Мышкин Михаил Юрьевич

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
2007–2014 Московская обл., г.Долгопрудный МФТИ (ГУ) Бакалавриат, Магистратура
2014–2018 Московская обл., г.Долгопрудный МФТИ (ГУ) аспирантура

Избранные публикации

  1. Männikkö R., Shenkarev Z.O., Thor M.G., Berkut A.A., Myshkin M.Y., Paramonov A.S., Kulbatskii D.S., Kuzmin D.A., SampedroCastañeda M., King L., Wilson E.R., Lyukmanova E.N., Kirpichnikov M.P., Schorge S., Bosmans F., Hanna M.G., Kullmann D.M., Vassilevski A.A. (2018). Spider toxin inhibits gating pore currents underlying periodic paralysis. Proc. Natl. Acad. Sci. U.S.A. 115 (17), 4495–4500 [+]

    Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel Na1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

    ID:2084
  2. Paramonov A.S., Lyukmanova E.N., Myshkin M.Y., Shulepko M.A., Kulbatskii D.S., Petrosian N.S., Chugunov A.O., Dolgikh D.A., Kirpichnikov M.P., Arseniev A.S., Shenkarev Z.O. (2017). NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel. Biochim. Biophys. Acta 1859 (3), 493–506 [+]

    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 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 (13)C,(15)N-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 Mn(2+) titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. (15)N 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. These results validate structural studies of isolated VSDs of Na(+) channels and show possible pitfalls in application of this 'divide and conquer' approach.

    ID:1921
  3. Panteleev P.V., Myshkin M.Y., Shenkarev Z.O., Ovchinnikova T.V. (2017). Dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity. Biochem. Biophys. Res. Commun. 482 (4), 1320–1326 [+]

    The β-hairpin antimicrobial peptides arenicins from marine polychaeta Arenicola marina exhibit a broad spectrum of antimicrobial activity and high cytotoxicity. In this study the biological activities of arenicin-1 and its therapeutically valuable analog Ar-1[V8R] were investigated. The peptide Ar-1[V8R] displays significantly reduced cytotoxicity against mammalian cells relative to the wild-type arenicin-1. At the same time, both peptides exhibit similar antibacterial activities and kinetics of bacterial membrane permeabilization. Comparative NMR analysis of the peptides spatial structures in water and membrane-mimicking environment showed that Ar-1[V8R] in contrast to arenicin has significantly lower dimerization propensity. Thus, dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity.

    ID:1792
  4. Мышкин М.Ю., Парамонов А.С., Кульбацкий Д.С., Люкманова Е.Н., Кирпичников М.П., Шенкарёв З.О. (2017). ПОДХОД “РАЗДЕЛЯЙ И ВЛАСТВУЙ” ДЛЯ СТРУКТУРНЫХ ИССЛЕДОВАНИЙ МУЛЬТИДОМЕННЫХ ИОННЫХ КАНАЛОВ НА ПРИМЕРЕ ИЗОЛИРОВАННЫХ ПОТЕНЦИАЛ-ЧУВСТВИТЕЛЬНЫХ ДОМЕНОВ КАНАЛОВ Kv2.1 И Nav1.4 ЧЕЛОВЕКА1. Биоорг. хим. 43 (6), 608–619 [+]

    Потенциал-зависимые K+- и Na+-ионные каналы вовлечены в широкий спектр физиологических
    процессов, включая возбудимость сердечных, мышечных и нервных клеток, а также секрецию гор-
    монов и нейромедиаторов. Эти каналы имеют модульную структуру и состоят из пяти мембранных
    доменов: четырех потенциал-чувствительных доменов (ПЧД) и одного порового домена. На ПЧД раз-
    личных каналов локализованы уникальные сайты связывания с лигандами, поэтому ПЧД рассматри-
    ваются в качестве перспективных фармакологических мишеней. Модульная организация ионных ка-
    налов позволяет ставить задачи по структурным ЯМР-исследованиям изолированных ПЧД отдельно
    от поры. В настоящей работе рассмотрена возможность таких исследований на примере ПЧД канала
    Kv2.1 человека и первого ПЧД канала Nav1.4 человека. Разработаны сопряженные системы бескле-
    точного синтеза на основе бактериального экстракта S30 из E. coli, позволяющие получать милли-
    граммовые количества препаратов ПЧД, включая меченые стабильными изотопами аналоги. Важным
    этапом ЯМР-исследований является подбор мембраномоделирующей среды, обеспечивающей дол-
    говременную стабильность природной структуры мембранного белка в растворе и высокое качество
    ЯМР-спектров. Скрининг различных сред показал, что домены каналов Kv2.1 и Nav1.4 нестабильны
    в средах, содержащих фосфолипиды: мицеллах короткоцепочечного липида DC7PC и липид-детер-
    гентных бицеллах на основе цвиттер-ионных или анионных насыщенных липидов (DMPC и DMPG).
    Показано, что оптимальной средой для структурных ЯМР-исследований являются смеси цвиттер-
    ионного и слабокатионного детергентов (FOS-12/LDAO). Однако, несмотря на высокое качество
    спектров, образец ПЧД канала Nav1.4 в окружении FOS-12/LDAO необратимо агрегировал в течение
    нескольких дней. Вероятно, ПЧД K+- и Na+-каналов человека не являются полностью автономными
    мембранными доменами и для их стабилизации необходимы контакты с другими доменами канала.

    ID:1925
  5. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Kasheverov I.E., Chugunov A.O., Kulbatskii D.S., Myshkin M.Y., Utkin Y.N., Efremov R.G., Tsetlin V.I., Arseniev A.S., Kirpichnikov M.P., Dolgikh D.A. (2016). Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors. Toxicon 119, 274–9 [+]

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.

    ID:1598
  6. Berkut A.A., Peigneur S., Myshkin M.Y., Paramonov A.S., Lyukmanova E.N., Arseniev A.S., Grishin E.V., Tytgat J., Shenkarev Z.O., Vassilevski A.A. (2015). Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae. J. Biol. Chem. 290 (1), 492–504 [+]

    We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

    ID:1138