J_273_4470
PubMedSearch : Kasheverov_2006_FEBS.J_273_4470
PubMedID: 16956365

Title : Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits - Shelukhina_2006_Biochemistry.(Mosc)_71_749
Author(s) : Shelukhina IV , Kryukova EV , Skok MV , Lykhmus EY , Zhmak MN , Mordvintsev DY , Kasheverov IE , Tsetlin VI
Ref : Biochemistry (Mosc) , 71 :749 , 2006
Abstract : We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.
ESTHER : Shelukhina_2006_Biochemistry.(Mosc)_71_749
PubMedSearch : Shelukhina_2006_Biochemistry.(Mosc)_71_749
PubMedID: 16903829

Title : A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins - Mordvintsev_2005_Comput.Biol.Chem_29_398
Author(s) : Mordvintsev DY , Polyak YL , Levtsova OV , Tourleigh YV , Kasheverov IE , Shaitan KV , Utkin YN , Tsetlin VI
Ref : Comput Biol Chem , 29 :398 , 2005
Abstract : Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.
ESTHER : Mordvintsev_2005_Comput.Biol.Chem_29_398
PubMedSearch : Mordvintsev_2005_Comput.Biol.Chem_29_398
PubMedID: 16290328

Title : Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist - Vulfius_2005_Neurosci.Lett_373_232
Author(s) : Vulfius CA , Tumina OB , Kasheverov IE , Utkin YN , Tsetlin VI
Ref : Neuroscience Letters , 373 :232 , 2005
Abstract : Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.
ESTHER : Vulfius_2005_Neurosci.Lett_373_232
PubMedSearch : Vulfius_2005_Neurosci.Lett_373_232
PubMedID: 15619549

Title : E. coli-expressed extracellular domain of rat 7 nicotinic acetylcholine receptor. -
Author(s) : Dergousova NI , Azeeva EA , Kryukova EV , Shibanova ED , Kasheverov IE , Korotina AS , Utkin YN , Tsetlin VI
Ref : Cholinergic Mechanisms, CRC Press :547 , 2004

Title : A comparative study on selectivity of alpha-conotoxins GI and ImI using their synthetic analogues and derivatives - Kasheverov_2003_Neurochem.Res_28_599
Author(s) : Kasheverov IE , Zhmak MN , Maslennikov IV , Utkin YN , Tsetlin VI
Ref : Neurochem Res , 28 :599 , 2003
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.
ESTHER : Kasheverov_2003_Neurochem.Res_28_599
PubMedSearch : Kasheverov_2003_Neurochem.Res_28_599
PubMedID: 12675150

Title : Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor - Tsetlin_2002_Eur.J.Biochem_269_2801
Author(s) : Tsetlin VI , Dergousova NI , Azeeva EA , Kryukova EV , Kudelina IA , Shibanova ED , Kasheverov IE , Methfessel C
Ref : European Journal of Biochemistry , 269 :2801 , 2002
Abstract : Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA 98, 3567-3570] and glutathione S-transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem. 276, 15810-15815]. However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm). Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.
ESTHER : Tsetlin_2002_Eur.J.Biochem_269_2801
PubMedSearch : Tsetlin_2002_Eur.J.Biochem_269_2801
PubMedID: 12047391

Title : [Photoactivatable analogues of alpha-conotoxins GI and MI and their interaction with nicotinic acetylcholine receptor] - Kasheverov_2002_Bioorg.Khim_28_101
Author(s) : Kasheverov IE , Rozhkova AM , Zhmak MN , Utkin Iu N , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 28 :101 , 2002
Abstract : Two photoactivatable analogues of alpha-conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, alpha-conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N alpha of Gly1 or N epsilon of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native alpha-conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [125I]Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR cross-linking. All the AChR subunits were found to be cross-linked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the alpha-conotoxin molecule.
ESTHER : Kasheverov_2002_Bioorg.Khim_28_101
PubMedSearch : Kasheverov_2002_Bioorg.Khim_28_101
PubMedID: 11962231

Title : Photoactivatable alpha-conotoxins reveal contacts with all subunits as well as antagonist-induced rearrangements in the Torpedo californica acetylcholine receptor - Kasheverov_2001_Eur.J.Biochem_268_3664
Author(s) : Kasheverov I , Rozhkova A , Zhmak M , Utkin Y , Ivanov V , Tsetlin VI
Ref : European Journal of Biochemistry , 268 :3664 , 2001
Abstract : Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of alpha-conotoxin MI and L-benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI were synthesized. All these compounds, similarly to native alpha-conotoxins, completely displaced the radioiodinated MI or GI from the membrane-bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated alpha-bungarotoxin (alphaBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross-linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native alpha-conotoxins or alphaBgt. Both equilibrium binding of benzophenone-derivatized alpha-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for alpha-conotoxins and alphaBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable alpha-conotoxins and (c) enhancement of benzophenone-derivatized alpha-conotoxins cross-linking at additional (physostigmine-related) sites by alphaBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.
ESTHER : Kasheverov_2001_Eur.J.Biochem_268_3664
PubMedSearch : Kasheverov_2001_Eur.J.Biochem_268_3664
PubMedID: 11432732

Title : [alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers] - Utkin_1999_Bioorg.Khim_25_805
Author(s) : Utkin Iu N , Kasheverov IE , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 25 :805 , 1999
Abstract : The review is devoted to the competitive blockers of different nicotinic acetylcholine receptors, alpha-neurotoxins from snake venoms, and alpha-conotoxins from marine snails of the Conidae family. The relationship between the structure and function of these toxins is discussed. Recent data on the mechanism of alpha-neurotoxin and alpha-conotoxin interaction with the nicotinic acetylcholine receptor are presented.
ESTHER : Utkin_1999_Bioorg.Khim_25_805
PubMedSearch : Utkin_1999_Bioorg.Khim_25_805
PubMedID: 10645484

Title : Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23 - Utkin_1998_Eur.J.Biochem_253_229
Author(s) : Utkin YN , Krivoshein AV , Davydov VL , Kasheverov IE , Franke P , Maslennikov IV , Arseniev AS , Hucho F , Tsetlin VI
Ref : European Journal of Biochemistry , 253 :229 , 1998
Abstract : Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.
ESTHER : Utkin_1998_Eur.J.Biochem_253_229
PubMedSearch : Utkin_1998_Eur.J.Biochem_253_229
PubMedID: 9578481

Title : [Effective binding of alpha-bungarotoxin with the solubilized alpha-subunit of Torpedo californica acetylcholine receptor] - Utkin_1996_Bioorg.Khim_22_387
Author(s) : Utkin Iu N , Mund M , Hucho F , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 22 :387 , 1996
Abstract : alpha-Subunit of the Torpedo californica nicotinic acetylcholine receptor was isolated by preparative SDS-PAGE followed by reversed-phase HPLC on a C4 column in an acetonitrile-isopropanol gradient in water. After removal of the organic solvents and solubilization in beta-octylglucoside, the purified alpha-subunit binds alpha-bungarotoxin with high affinity (Kd 28 nM).
ESTHER : Utkin_1996_Bioorg.Khim_22_387
PubMedSearch : Utkin_1996_Bioorg.Khim_22_387
PubMedID: 8929226

Title : [Tachykinin receptors in the rat brain bind alpha-bungarotoxin] - Utkin_1989_Bioorg.Khim_15_1030
Author(s) : Utkin Iu N , Lazakovich EM , Kasheverov IE , Arkhipova SF , Tsetlin VI
Ref : Bioorganicheskaia Khimiia , 15 :1030 , 1989
Abstract : alpha-Bungarotoxin was found to inhibit effectively the binding of 125I-labelled substance P and eledoisin to membrane and to solubilize preparations of the rat brain. Other postsynaptic neurotoxins exerted similar but less pronounced influence on the interaction of tachykinins with their receptors. The obtained results suggest that some alpha-bungarotoxin-binding polypeptides in brain are components of tachykinin receptors.
ESTHER : Utkin_1989_Bioorg.Khim_15_1030
PubMedSearch : Utkin_1989_Bioorg.Khim_15_1030
PubMedID: 2480130

Title : A study on the rat brain receptors for tachykinins and muramyl peptides -
Author(s) : Utkin YN , Lazakovich EM , Kaydalov AA , Kasheverov IE , Tsetlin VI
Ref : J Protein Chem , 8 :368 , 1989
PubMedID: 2477015

Title : Interaction surfaces of neurotoxins and acetylcholine receptor - Tsetlin_1982_Toxicon_20_83
Author(s) : Tsetlin VI , Karlsson E , Utkin YN , Pluzhnikov KA , Arseniev AS , Surin AM , Kondakov VV , Bystrov VF , Ivanov VT , Ovchinnikov Yu A
Ref : Toxicon , 20 :83 , 1982
Abstract : Binding of neurotoxin II Naja naja oxiana derivatives containing one spin label at various positions (Leu 1, Glu 2, Lys 15, Lys 25, Lys 26, His 31, Lys 44 and Lys 46) to purified solubilized acetylcholine receptor protein (AchR) from Torpedo marmorata was studied by EPR techniques. AchR interaction with several dansylated neurotoxin II derivatives was followed by difference fluorescence spectroscopy. A series of neurotoxin II p-azidobenzoyl derivatives were prepared and in three of them modified lysine residues were identified. In combination, spectroscopic data and photolabeling implicate a considerable area of the neurotoxin in association with AchR. Rigidity of the neurotoxin II conformation allowed to regard its binding surface as a mould of the AchR corresponding site and to estimate the minimal size of the latter. Conformation of the long-chain neurotoxins and their binding to AchR are briefly discussed basing on the 1H and 19F NMR studies of neurotoxin I Naja naja oxiana, toxin 3 Naja naja siamensis and its acetylated or trifluoroacetylated derivatives, as well as on Achr interaction with the derivatives spin labeled at Lys 27 and His 71.
ESTHER : Tsetlin_1982_Toxicon_20_83
PubMedSearch : Tsetlin_1982_Toxicon_20_83
PubMedID: 7080049

Title : EPR and fluorescence study of interaction of Naja naja oxiana neurotoxin II and its derivatives with acetylcholine receptor protein from Torpedo marmorata -
Author(s) : Tsetlin VI , Karlsson E , Arseniev AS , Utkin YN , Surin AM , Pashkov VS , Pluzhnikov KA , Ivanov VT , Bystrov VF , Ovchinnikov YA
Ref : FEBS Letters , 106 :47 , 1979
PubMedID: 227728