Shenkarev ZO

References (10)

Title : NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel - Paramonov_2017_Biochim.Biophys.Acta_1859_493
Author(s) : Paramonov AS , Lyukmanova EN , Myshkin MY , Shulepko MA , Kulbatskii DS , Petrosian NS , Chugunov AO , Dolgikh DA , Kirpichnikov MP , Arseniev AS , Shenkarev ZO
Ref : Biochimica & Biophysica Acta , 1859 :493 , 2017
Abstract : Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The alpha-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15N relaxation data revealed characteristic pattern of mus-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels. These results validate structural studies of isolated VSDs of Na+ channels and show possible pitfalls in application of this 'divide and conquer' approach.
ESTHER : Paramonov_2017_Biochim.Biophys.Acta_1859_493
PubMedSearch : Paramonov_2017_Biochim.Biophys.Acta_1859_493
PubMedID: 28065835

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 : Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors - Lyukmanova_2016_Sci.Rep_6_30698
Author(s) : Lyukmanova EN , Shulepko MA , Shenkarev ZO , Bychkov ML , Paramonov AS , Chugunov AO , Kulbatskii DS , Arvaniti M , Dolejsi E , Schaer T , Arseniev AS , Efremov RG , Thomsen MS , Dolezal V , Bertrand D , Dolgikh DA , Kirpichnikov MP
Ref : Sci Rep , 6 :30698 , 2016
Abstract : Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the alpha3beta2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved beta-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at alpha4beta2 and alpha3beta2-nAChRs (IC50 ~0.17 and >3 muM, respectively) expressed in Xenopus oocytes. In contrast, at alpha7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 muM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with alpha3beta2-nAChRs, while it inhibited cell growth via alpha7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at alpha7 and alpha3beta2-nAChRs.
ESTHER : Lyukmanova_2016_Sci.Rep_6_30698
PubMedSearch : Lyukmanova_2016_Sci.Rep_6_30698
PubMedID: 27485575

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 : 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 : 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 : 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 : 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