Внутриклеточные перемещения белков антиоксидантного комплекса. Новая система локализационных репортеров тема диссертации и автореферата по ВАК РФ 03.01.03, кандидат биологических наук Чумаков, Степан Петрович

• Чумаков, Степан Петрович
• кандидат биологических наук
• 2010

Оглавление диссертации кандидат биологических наук Чумаков, Степан Петрович

Оглавление.

Список используемых сокращений.

Глава 1. Организация и регуляция транспорта между цитоплазмой и ядром.

Введение диссертации (часть автореферата) на тему «Внутриклеточные перемещения белков антиоксидантного комплекса. Новая система локализационных репортеров»

Кариоферины и их роль в организации ядерно-цитоплазматического транспорта.7

ГТФаза Ran и Ran-зависимый транспорт.11

Транспорт рецепторов стероидных гормонов.16

Нукпеопорины, их строение и функции.17

Транспортные функции Nup98.22

Модели функционирования NPC.23

Транслокация трансмембранных белков на внутреннюю ядерную мембрану.27

Иерархическая регуляция ядерного транспорта.28

Регуляция на уровне отдельного груза.29

Внутримолекулярное маскирование NLS/NES.29

Межмолекулярное маскирование NLS/NES.30

Модуляция аффинности к кариоферинам посттранскрипционной модификацией.31

Модуляция аффинности к кариоферинам путем посттрансляционной модификации.32

Удержание в цитоплазме или ядре.32

Регуляция на уровне транспортных рецепторов.33

Модуляция экспрессии компонентов нукпеоцитоплазматической транспортной системы.33

Роль импортинов в гаметогенезе.34

Роль экспортинов в процессе развития организма.35

Регуляция на уровне компонентов NPC.36

Роль нукпеопоринов и других модуляторов ядерного транспорта в процессе развития.36

Роль нукпеопоринов в митозе.36

Роль нукпеопоринов в онкогенезе и развитии вирусной инфекции.37

Глава 2. Материалы и методы.39

Используемые клеточные линии, их культивирование и обработка.39

Селекция клеточных культур после трансдукции лентивирусными конструктами 40

Измерение концентрации белка по методу Бредфорд.40

Фракционирование белков в полиакриламидном геле.40

Перенос белков из гелей на фильтры и иммунодетекция.40

Получение компетентных клеток E.coli и трансформация.41

Препаративное и аналитическое выделение плазмидной ДНК.41

Обработка ДНК рестрикционными эндонукпеазами.42

Фракционирование и извлечение ДНК из агарозных гелей.42

Реакция лигирования.42

Трансфекция лентивирусных и плазмидных конструктов и получение клеточных линий.42

Иммунофлуоресценция.43

Определение активности (3-галактозидазы при помощи системы GalScreen®.44

Определение уровней внутриклеточных ROS на FACS при помощи DCF-DA.44

Получение экспрессионных лентивирусных конструктов.44

Этапы клонирования ш-конструкта.44

Этапы клонирования а-конструктов.49

Получение а-конструктов, экспрессирующих мутантные.50 варианты Sesn2 с перманентно-активированными и перманентнорепрессированными сайтами фосфорилирования.50

Получение конструктов, одновременно экспрессирующих несколько shPHK, специфичные к различным генам.52

Глава 3. Резульаты и обсуждение.54

Принцип действия и структура локализационного репортера.54

Конструирование вектора для экспрессии ш-пептида.55

Конструирование векторов для экспрессии белков, слитых с а-пептидом.56

Получение клонов oo-nuc-bleo.58

Отработка функционирования репортера на примере белка F0X03A.60

Влияние обработки hTNF и культивации в среде без сыворотки на изменение внутриклеточной локализации белка Sesn2.63

Перемещения Sesn2 в митохондрии и к плазматической мембране при окислительном стрессе.65

Влияние сульфиредоксина на передислокацию Sesn2.67

Переход Sesn2 в ядро под действием РМА.68

Исследование механизмов PMA-индуцированной передислокации Sesn2.70

Влияние сайтов фосфорилирования белковой молекулы Sesn2 на динамику ядерной транслокации.72

Заключение.75

Выводы.75

Список литературы.76

Список используемых сокращений

AFMAtomic Force MicroscopyАтомно-силовая микроскопия

Ala Alanine Алании

APC Anaphase Promoting Complex Циклосома

BSA Bovine Serum Albumin Бычий сывороточной альбумин

CKII Casein Kinase II Казеинкиназа II

CTE Constitutive Transport Element Конститутивный транспортный элемент

Cys Cysteine Цистеин

DCF 2,,7'-dichlorofluorescin 2',7'-дихлорфлуоресцин

DMEM Dulbecco's Modified Eagle's Среда Игла в модификации

Medium Дульбеко

DMSO Dimethyl Sulfoxide Диметилсульфоксид

DPI Diphenyleneiodonium chloride Дифенилиод хлорид

EBSS Earle's Balanced Salt Solution Сбалансированный солевой раствор

Эрла

EGF Epidermal Growth Factor Эпидермальный ростовой фактор

EGFP Enhanced Green Fluorescent Модифицированный зеленый

Protein флуоресцентный белок

EGFR Epidermal Growth Factor Receptor Рецептор эпидермального ростового фактора

ER Endoplasmic Reticulum localization Сигнал эндоплазматической signal локализации

FACS Fluorescence-Activated Cell Sorter Проточный цитофлуориметр

FCS Fetal Calf Serum Фетальная телячья сыворотка

FG Fenilalanine-Glycine Фенилапанин-глициновый домен; повтор)

FITC Fluorescein isothiocyanate Флуоресцин изотиоцианат

FRET Fluorescence Resonance Energy Резонансный перенос энергии

Transfer флуоресценции

GAG Group-specific antigen Антиген Групповой специфичности

GFP Green Fluorescent Protein Зеленый флуоресцентный белок

HIV Human Immunodeficiency Virus Вирус иммунодефицита человека hTNF Human Tumor Necrosis Factor Фактор некроза опухолей человека

IGF1 Insulin-like Growth Factor Инсулин-подобный фактор роста

INM Inner Nuclear Membrane Внутренняя ядерная мембрана

Kaps Karyopherins Кариоферины

LB Lysogeny Broth Лизогенный бульон

LTR Long Terminal Repeat Длинный концевой повтор

Mem Membrane localization signal Сигнал локализации на плазматической мембране

Mit Mitochondrial localization signal Сигнал митохондриапьной локализации

MOPS 3-(N-morpholino) propanesulfonic 3-(Ы-морфолино)пропансульфоновая acid кислота

NAC N-acetyl-cysteine N-346™ л-ци стеи н

NES Nuclear Export Signal Сигнал ядерного экспорта

NF-AT2 Nuclear factor of activated Ядерный фактор активированных

T-lymphocytes 2 Т-лимфоцитов N92

NLS Nuclear Localization Signal Сигнал ядерной локализации

NOX NADPH Oxidase 4 НАДФ-Н оксидаза

NPC Nuclear Pore Complex Комплекс белков ядерной поры

NTD N-Terminal Domain N-концевой домен

NUC Nuclear Localization Signal Сигнал ядерной локализации

Nup Nucleoporin Нукпеопорин

ONM Outer Nuclear Membrane Внешняя ядерная мембрана

PBS Phosphate Buffered Saline Фосфатный Буферный Раствор

Phe Phenylalanine Фенилаланин

PI3K Phosphoinositide 3-kinase Фосфатидилинозитол-З-киназа

PKA Protein Kinase A Протеинкиназа А

PKB Protein Kinase B Протеинкиназа В

PKC Protein Kinase C Протеинкиназа С

PMA Phorbol 12-myristate 13-acetate Форбол-12-миристрат-13-ацетат

PoM Pore Membrane Поровая мембрана

PTK Phosphotyrosin Kinase Фосфотирозинкиназа

PVDF Polyvinylidene Fluoride Фторопласт

RanBPI Ran-Binding Proteinl Ran-связывэющий белок1

RanGAP Ran GTPase Activating Protein Белок-активатор ГТФазы Ran

RanGEF Ran Guanidine Exchange Factor Фактор гуанидинового обмена Ran

Ran-GTP Ran-Guanidinediphosphate Ran-гуанидиндифосфат

Ran-GTP Ran-Guanidinetriphosphate Ran-гуанидинтрифосфат

Rev Revertase Ревертаза

ROS Reactive Oxygen Species Соединения активного кислорода

Ser Serine Серин shRNA Short hairpin RNA Короткая шпилечная РНК

SOB Super Optimal Broth Супер-оптимальный питательный бульон

TBS Tris-Buffered Solution Трис-буфер

Thr Threonine Треонин

TRITC Rhodamine Родамин

Tvr Tyrosine Тирозин

UTR Untranslated Region Нетранслируемая область мРНК

VSVG Vesicular Stomatitis Virus glycoprotein Гликопротеин Вируса Везикулярного Стоматита

ГР Глкжокортикоидный рецептор гяРНП Гетерогенная ядерная рибонуклеопротеиновая частица

ДНК Дезоксирибонуклеиновая кислота мРНК Матричная рибонуклеиновая кислота

НТФ Нукпеотидтрифосфат

ПЦР Полимеразная Цепная Реакция

РНК Рибонуклеиновая кислота тРНК Транспортная рибонуклеиновая кислота

ЭДТА Этилендиаминтетрауксусная кислота

Заключение диссертации по теме «Молекулярная биология», Чумаков, Степан Петрович

Выводы

1. Сконструирована репортерная система для определения внутриклеточных перемещений исследуемого белка, основанная на эффекте а-комплементации 3-галактозидазы

2. Получены клоны клеток линии RKO, стабильно экспрессирующие компоненты локализационной системы. Возможности репортерной системы испытаны на примере флуоресцентного белка GFP и транскрипционного фактора F0X03A.

3. При исследовании антиоксидантного белка Sesn2 установлено, что повышение внутриклеточных уровней ROS приводит к перемещению Sesn2 из цитоплазмы в ядро, что может способствовать защите генома от окислительных повреждений.

4. Установлено, что активация протеинкиназы С вызывает переход Sesn2 из цитоплазмы в ядро, а ее ингибирование предотвращает такой переход. Фосфорилирование сайтов РКС в структуре Sesn2 усиливает его переход в ядро в ответ на стресс, а предотвращение фосфорилирования по этим сайтам снижает количество Sesn2 в ядре.

5. Предложена модель, объясняющая механизм перемещения Sesn2 в ядро в ответ на внешние стимулы, Под влиянием активации мембранных рецепторов происходит стимуляция протеинкиназы С, которая, в свою очередь, стимулирует активность NOX и продукцию соединений активного кислорода. Одновременно протеинкиназа С стимулирует переход Sesn2 в ядро, тем самым настраивая ядерные ан-тиоксидантные системы перед ожидаемым повышением уровня соединений активного кислорода.

Заключение

Разработанная система локализационных репортеров является мощным инструментом для определения изменений внутриклеточной локализации белков. Широкий динамический диапазон и высокая чувствительность позволяют использовать локализационный репортер для определения небольших, относительно общего количества, перемещений исследуемого белка внутри клетки. Изучение внутриклеточной динамики белка Sesn2, являющегося компонентом клеточной защиты от переокисления, показало, что в ответ на окислительный стресс, комплекс белков, в который входит Sesn2, передислоцируется из цитоплазмы в ядро. Возможно, транслокация в ядро способствует повышению устойчивости компонентов ядра к окислению под действием сигнальных молекул Н202. Важную роль в процессе перехода белка Sesn2 из цитоплазмы в ядро играет протеинкиназа С. Sesn2 содержит три сайта фосфорилирования при помощи РКС. Фосфорилирование этих сайтов увеличивает интенсивность перехода Sesn2 из цитоплазмы в ядро в ответ на окислительный стресс, однако само по себе фосфорилирование недостаточно для транслокации Sesn2 в ядро. Весьма вероятно, что для перемещения требуется также фосфорилирование других белков, возможно входящих в комплекс с Sesn2. В настоящий момент детали процесса, приводящего к перемещению Sesn2 в ядро, неизвестны и требуют дальнейшего изучения.

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