Лаборатория инженерии белка

Отдел биоинженерии

Руководитель: Долгих Дмитрий Александрович, д. б. н.
dolgikh@nmr.ru+7(495)336-80-11

www.nmr.ru/peg/

белковая инженерия, мембранные белки, de novo дизайн белковых молекул, структурно-функциональные исследования белков, получение и исследование природных и модифицированных рекомбинантных белков для медицины и биотехнологии

Лаборатория занимается конструированием и исследованием различных рекомбинантных  белков, прежде всего, представляющих интерес для медицины и биотехнологии. В частности, сотрудники изучают токсины, антитела, циткины, мембранные белки (рецепторы, транспортеры, ионные каналы и другие).

Группа Екатерины Люкмановой исследует «трехпетельные» белки человека семейства Ly6/uPAR и родственные им нейротоксины из яда змей. В центре внимания находятся молекулярные механизмы взаимодействия изучаемых белков с их рецепторами. Коллектив группы владеет передовыми технологиями рекомбинантной продукции «трехпетельных» белков, недоступных из природных источников. Это позволило впервые исследовать пространственную структуру и провести ряд функциональных исследований таких белков человека как Lynx1 (фактор регуляции нейропластичности), а также SLURP-1 и SLURP-2 (ауто/паракринные регуляторы эпителиальных клеток). Эти белки в будущем могут быть использованы в качестве прототипов для препаратов, улучшающих когнитивные функции, противораковых препаратов, а также лекарств, направленных на лечение ряда кожных заболеваний.

В настоящее время активно ведется исследование новых малоизученных «трехпетельных» белков Lypd6 и Lypd6B. Кроме того, группа сотрудничает с группой структурной биологии ионных каналов ИБХ РАН в исследовании потенциалозависимых ионных каналов человека.

Группа Теймура Алиева занимается дизайном и усовершенствованием (гуманизация, увеличение аффинности) рекомбинантных моноклональных антител, разработкой методов их экспрессии в клетках млекопитающих. Такие антитела могут быть использованы для терапии и диагностики аутоиммунных, онкологических и инфекционных заболеваний.

В Лаборатории получают антитела против вируса гриппа А с использованием иммуноглобулина А в попытке создать универсальное профилактическое средство против всех подтипов вируса А. Помимо этого, сотрудники получают уникальные антитела для создания средства профилактики и терапии на ранних стадиях лихорадки Эбола.

Кроме того, Лаборатория занимается созданием искусственных связывающих белков, обладающих функциями антител. Для отбора белков разрабатывается система клеточного дисплея белкового домена фибронектина. В частности, для этих целей используется белок внешней мембраны микроорганизма, выделенного из вечной мерзлоты, – аутотранспортер Psychrobacter cryohalolentis. Другим интересным объектом является протеородопсин Exiguobacterium sibiricum, впервые изученный сотрудниками Лаборатории. Совместно с Институтом структурной биологии в Гренобле исследована пространственная структура этого белка.

Еще одна группа под руководством Марине Гаспарян разработала и получила уникальный рецептор – селективный мутантный вариант противоопухолевого цитокина TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) – DR5-B, который на порядок эффективнее убивает опухолевые клетки по сравнению с TRAIL дикого типа как отдельно, так и в комбинации с химиопрепаратами. Препарат DR5-B в доклинических испытаниях показал отсутствие токсичности, а значит, может рассматриваться как эффективное средство для терапии опухолевых заболеваний различного происхождения.

Группа Риты Чертковой проводит исследования функциональных активностей белка-переносчика электронов – цитохрома с из различных организмов. С помощью мутагенеза удалось установить аминокислотные остатки, которые отвечают за проявление активности этого белка в апоптозе, а также остатки, отвечающие за связывание с белковыми редокс-партнерами, благодаря чему осуществляется перенос электронов в дыхательной цепи. Проводятся исследования механизмов меж- и внутримолекулярного электронного транспорта цитохрома с, результаты которых могут быть использованы для получения электронных устройств с молекулами белка в виде монослоя на проводящих поверхностях.

На протяжении многих лет в Лаборатории создаются и исследуются искусственные белки на основе альбебетина – первого искусственного белка, созданного под заданную структуру. В результате были получены варианты альбебетина, обладающие противовирусной, антипролиферативной и инсулин-подобной активностью. Кроме того, альбебетин и его биологически активные варианты использовали в качестве модельных белков для исследования закономерностей формирования амилоидных фибрилл, лежащих в основе болезни Альцгеймера.

Исследования проводятся в сотрудничестве с Биологическим факультетом МГУ, НИИ гриппаВсероссийским научным центром молекулярной диагностики и леченияИнститутом белка РАНИнститутом физико-химических и биологических проблем РАНПочвенным институтом им. В.В. ДокучаеваУниверситетом КопенгагенаУниверситетом Калифорнии в Ирвайне и др.

Лаборатория инженерии белка создана в 2002 году как самостоятельное научно-исследовательское структурное подразделение ИБХ РАН на основе лаборатории химии генов и группы белковой инженерии лаборатории спектрального анализа. Лабораторию основал академик Михаил Петрович Кирпичников, который сейчас является деканом Биологического факультета МГУ и руководителем отдела биоинженерии ИБХ РАН. За пионерские работы по конструированию искусственных белков с заданной структурой и биологической активностью, положившие начало развитию молекулярной биоинженерии в нашей стране, М.П.Кирпичникову и Д.А.Долгих была присуждена Государственная премия РФ в области науки и техники за 1999 г. (совместно с О.Б. Птициным и А.В. Финкельштейном из Института белка РАН).

Сейчас Лабораторией руководит профессор Дмитрий Александрович Долгих. Он специализируется в области структурно-функциональных исследований, дизайна и биоинженерии белков. Под его руководством сотрудники Лаборатории провели фундаментальные исследования структуры и функции целого ряда белков, представляющих первостепенный научный и научно-прикладной интерес, в том числе цитокинов, биологически активных искусственных белков, нейротоксинов, цитохрома с, ионных каналов, антител.

Лаборатория инженерии белка ИБХ РАН проводит фундаментальные и социально ориентированные прикладные исследования в области генетической и белковой инженерии, в том числе:

  • получение и исследование рекомбинантных белков, представляющих биомедицинский интерес; прежде всего, мембранных и мембранно-активных белков;
  • изучение структурно-функциональных отношений в белках;
  • конструирование de novo белков для исследования принципов их структурной организации;
  • создание новых, не существующих в природе белков, обладающих заданными полезными свойствами.

К основным достижениям лаборатории инженерии белка с момента ее создания относятся следующие работы:

  • На основе белка de novo альбебетина с заданной структурой были сконструированы искусственные белки, моделирующие противовирусный центр связывания aльфа2-интерферона человека и обладающие выраженной противовирусной активностью, сравнимой с активностью aльфа2-интерферона.
  • Была создана уникальная высокоэффективная экспрессирующая система для получения нейротоксина II из яда кобры Naja oxiana, в результате чего были проведены структурно-функциональные исследования и сконструированы мутантные варианты нейротоксина II c измененной специфичностью взаимодействия с рецепторами и предложена модель взаимодействия нейротоксина с липидным окружением рецептора.
  • Были сконструированы и получены мутантные варианты цитохрома с, лишенные апоптозной активности, но сохраняющие функцию переносчика электрона в дыхательной цепи и антиоксидантные свойства цитохрома с.
  • Была получена новая холодоактивная эстераза Грам-отрицательной бактерии Psychrobacter cryohalolentis, выделенной из вечной мерзлоты, определена ее пространственная структура и функциональные свойства.
  • Был получен бактериородопсин Exiguobacterium sibiricum – новый уникальный представитель семейства ретиналь-содержащих белков, осуществляющих трансмембранный перенос протонов, и выявлены основные этапы этого процесса, а также определена его пространственная структура и показано, что он представляет собой новый тип протонного насоса: его протон-акцепторный участок включает сопряженные остатки His57 и Asp85, а донором протонов для основания Шиффа является остаток лизина.
  • Были получены рекомбинантные нейромодуляторы Lynx1 и SLURP-1 человека – уникальные белки-регуляторы никотинового ацетилхолинового рецептора и проведено исследование их физико-химических свойств; была определена пространственная структура Lynx1.
  • Была разработана бесклеточная система синтеза интегральных мембранных белков, основанная на использовании мембраномиметиков (мицеллы детергентов, бицеллы, липосомы и липид-белковые нанодиски), которая позволяет получать структурированные и функционально активные мембранные белки. С помощью этой уникальной системы мы планируем в ближайшее время получить ряд мембранных белков, исследование которых позволит создавать новые высокоэффективные препараты для медицины и биотехнологии.
Ф.И.О.ДолжностьКонтакты
Долгих Дмитрий Александрович, д. б. н.рук. подр.dolgikh@nmr.ru+7(495)336-80-11
Вульфсон Андрей Николаевич, к. х. н.в.н.с.andreywulfson@mail.ru+7(495)330-72-74
Некрасов Алексей Норбертович, к. ф.-м. н.с.н.с.an_nekrasov@mail.ru
Гаспарян Марине Эдуардовна, к. б. н.с.н.с.marine_gasparian@yahoo.com+7(495)335-28-88
Черткова Рита Валерьевна, к. б. н.с.н.с.cherita@inbox.ru+7(495)335-28-88
Литвинов Иван Сергеевич, к. б. н.с.н.с.litvinov@mail.ibch.ru+7(495)3307265
Петровская Лада Евгеньевна, к. х. н.с.н.с.lpetr65@yahoo.com+7(495)330-69-83
Шингарова Людмила Николаевна, к. х. н.с.н.с.lshing@mx.ibch.ru+7(495)330-69-83
Шулепко Михаил Анатольевич, к. б. н.н.с.misha97@rambler.ru+7(495)330-69-83
Болдырева Елена Филипповнан.с.+7(495)330-66-29
Панина Анна Алексеевнан.с.+7()3306638
Яголович Анна Валерьевна, к. б. н.н.с.anne-gor2002@yandex.ru+7(926)3780155
Кульбацкий Дмитрий Сергеевичм.н.с.d.kulbatskiy@gmail.com
Топорова Виктория Александровнам.н.с.toporova-viktorija@rambler.ru+7(495)330-69-83
Балабашин Дмитрий Сергеевичм.н.с.
Гапизов Султан Шахбановичм.н.с.
Ларина Мария Викторовнам.н.с.
Бычков Максим Леонидовичасп.mlb@live.ru
Маркова Татьяна Геннадиевнатех.-лаб.+7()3307265

Ранее здесь работали:

Колосов Михаил Николаевич, академикрук. подр.
Люкманова Екатерина Назымовна, к. б. н.с.н.с.ekaterina-lyukmanova@yandex.ru
Шульга Александр Николаевичс.н.с.
Шульга Алексей Анатольевич, к. б. н.с.н.с.schulga@gmail.com
Зырянова Ирина Михайловна, к. б. н.н.с.zyrianova@yandex.ru
Гончарук Марина Валерьевна, к. б. н.н.с.m.s.goncharuk@gmail.com
Гончарук Сергей Александрович, к. б. н.н.с.ms.goncharuk@gmail.com
Брянцева Татьяна Владимировнам.н.с.tato-tato@yandex.ru
Ким асп.
Охрименко Иван студ.i.s.okhrimenko@yandex.ru
Нуреев Михаил инженерmnureev@mail.ru
Добрынина Татьяна Владимировнаинж.-иссл.

Избранные публикации

  1. Valieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. (2016). Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat. Struct. Mol. Biol. , [+]

    DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

    ID:1625
  2. Feldman T.B., Smitienko O.A., Shelaev I.V., Gostev F.E., Nekrasova O.V., Dolgikh D.A., Nadtochenko V.A., Kirpichnikov M.P., Ostrovsky M.A. (2016). Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin. J. Photochem. Photobiol. B, Biol. 164, 296–305 [+]

    Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

    ID:1612
  3. Siletsky S.A., Mamedov M.D., Lukashev E.P., Balashov S.P., Dolgikh D.A., Rubin A.B., Kirpichnikov M.P., Petrovskaya L.E. (2016). Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum. Biochim. Biophys. Acta 1857 (11), 1741–1750 [+]

    A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.

    ID:1662
  4. Thomsen M.S., Arvaniti M., Jensen M.M., Shulepko M.A., Dolgikh D.A., Pinborg L.H., Härtig W., Lyukmanova E.N., Mikkelsen J.D. (2016). Lynx1 and Aβ1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes. Neurobiol. Aging 46, 13–21 [+]

    Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease.

    ID:1667
  5. Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Y.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2016). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol , [+]

    Разработан комплексный подход к поиску, исследованию и конструированию  пептидных блокаторов калиевого канала Kv1.6. Подход основан на применении разработанной нами биоинженерной аналитической системы для изучения связывания блокаторов с гибридным каналом KcsA-Kv1.6 методом конфокальной микроскопии и молекулярного моделирования комплексов пептидных блокаторов с каналом Kv1.6. Используя разработанный подход, охарактеризована аффинность ряда пептидных блокаторов к каналу Kv1.6, построены молекулярные модели их комплексов, описан интерфейс взаимодействия и аминокислотные остатки, влияющие на селективность  взаимодействия блокаторов с каналом Kv1.6.

    ID:1611
  6. Pletneva N.V., Pletnev S., Pakhomov A.A., Chertkova R.V., Martynov V.I., Muslinkina L., Dauter Z., Pletnev V.Z. (2016). Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. Acta Crystallogr D Struct Biol 72 (Pt 8), 922–32 [+]

    The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

    ID:1587
  7. Petrovskaya L.E., NovototskayaVlasova K.A., Spirina E.V., Durdenko E.V., Lomakina G.Y., Zavialova M.G., Nikolaev E.N., Rivkina E.M. (2016). Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library. FEMS Microbiol. Ecol. 92 (5), fiw046 [+]

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

    ID:1668
  8. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    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-α7-nAChRs antibodies revealed α7 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 α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-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 α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

    ID:1420
  9. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561
  10. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Paramonov A.S., Chugunov A.O., Janickova H., Dolejsi E., Dolezal V., Utkin Y.N., Tsetlin V.I., Arseniev A.S., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2015). Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290 (39), 23616–30 [+]

    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.

    ID:1394
  11. Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (2015). Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells. Biochemistry Mosc. 80 (8), 1080–91 [+]

    Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

    ID:1482
  12. Bychkov M.L., Gasparian M.E., Dolgikh D.A., Kirpichnikov M.P. (2014). Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS ONE 9 (10), e109756 [+]

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.

    ID:1296
  13. Manni S., Mineev K.S., Usmanova D., Lyukmanova E.N., Shulepko M.A., Kirpichnikov M.P., Winter J., Matkovic M., Deupi X., Arseniev A.S., BallmerHofer K. (2014). Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation. Structure 22 (8), 1077–89 [+]

    Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

    ID:1104
  14. Shenkarev Z.O., Lyukmanova E.N., Butenko I.O., Petrovskaya L.E., Paramonov A.S., Shulepko M.A., Nekrasova O.V., Kirpichnikov M.P., Arseniev A.S. (2013). Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins. Biochim. Biophys. Acta 1828 (2), 776–84 [+]

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

    ID:802
  15. Lyukmanova E.N., Shenkarev Z.O., Khabibullina N.F., Kulbatskiy D.S., Shulepko M.A., Petrovskaya L.E., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P. (2012). N-terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems. Acta Naturae 4 (4), 58–64 [+]

    G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

    ID:801
  16. Ostapchenko V.G., Gasparian M.E., Kosinsky Y.A., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2012). Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit. J. Biomol. Struct. Dyn. 30 (1), 62–73 [+]

    Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

    ID:809
  17. Lyukmanova E.N., Shenkarev Z.O., Khabibullina N.F., Kopeina G.S., Shulepko M.A., Paramonov A.S., Mineev K.S., Tikhonov R.V., Shingarova L.N., Petrovskaya L.E., Dolgikh D.A., Arseniev A.S., Kirpichnikov M.P. (2011). Lipid-protein nanodisks for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes. Biochim. Biophys. Acta , [+]

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodisks (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodisks resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

    ID:541
  18. Bocharov E.V., Mayzel M.L., Volynsky P.E., Mineev K.S., Tkach E.N., Ermolyuk Y.S., Schulga A.A., Efremov R.G., Arseniev A.S. (2010). Left-handed dimer of EphA2 transmembrane domain: Helix packing diversity among receptor tyrosine kinases. Biophys. J. 98 (5), 881–9 [+]

    The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

    ID:320
  19. Volynsky P.E., Mineeva E.A., Goncharuk M.V., Ermolyuk Y.S., Arseniev A.S., Efremov R.G. (2010). Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane alpha-helical dimers: ephrin receptor A1. Phys Biol 7, 16014 [+]

    Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins.

    ID:312
  20. Goncharuk S.A., Shulga A.A., Ermolyuk Y.S., Kuzmichev P.K., Sobol V.A., Bocharov E.V., Chupin V.V., Arseniev A.S., Kirpichnikov M.P. (2009). Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. Biochemistry Mosc. 74 (12), 1344–9 [+]

    Описан эффективный способ получения мембранного белка KCNE3, а также его изотопно-меченых производных (15N-, 13C-), в количествах, достаточных для проведения структурно-функциональных исследований. Очищенный белковый препарат в составе мицелл различных детергентов охарактеризован методами динамического светорассеяния, КД-спектроскопии и ЯМР-спектроскопии. Показано, что в мицеллах DPC/LDAO белок находится в мономерной форме и принимает преимущественно альфа-спиральную конформацию. Наличие кросс-пиков от всех глицинов в 15N-HSQC–спектре ЯМР, а также относительно небольшая ширина линий (~20 Гц) подтверждают высокое качество полученного образца и возможность получения структурно-динамической информации о KCNE3 методом гетероядерной спектроскопии ЯМР высокого разрешения.

    ID:273
  21. Lesovoy D.M., Bocharov E.V., Lyukmanova E.N., Kosinsky Y.A., Shulepko M.A., Dolgikh D.A., Kirpichnikov M.P., Efremov R.G., Arseniev A.S. (2009). Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor. Biophys. J. 97 (7), 2089–97 [+]

    The action of three-finger snake alpha-neurotoxins at their targets, nicotinic acetylcholine receptors (nAChR), is widely studied because of its biological and pharmacological relevance. Most such studies deal only with ligands and receptor models; however, for many ligand/receptor systems the membrane environment may affect ligand binding. In this work we focused on binding of short-chain alpha-neurotoxin II (NTII) from Naja oxiana to the native-like lipid bilayer, and the possible role played by the membrane in delivering the toxin to nAChR. Experimental (NMR and mutagenesis) and molecular modeling (molecular-dynamics simulation) studies revealed a specific interaction of the toxin molecule with the phosphatidylserine headgroup of lipids, resulting in the proper topology of NTII on lipid bilayers favoring the attack of nAChR. Analysis of short-chain alpha-neurotoxins showed that most of them possess a high positive charge and sequence homology in the lipid-binding motif of NTII, implying that interaction with the membrane surrounding nAChR may be common for the toxin family.

    ID:319
  22. Schulga A.A., Mechev P.V., Kirpichnikov M.P., Skryabin K.G., Deyev S.M. (2009). Construction of the plasmid-free strain for human growth hormone production. Biochimie 128-129, 148–53 [+]

    The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

    ID:1669
  23. Bocharov E.V., Mayzel M.L., Volynsky P.E., Goncharuk M.V., Ermolyuk Y.S., Schulga A.A., Artemenko E.O., Efremov R.G., Arseniev A.S. (2008). Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1. J. Biol. Chem. 283 (43), 29385–95 [+]

    Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.

    ID:317
  24. Bocharov E.V., Mineev K.S., Volynsky P.E., Ermolyuk Y.S., Tkach E.N., Sobol A.G., Chupin V.V., Kirpichnikov M.P., Efremov R.G., Arseniev A.S. (2008). Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J. Biol. Chem. 283 (11), 6950–6 [+]

    Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

    ID:314

Долгих Дмитрий Александрович

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 33, комн. 424
  • Тел.: +7(495)336-80-11
  • Эл. почта: dolgikh@nmr.ru

Доклиническое исследование мутантного варианта цитокина TRAIL с повышенной селективностью к «рецептору смерти» DR5 (2016-03-29)

Гаспарян М.Э., Бычков М.Л., Яголович А.В., Долгих Д.А., Кирпичников М.П. (ИБХ)

Налобин Д.С., Калабушев С.Н., Ахаев Д.Н. (Биофак МГУ)

Проведено доклиническое исследование препарата на основе мутантного варианта противоопухолевого цитокина TRAIL, обладающего повышенной селективностью к рецептору смерти DR5. Вариант DR5-B отличается от белка TRAIL дикого типа шестью заменами; он связывается с рецептором DR5, проводящим сигнал апоптоза, столь же эффективно, что и белок дикого типа, но значительно хуже связывается с ингибирующими TRAIL-индуцируемый апоптозный сигнал рецепторами-ловушками. Проведенные нами эксперименты на клеточных культурах показали, что DR5-B убивает опухолевые клетки различного происхождения (как отдельно, так и в комбинации с химиопрепаратами) в 2-10 раз эффективнее, чем TRAIL дикого типа, который недавно прошедший клинические испытания в США и показал весьма ограниченный терапевтический эффект. Полученные результаты позволяют сделать вывод, что этот мутантный вариант (DR5-B) не токсичен и может стать эффективным препаратом для лечения многих видов рака, при которых наблюдается повышенная экспрессия рецептора DR5. (Совместно с лабораторией прототипирования и испытаний биотехнологических разработок Биологического факультета МГУ).

Библиография

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.

Публикации

  1. Gasparian M.E., Bychkov M.L., Yagolovich A.V., Dolgikh D.A., Kirpichnikov M.P. (2015). Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells. Biochemistry Mosc. 80 (8), 1080–91 [+]

    Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

    ID:1482
  2. Bychkov M.L., Gasparian M.E., Dolgikh D.A., Kirpichnikov M.P. (2014). Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4. PLoS ONE 9 (10), e109756 [+]

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.

    ID:1296