Bioengineering department

All publications (show selected)

Mikhail Kirpichnikov

Structures of the complexes of the extracellular domain a7-nAChR with neurotoxins a-Bgtx and WTX were determined by cryo-EM

Laboratory of structural biology of ion channels,  Group of in silico analysis of membrane proteins structure,  Laboratory of bioengineering of neuromodulators and neuroreceptors

Using cryo-electron microscopy, images of complexes of the a7-nAChR extracellular domain with neurotoxins a-Bgtx and WTX were obtained, which made it possible in both cases to determine the structures of the complexes with an average resolution of 3.5 A and 6.0 A, respectively. Additional data on the interaction of the WTX toxin with the a7-nAChR membrane environment and the receptor itself were obtained by NMR spectroscopy and site-directed mutagenesis. The data obtained allowed to build a model of the complex of the full-length a7-nAChR receptor with the WTX toxin in the membrane environment using computer modelling, which in turn made it possible to describe a new type of neurotoxin/receptor interaction, that was not previously described. In the work, a new method for confirmation of the receptor-ligand complex formation using fluorescently labeled ligands and size-exclusion chromatography was also developed.

Publications

  1. Kulbatskii DS, Shulepko MA, Sluchanko NN, Yablokov EO, Kamyshinsky RA, Chesnokov YM, Kirpichnikov MP, Lyukmanova EN (2020). Efficient screening of ligand-receptor complex formation using fluorescence labeling and size-exclusion chromatography. Biochem Biophys Res Commun ,

The role of natural mutations of the human protein SLURP-1 in the pathogenesis of Mal de Meleda skin disease has been determined

Laboratory of structural biology of ion channels,  Group of in silico analysis of membrane proteins structure,  Laboratory of bioengineering of neuromodulators and neuroreceptors

Mal de Meleda (MDM) is recessively inherited palmoplantar keratoderma associated with mutations in a gene encoding SLURP-1 protein. SLURP-1 controls growth, differentiation, and apoptosis of keratinocytes by interaction with α7 type nicotinic acetylcholine receptors (α7-nAChRs). SLURP-1 has a three-finger structure with β-structural core (‘head’) and three prolonged loops (fingers). To determine a role of SLURP-1 mutations, we produced 24 mutant variants of the protein with substitutions in different parts of the SLURP-1 molecule, including natural variants involved in MDM development. All mutants except R71/H/P/A, T52/A, R96/P, and L98/P were successfully refolded. Antiproliferative activity of the mutants was studied on Het-1A keratinocytes. Mutations in the loops I and III resulted in the partial or complete inactivation of SLURP-1, while majority of mutations in the loop II increased SLURP-1 antiproliferative activity. Substitutions of R96 and L98 residues located in the protein ‘head’ by alanine resulted in appearance of proapoptotic activity of SLURP-1. Our results agree with observed diversity of the MDM phenotypes. Using the obtained functional data, model of the SLURP-1/α7-nAChR complex was built. Our study provides new functional and structural information about a role of the SLURP-1 mutations in MDM pathogenesis and predicts new SLURP-1 variants, which could drive the disease.

Acid-sensitive channels ASIC1 are a molecular target of three-finger toxins from black mamba venom (Dendroaspis polylepis) - mambalgins. Mambalgins effectively inhibit homo- and heteromeric receptors containing the ASIC1a subunit; however, the possibility of their use as antitumor agents has not been previously studied. It was shown that ASIC1a mRNA is expressed in cells of chronic myelogenous leukemia and gliomas, but not in normal cells. Mambalgin-2 inhibited the growth of U251 MG and A172 glioma cells with EC50 in the nanomolar range, without affecting the proliferation of normal astrocytes. Interestingly, mambalgin-2 mutants did not affect the growth of glioma cells, indicating ASIC1a as the main molecular target of mambalgin-2 in glioma cells. Mambalgin-2 caused cell cycle arrest, inhibited phosphorylation of cyclin D1 and cyclin-dependent kinases (CDK), and induced apoptosis in glioma cells. In addition, mambalgin-2 inhibited the growth of the primary cell culture obtained from a patient with glioblastoma. Our data point on mambalgin-2 as a promising prototype of drugs for the targeted treatment of oncological diseases, and channels containing the ASIC1a subunit as a new target of targeted therapy.

The genetically modified receptor-selective variant DR5 of cytokine TRAIL demonstrated increased antitumor activity and an improved pharmacokinetic profile

Laboratory of molecular immunology,  Laboratory of protein engineering

A genetically modified receptor-selective variant DR5-B of the antitumor cytokine TRAIL was developed, which selectively binds only to the apoptosis-inducing death receptor DR5, without affinity for the receptor DR4 and decoy receptors DcR1, DcR2 and OPG. DR5-B showed increased proapoptotic activity in tumor cells both separately and in combination with chemotherapy drugs. DR5-B inhibited tumor growth in HCT116 and Caco-2 xenografts, and its efficacy was 2.5-3 times higher than wild type TRAIL. DR5-B also significantly increased the animal survival. The pharmacokinetic parameters of DR5-B were comparable to those of TRAIL, except that the half-life was 3.5 times higher. Thus, DR5-B can be considered as an effective agent for the treatment of tumor diseases.

New fluorescent probes based on hongotoxin for the search and studying potassium Kv1.1-and Kv1.3-channel blockers

Department of structural biology,  Laboratory of optical microscopy and spectroscopy of biomolecules,  Group of nanobioengineering

Using bioengineering methods, new genetically encoded fluorescent ligands have been obtained based on hongotoxin 1 and the red fluorescent protein tagRFP localized at the N- or C-terminus of the chimeric protein: RFP-L1-HgTx1 and HgTx1-L3-RFP.

The dissociation constants of the complexes of RFP-L1-HgTx1 and HgTx1-L3-RFP with the binding sites of the voltage-gated potassium channels Kv1.x (x = 1, 3) are in the nanomolar concentration range.

RFP-L1-HgTx1 and HgTx1-L3-RFP can be used as part of bioengineered cellular systems based on hybrid channels KcsA-Kv1.x (x = 1, 3) to search and study blockers of Kv1.1 and Kv1.3 channels that are of interest for the treatment of diseases accompanied by impaired activity of potassium channels.

The work was carried out with participation of A.L.Primak and M.A.Skutel, the students of the Faculty of Biology, Lomonosov Moscow State University. The study was supported by grant RSF 19-74-30014.

Caloric restriction triggers morphofunctional remodeling of astrocytes and enhances synaptic plasticity in the mouse hippocampus

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Extrasynaptic Signalling

Calorie-restricted (CR) diet has multiple beneficial effects on brain function. Here we report morphological and functional changes in hippocampal astrocytes in 3-months-old mice subjected to 1 month of the diet. Whole-cell patch-clamp recordings were performed in the CA1 stratum (str.) radiatum astrocytes of hippocampal slices. The cells were also loaded with fluorescent dye through the patch pipette. CR did not affect the number of astrocytic branches but increased the volume fraction (VF) of distal perisynaptic astrocytic leaflets. The astrocyte growth did not lead to a decrease in the cell input resistance, which may be attributed to a decrease in astrocyte coupling through the gap junctions. Western blotting revealed a decrease in the expression of Cx43 but not Cx30. Immunocytochemical analysis demonstrated a decrease in the density and size of Cx43 clusters. Cx30 cluster density did not change, while their size increased in the vicinity of astrocytic soma. CR shortened K+ and glutamate transporter currents in astrocytes in response to 5 × 50 Hz Schaffer collateral stimulation. However, no change in the expression of astrocytic glutamate transporter 1 (GLT-1) was observed, while the level of glutamine synthetase (GS) decreased. These findings suggest that enhanced enwrapping of synapses by the astrocytic leaflets reduces glutamate and K+ spillover. Reduced spillover led to a decreased contribution of extrasynaptic N2B containing N-methyl-D-aspartate receptors (NMDARs) to the tail of burst-induced EPSCs. The magnitude of long-term potentiation (LTP) in the glutamatergic CA3–CA1 synapses was significantly enhanced after CR. This enhancement was abolished by N2B-NMDARs antagonist. Our findings suggest that astrocytic morphofunctional remodeling is responsible for enhanced synaptic plasticity, which provides a basis for improved learning and memory reported after CR.

For the first time, toxin-binding site in the voltage-sensitive domain III of the human Nav1.4 channel is found

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Studying the interaction of ion channels with ligands modulating their activity is extremely important from the point of view of understanding the mechanisms of functioning and the development of new drugs for the treatment of different channelopaties. NMR spectroscopy was used to study the interaction of the voltage-sensitive domain from the third subunit of the human Na+ channel Nav1.4 (VSD-III) with the spider toxin Hm-3. Despite the fact that the presence of a toxin binding site on this domain was not previously assumed due to the interaction of the domain with the beta subunit of the channel, we for the first time revealed the presence of such site, and also evaluated the energetics of the interaction toxin/domain. It was shown that the affinity of Hm-3 for VSD-III is higher than for the “canonical” binding sites on the second and first channel domains.

 

Structures and dynamics of four human three-thinger proteins were determined by NMR

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of structural biology of ion channels

Three-finger proteins with a characteristic structure, including a Cys-stabilized β-structural core and three extended loops, are found in many vertebrates, as well as in arthropods and echinoderms. The most studied representatives of these proteins are snake neurotoxins. However, three-finger proteins from higher mammals, including humans, are of particular interest because they act as modulators of nicotinic acetylcholine receptors (nAChR).

A comprehensive analysis of the structural and dynamic properties of a number of human three-finger proteins acting on nAChRs was carried out using NMR methods. Among them both water-soluble proteins (SLURP-1, SLURP-2) and isolated three-finger LU domains of GPI-anchored proteins (Lypd6, Lypd6b, Lynx1, Lynx2), as well as snake neurotoxins (“weak” WTX toxin and neurotoxin NTII) were represented. For some proteins, the three-dimensional structure and dynamics were determined for the first time (SLURP-1, Lypd6, Lypd6b, Lynx2).

Two types of the β-structure topology were found: one wide β-sheet for Lypd6 and Lypd6b and two β-layers for other proteins. α-Helical segments, which previously were rarely observed in the three-finger proteins, were found in the loops I and III of Lypd6, Lypd6b, and Lynx2. The dependence of the modulator/receptor interaction mode from the spatial structure features, such as the distribution of charged and hydrophobic residues on the surface, has been shown. Significant conformational plasticity of three-finger proteins was shown: the loops were highly mobile at picosecond-nanosecond

Soluble variant of human Lynx1 positively modulates synaptic plasticity and ameliorates cognitive impairment associated with α7-nAChR dysfunction

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of structural biology of ion channels

Lynx1 is a GPI-tethered protein colocalized with nicotinic acetylcholine receptors (nAChRs) in the brain areas important for learning and memory. Previously we demonstrated that at low micromolar concentrations the water-soluble Lynx1 variant lacking GPI-anchor (ws-Lynx1) acts on α7-nAChRs as a positive allosteric modulator. We hypothesized that ws-Lynx1 could be used for improvement of cognitive processes dependent on nAChRs. Here we showed that 2 µM ws-Lynx1 increased the acetylcholine-evoked current at α7-nAChRs in the rat primary visual cortex L1 interneurons. At higher concentrations ws-Lynx1 inhibits α7-nAChRs expressed in X. laevis oocytes with IC50 ~50 µM. In C57BL/6 mice, ws-Lynx1 penetrated the blood-brain barrier upon intranasal administration and accumulated in the cortex, hippocampus, and cerebellum. Chronic ws-Lynx1 treatment prevented the olfactory memory and motor learning impairment induced by the α7-nAChRs inhibitor methyllycaconitine (MLA). Enhanced long-term potentiation (LTP) and increased paired-pulse facilitation ratio were observed in the hippocampal slices incubated with ws-Lynx1 and in the slices from ws-Lynx1-treated mice. LTP blockade observed in MLA-treated mice was abolished by ws-Lynx1 co-administration. To understand the mechanism of ws-Lynx1 action, we studied the interaction of ws-Lynx1 and MLA at α7-nAChRs, measured the basal concentrations of endogenous Lynx1 and the α7 nAChR subunit and their association in the mouse brain . Our findings suggest that endogenous Lynx1 limits α7-nAChRs activation in the adult brain. Ws-Lynx1 partially displaces Lynx1 causing positive modulation of α7-nAChRs and enhancement of synaptic plasticity. Ws-Lynx1 and similar compounds may constitute useful hits for treatment of cognitive deficits associated with the cholinergic system dysfunction.

Cytochrome C mutant variants immobilized on a gold surface as a monolayer provides a realistic path to protein-based bioelectronics

Laboratory of protein engineering

Abstract: A sample-type protein monolayer, that can be a stepping stone to practical devices, can behave as an electrically driven switch. This feat is achieved using a redox protein, cytochrome C (CytC), with its heme shielded from direct contact with the solid-state electrodes. Ab initio DFT calculations, carried out on the CytC–Au structure, show that the coupling of the heme, the origin of the protein frontier orbitals, to the electrodes is sufficiently weak to prevent Fermi level pinning. Thus, external bias can bring these orbitals in and out of resonance with the electrode. Using a cytochrome C mutant for direct S–Au bonding, approximately 80% of the Au–CytC–Au junctions show at greater than 0.5 V bias a clear conductance peak, consistent with resonant tunneling. The on–off change persists up to room temperature, demonstrating reversible, bias-controlled switching of a protein ensemble, which, with its built-in redundancy, provides a realistic path to protein-based bioelectronics.

Publications

  1. Cahen D, Fereiro JA, Kayser B, Romero-Muñiz C, Vilan A, Dolgikh DA, Chertkova RV, Cuevas JC, Zotti LA, Pecht I, Sheves M (2019). Solid-state Protein-based Reversible Biased-induced Tunneling Current Switch. Angew Chem Int Ed Engl 58 (34), 11852–11859

Nicotinic acetylcholine receptors (nAChR) play an important role in the physiology of epithelial cells, and their activation contributes to the development of carcinomas. Natural modulators of nicotinic acetylcholine receptors may become promising prototypes of new antitumor agents.

Recombinant analogs of human three-finger proteins ws-Lynx1 and rSLURP-1 were shown to inhibit the growth of lung carcinoma and melanoma cells. Ws-Lynx1 in A549 cells stimulates antiproliferative and proapoptotic signaling cascades associated with activation of α7-nAChR. rSLURP-1 inhibits nicotine-induced lung carcinoma cell growth, and also abolishes nicotine-induced increase in the α7-nAChR expression and decrease in the PTEN tumor suppressor gene expression. In addition, rSLURP-1 inhibits the growth of multicellular spheroids from cells of various carcinomas. The combined use of rSLURP-1 with other antitumor drugs (gefitinib, bortezomib, doxorubicin) leads to a complete stop in the growth of spheroids.

Thus, ws-Lynx1 and rSLURP-1 are promising prototypes for the development of new drugs for the cancer treatment.

Combinatorial selective incorporation of stable13C and 15N isotopes facilitates NMR spectra analysis and allows mapping of the binding interfaces between membrane receptors and their ligands

Group of in silico analysis of membrane proteins structure,  Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Combinatorial incorporation of stable 13C and 15N isotopes into protein molecules can significantly simplify the analysis of NMR spectra. For the first time, the problem was solved and the CombLabel algorithm was developed for calculating combinatorial 13C and 15N  labeling schemes with a minimum price. The application of the program allowed to assign 50% of the NMR signals of the backbone of the second voltage-sensing domain of human sodium channel Nav1.4 (VSD-II). Leak currents through mutant variants of Nav1.4 containing Arg675Gly mutation in VSD-II lead to the development of a hereditary disease – normokalemic periodic paralysis. Hm-3 toxin from the venom of spider Heriaeus melloteei is able to block leak currents in VSD-II. By the means of NMR spectroscopy the interaction interface between VSD-II and Hm-3 toxin was determined. According to the model of the VSD-II/Hm-3 complex, based on the NMR data, the toxin binds to the extracellular S1-S2 loop, destabilizing the state of the domain, at which leak currents are observed. Using the example of the complexes of Hm-3 toxin with VSD-I and VSD-II of the Nav1.4 channel, it has been shown that arachnid toxins can interact differently with different domains within the same sodium channel.

The physiological effect of two bisbenzylisoquinoline alkaloids having activity on ASIC1a

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Biological Testing,  Laboratory of neuroreceptors and neuroregulators

The ASIC1a is the most sensitive subtype of acid-sensing ion channel in the cell membrane, and it plays an important role in the excitation of neurons of CNS. Long time the ligands to this ASIC subtype are under intense attention for the development of drugs for pain relief, as well as protectors from strokes and neurodegenerative diseases. In in vitro experiments on heterologically expressed ASIC1a channels, the action of two bisbenzylisoquinoline alkaloids from plants was studied by electrophysiological method of two-electrode potential fixing on oocyte cells.

The alkaloid lindoldhamine extracted from the leaves of Laurus nobilis L. significantly inhibited the ASIC1a channel’s response to physiologically-relevant stimuli of pH 6.5–6.85 with IC50 range 150–9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund’s adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid. Thus, it was shown not only a prospective of plant alkaloids using for a pain relief, but was indirectly confirmed the involvement of the ASIC1a channels of the peripheral nervous system in the generation of a pain response to mild acidification.

The structural analogue named daurisoline, unlike lindoldamine, did not inhibit the activation of the ASIC1a channel by protons, but produced the second peak component of the ASIC1a current. This second peak manifested with a 2.5 seconds delay after the first fast respond followed by completely desensitization with the same kinetics as the main peak. The presence of second current components was specific characteristic of ASIC2 and ASIC3 subtypes early, but this component is sustained, that last all time while the acid stimulus presented. The discovery of the second component of ASIC1a current allows us to declare the common mechanism of opening and desensitization for all ASICs, which will be interesting to determine in further experiments.

Data of spFRET analysis support  the hypothesis that  anticancer drug curaxin, namely, its  CBL0137 derivative, can  affect long-distance enhancer-promoter communication (EPC) in chromatin by disrupting nucleosome structure or affecting the structure and dynamics of the linker DNA supporting efficient EPC (Kantidze et al., Nat Commun., 2019,10(1):1441). The data indicate also that CBL0137 attracts human FACT (protein factor that FAcilitates Chromatin Transcription) to nucleosomes, mediates hFACT-induced scaled, partially reversible nucleosome unfolding (or uncoiling of the nucleosomal DNA) and traps hFACT on nucleosomes. This curaxin-dependent FACT trapping can be a reason of hFACT redistribution from the transcribed chromatin regions to other genomic loci and contribute to the anticancer action of curaxins (Chang et al. Science Advances, 2018, 4 (11), eaav2131).

The studies were performed jointly with the specialists from the Institute of Gene Biology RAS (Kantidze O.L., Luzhin A.V., Golov A.K., Velichko A.K.),   Biology Faculty of Lomonosov Moscow State University (Valieva M.E., Lyubitelev A.V., Razin S.V.), Fox Chase Cancer Center, USA (Nizovtseva E.V., Studitsky V.M., Kulaeva O.I., Chang H.-W.), Roswell Park Comprehensive Cancer Center, USA (Gurova K.V., Safina A., Wang J.,), Eunice Kennedy Shriver National Institute for Child Health and Human Development, USA (Chereji R.V.), Rutgers University, USA (Morozov A.V.).

Publications

  1. Kantidze OL, Luzhin AV, Nizovtseva EV, Safina A, Valieva ME, Golov AK, Velichko AK, Lyubitelev AV, Feofanov AV, Gurova KV, Studitsky VM, Razin SV (2019). The anti-cancer drugs curaxins target spatial genome organization. Nat Commun 10 (1), 1441
  2. Chang HW, Valieva ME, Safina A, Chereji RV, Wang J, Kulaeva OI, Morozov AV, Kirpichnikov MP, Feofanov AV, Gurova KV, Studitsky VM (2018). Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins. Sci Adv 4 (11), eaav2131

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

Laboratory of optical microscopy and spectroscopy of biomolecules,  Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology,  Group of nanobioengineering

Оksana V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling), A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Functional studies of retinal protein from Exiguobacterium sibiricum (ESR)

Laboratory of protein engineering

A retinal protein from Exiguobacterium sibiricum (ESR) is a light-dependent proton pump which possesses a unique structural feature – a lysine residue at a site for the internal proton donor for the Schiff base. For the first time, ESR derivatives with synthetic retinal analogs were produced and studied. With the use of direct electrometric approach, the kinetics of light-induced transmembrane electrical potential difference generation in proteoliposomes reconstituted with ESR and its mutant variant K96A were examined. We have demonstrated that at neutral and alkaline pH wt ESR produces positive photoelectric response corresponding to the transfer of a proton to the medium. Mutation of K96 results in the lack of millisecond electrogenic phase related to the Schiff base reprotonation. As a result, we have shown that reduced efficiency of proton transport in K96A is explained not only by slowing of the photocycle but also by increased probability of back reactions.

In collaboration with Biological Faculty MSU, IBCP RAS and MIPT

Publications

  1. Siletsky SA, Mamedov MD, Lukashev EP, Balashov SP, Dolgikh DA, Rubin AB, Kirpichnikov MP, Petrovskaya LE (2018). Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR, a light-driven proton pump from Exiguobacterium sibiricum. BIOCHIM BIOPHYS ACTA 1860 (1), 1–11
  2. Belikov NE, Melnikova IA, Demina OV, Petrovskaya LE, Kryukova EA, Dolgikh DA, Kuzmichev PK, Chupin VV, Lukin AY, Shumsky AN, Chizhov I, Levin PP, Kirpichnikov MP, Varfolomeev SD, Khodonov AA (2018). The effect of the chromophoric group modification on the optical properties of retinal proteins. MENDELEEV COMMUN 28 (4), 406–408

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells.

The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action.

Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Laboratory of Molecular Instruments for Neurobiology is known for systematic study of Arthropods’ venoms and derived peptides that specifically target various ion channels. Scorpions’ venom is abundant with potassium channels (Kv) blockers, and this diversity was described in previously released in Kalium database.

In cooperation with Laboratory of optical microscopy and spectroscopy of biomolecules and Group of nanobioengineering an unique screening system permitted identification in the Mesobuthus eupeus scorpion venom of Kv1.2 blocker: peptide MeKTx11-1 binging with high affinity (IC50 ≈0,2 nM) and specificity (effect on Kv1.1, 1.3 and 1.6 emerges at >100-fold higher concentrations). This peptide differs from the related MeKTx11-3 by just two residues, possessing substantially lower Kv1.2-specificity.

Finally, Group of in silico analysis of membrane proteins structure conducted a molecular modeling study of these two peptides interacting with Kv1.2 channel, immersed into an explicit lipid bilayer. This study uncovered mechanism of selective action of MeKTx11-1 peptide. The developed analysis technique will be of use for future design of selective ligands of Kv and other channels, which may be applied in fundamental studies of molecular basis of nervous system function and as drugs prototypes.

A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules), О.V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Scorpion venom is rich in peptide blockers of voltage-gated potassium channels (KV), and we have reflected this diversity previously in Kalium, a database dedicated to such peptides. A high-affinity and selective blocker of KV1.2 channels, characteristic of the human central nervous system, was obtained from the venom of the scorpion Mesobuthus eupeus. Using molecular modeling and site-directed mutagenesis, the mechanism of selective interaction between the toxin and channels was investigated.

Efremenko A.V., Sharonov G.V., Feofanov A.V. (Laboratory of optical microscopy and spectroscopy of biomolecules), Lyukmanova E.N., Bychkov M.L., Shulepko M.A., Kulbatskii D.S., Dolgikh D.A., Kirpichnikov M.P. (Group of bioengineering of neuromodulators and neuroreceptors), Shenkarev Z.O. (Group of structural biology of ion channels).

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells. The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action. Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Interaction of gating modifier toxin Hm-3 with voltage-sensing domains of Nav1.4 sodium channel: structural view on the membrane-mediated binding

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of structural biology of ion channels

Voltage-gated Na+ channels (Nav) are essential for the functioning of cardiovascular, muscular, and nervous systems. Certain mutations trigger a leak current through voltage-sensing domains (VSDs) of Nav leading to various diseases. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the S4 segments of VSDs in the human skeletal muscle channel NaV1.4. The gating modifier toxin Hm-3 (crab spider Heriaeus melloteei) inhibits leak currents through such mutant channels. To investigate molecular basis of Hm-3 interaction with NaV1.4 channel, we studied isolated VSD-I by NMR spectroscopy in membrane mimicking environment. Hm-3/VSD-I complex was modeled using protein-protein docking guided by NMR restrains. The toxin initially anchors onto the membrane surface and then forms the complex with the S3b-S4 loop of the VSD-I. The Hm-3 binding blocks movement of the voltage-sensor helix S4 and induces some allosteric changes that prevent development of gating-pore currents. Our report is the first NMR study of structural interactions between gating modifier toxins and Nav channels.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Toxin from the venom of the crab spider Heriaeus melloteei may serve as a hit in drug discovery for hypokalemic periodic paralysis type 2; there is no reliable medication for all cases of this disease. It is caused by mutations in the gene encoding voltage-gated sodium channels NaV1.4, characteristic of skeletal muscles. As a result of the mutations, these channels conduct aberrant currents, the muscles are unable to respond to the signals of the nervous system, and weakness develops followed by paralysis. Hm-3 toxin was found to be able to selectively inhibit such currents through voltage-sensing domain I of mutant channels. Read more in the press release on the IBCh website.

Publications

  1. Männikkö R, Shenkarev ZO, Thor MG, Berkut AA, Myshkin MY, Paramonov AS, Kulbatskii DS, Kuzmin DA, Castañeda MS, King L, Wilson ER, Lyukmanova EN, Kirpichnikov MP, Schorge S, Bosmans F, Hanna MG, Kullmann DM, Vassilevski AA (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc Natl Acad Sci U S A 115 (17), 4495–4500

Fragment (76)PGTKMIFA(83) of horse cytochrome c plays a key role in its electron transport activity

Laboratory of protein engineering

The influence of conformation of the loop fragment P76GTKMIFA83 of horse cytochrome c on its electron transport activity was studied. Based on the information structure analysis, a number of variants of cytochrome c with multiple substitutions in the site P76GTKMIFA83, were obtained, aimed at lowering its conformational mobility. Succinate:cytochrome c-reductase and cytochrome c-oxidase activity of rat liver mitoplasts in the presence of cytochrome c variants was studied. It was shown that the ability to transfer electron of mutant variants has been decreased to different extent. According to the Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data, a decrease in the electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

Publications

  1. Chertkova RV, Brazhe NA, Bryantseva TV, Nekrasov AN, Dolgikh DA, Yusipovich AI, Sosnovtseva O, Maksimov GV, Rubin AB, Kirpichnikov MP (2017). New insight into the mechanism of mitochondrial cytochrome c function. PLoS One 12 (5), e0178280

An efficient method for production of recombinant α-КТх peptides – the blockers of potassium channels

Laboratory of optical microscopy and spectroscopy of biomolecules,  Group of nanobioengineering

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides: high yield of the target peptides (12-22 mg/l culture); retaining the native amino acid sequence of α-КТх peptides; high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds; simple and reliable procedure of peptide isolation and purification. The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical value.

AN EFFICIENT METHOD FOR PRODUCTION OF RECOMBINANT α-КТХ PEPTIDES – THE BLOCKERS OF POTASSIUM CHANNELS

Group of nanobioengineering,  Laboratory of optical microscopy and spectroscopy of biomolecules

O.V.Nekrasova, K.S.Kudryashova, S.A.Yakimov, M.P.Kirpichnikov

A.V.Feofanov

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides:

  • high yield of the target peptides (12-22 mg/l culture);
  • retaining the native amino acid sequence of α-КТх peptides;
  • high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds;
  • simple and reliable procedure of peptide isolation and purification.

The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical significance.

Secondary structure and dynamics of the voltage-sensing domain of second pseudosubunit of human skeletal muscle sodium channel Nav1.4

Laboratory of biomolecular NMR-spectroscopy,  Laboratory of biomolecular modeling,  Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of structural biology of ion channels

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues. This complexity significantly impedes structural studies of full-sized Na+ channels. The isolated voltage-sensing domain (VSD-II) of human skeletal muscle Nav1.4 channel was studied by NMR in membrane mimicking environment. Secondary structure of VSD-II showed similarity with the bacterial Na+ channels. Fragment of S4 helix between the first and second conserved Arg residues probably adopts 3/10-helical conformation. 15N-relaxation data revealed characteristic pattern of μs-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.

Development of integrated transcriptomic and proteomic approach to search for blockers of potassium channels in animal venoms

Group of nanobioengineering

Authors: 

Kuzmenkov A.I. , Vassilevski A.A., Grishin Eu.V.

Department of molecular neurobiology

Kudryashova K.S., Nekrasova O.V., Kirpichnikov M.P. 

Bioengineering Department  

Feofanov A.V. 

Laboratory of optical microscopy and spectroscopy of biomolecules

Annotation: 

An original approach was developed to search for new ligands of potassium channels. It combines the bioengineering cellular test system and transcriptomic and proteomic analysis of animal venoms. Using this approach eight high-affinity peptide blockers of voltage-gated potassium channel Kv1.1 (including five new peptides) were found in the venom of the scorpion Mesobuthus eupeus. The proposed approach is a versatile and effective tool for directed search for  blockers of potassium channels in natural venoms.

Preclinical studies of the cytokine TRAIL mutant variant possessing high selectivity to the “death receptor” DR5

Laboratory of protein engineering

Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (Institute of Bioorganic Chemistry);

Nalobin D.S., Kalabushev S.N., Ahaev D.N. (Biological Faculty, MSU)

A preclinical study of the mutant variant of the antitumor cytokine TRAIL, which has a higher selectivity for the death receptor DR5, has been done. Variant DR5-B differs from the wild-type TRAIL by six substitutions of amino acid residues; it binds to the apoptosis conductive signal receptor DR5 as efficiently as the wild-type protein, but its affinity to apoptosis inhibitory decoy receptors DcR1 and DcR2 is significantly lower in comparison to TRAIL. Our experiments in cell cultures demonstrated that DR5-B kills tumor cells of different origin (either alone or in combination with chemotherapy) 2-10 times more effectively than wild-type TRAIL, which has recently passed clinical trials in the United States and has shown very limited therapeutic effect. Our results suggest that the mutant version of TRAIL (DR5-B) is not toxic and can be apply for an effective treatment of different types of cancer in which there is increased expression of DR5 receptor. (In collaboration with the Laboratory of prototyping and testing of biotechnological development of the Biological Faculty of Moscow State University).

Bibliography

M.E.Gasparian, M.L.Bychkov, A.V.Yagolovich, D.A.Dolgikh, M.P.Kirpichnikov. Mutations enhancing selectivity of antitumor cytokine TRAIL to DR5 receptor increase its cytotoxicity against tumor cells. Biochemistry (Moscow), 2015, 80: 1080–1091.

Bychkov M.L., Gasparian, M.E., Dolgikh, D.A., Kirpichnikov, M.P. Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS One, 2014, 9, e109756.