Определение функциональной значимости взаимодействия катенина р120 с метил-ДНК связывающим белком Каизо тема диссертации и автореферата по ВАК РФ 03.00.26, кандидат биологических наук Айтхожина, Дана Сабировна

• Айтхожина, Дана Сабировна
• кандидат биологических наук
• 2006

Оглавление диссертации кандидат биологических наук Айтхожина, Дана Сабировна

Используемые сокращения.

Введение.

1. Обзор литературы.

1.1 Белки MBD семейства.

1.2. Белок Канзо и его особенности.

1.3 ДНК- связывающий домен цинковые пальцы.

1.4 р120- катенин и его особенности.

1.5 Белки NF-kB и IkB семейств: роль и регуляция.

1.6 Регуляция ядерного транспорта: NLS, NES и их регуляция.

1.7 Дрожжевая другибридная система.

2. Материалы и методы.

2.1 Методики.

2.2 Полимеразная цепная реакция.

2.3 Расщепление ДНК эндонуклеазами рестрикции.

2.4 Бактериальные штаммы и плазмиды.

2.5 Конструкции.

2.5.1 Конструкции, использованные в экспериментах по определению клеточной локализации Каизо.

2.5.2 Конструкции для поиска сигнала ядерной локализации.

2.5.3 Конструкции для дрожжевой двугибридной системы.

2.5.4 Конструкции, использованные в экспериментах по иммунопреципитации.

2.5.5 Получение конструкций, кодирующих функциональные домены Каизо с N- концевым FLAG и С- концевым НА пептидами.

2.5.6. Получение конструкций для эксперимента no in vitro фосфорилированию

Каизо.

2.6 Работа с дрожжами.

2.6.1 Культивирование дрожжей.

2.6.2 Получение дрожжевых компетентных клеток.

2.6.3 Трансформация дрожжевых компетентных клеток.

2.6.4 Маттинг.

2.7 Выделение ядерных белковых экстрактов.

2.8 Гель- фильтрационная хроматография.

2.9 Вестерн блот- гибридизация со специфическими антителами.

2.10 Иммунопреципитация и разделение белков в денатурирующем SDS- ПААГ.

2.11 Клеточные линии и трансфекции.

2.12 Иммунофлуоресцентный анализ.

2.13 Экспрессия рекомбинантных белков.

2.14 Эксперимент по задержке ДНК- белковых комплексов в геле.

2.15 Анализ статуса фосфорилирования белка Каизо.

2.15.1 Дефосфорилирование с помощью А,- фосфатазы.

2.15.2 Мечение белков 32Р in vivo.

2.15.3 Получение пептидов при помощи обработки трипсином.

2.15.4 Картирование фосфорилированных пептидов.

2.15.5 Анализ фосфорилирования аминокислот.

2.15.6 Анализ статуса фосфорилирования функциональных доменов белка

Каизо.

2.15.7. In vitro фосфорилирование Каизо.

2.16 Метилирование ДНК с помощью SssI- метилазы.

2.17 Тест на способность Каизо функционировать как метил- зависимый репрессор.

2.18 Тест на способность Каизо функционировать как метил- зависимый активатор.

3. Результаты и их обсуждение.

3.1 Каизо и р120 находятся в разных белковых комплексах в ядерных экстрактах клеток эритролейкемии человека К562.

3.2 Каизо подвергается пост- трансляционной модификации.

3.3 Каизо фосфорилирован по остаткам серина.

3.4 Каизо содержит 2 или 3 сайта фосфорилирования.

3.5 Каизо фосфорилирован по остаткам серина, расположенным в BTB/POZ домене.

3.6 МарКарКЗ киназа не фосфорилирует Каизо in vitro.

3.7 Фосфорилирование белка Каизо по остаткам серина, расположенным в BTB/POZ домене не препятствует связыванию Каизо с р120.

3.8 В дифференцированных клетках линии РС12 Каизо распределен по всей клетке.

3.9 Клеточная локализация Каизо регулируется ингибитором NF-kB (IkB).

3.10 Характеристика сигнала ядерной локализации Каизо.

3.11 Анализ взаимодействия Каизо с р120 в дрожжевой двугибридной системе.

3.12 р120 способен маскировать сигнал ядерной локализации белка Каизо.

3.13 Влияние р120 на функциональную активность белка Каизо.

3.14 Уровень и клеточная локализация катеннна р120 не изменены при генетическом нокауте Каизо.

4. Выводы.

Введение диссертации (часть автореферата) на тему «Определение функциональной значимости взаимодействия катенина р120 с метил-ДНК связывающим белком Каизо»

Одним из ключевых направлений молекулярной биологии является исследование строения и функций белков, связанных с злокачественным перерождением клеток. Поэтому изучение взаимодействия двух белков непосредственно вовлеченных в опухолеобразование кажется вдвойне интересным.

Р", у- катенины, а также р120- это семейство многофункциональных белков, содержащих в своем составе так называемый Arm- домен, состоящий из 10 или более Arm повторов. Эти белки входят в состав мультибелковых катенин- кадхериновых комплексов, основным составляющим которых является трансмембранный гликопротеин Е-кадхерин, обеспечивающий межклеточную систему адгезии эпителиальных клеток, посредством взаимодействий своих внеклеточных доменов. Нарушение функционирования этой системы, связанное с дефектами каких- либо компонентов связывают с метастатическим фенотипом в 50% случаев карцином человека. Ключевая роль катенина р120 заключается в его способности посттрансляционно стабилизировать Е- кадхерин и влиять на силу адгезии клеток, а также регулировать динамику цитоскелета.

Каизо, новый член семейства BTB/POZ домен содержащих транскрипционных факторов, был впервые охарактеризован как партнер р120 в дрожжевой двугибридной системе. У позвоночных, большинство BTB/POZ белков (HIC-1, BcI-6, PLZF, ZF5, MIZ-1) являются регуляторами транскрипции и ассоциируются либо с ростом, либо подавлением роста опухолей. Лучше всего это показано на примере Вс1-6- белка, прямо ответственного за возникновение лимфом В- клеток. Вс1-6 является транскрипционным репрессором различных генов, включая циклины D1 и D2.

Каизо также является репрессором транскрипции, причем Каизо специфически связывает метилированную ДНК посредством трех доменов «цинковые пальцы» С2Н2 типа. Как было показано мыши, с генетически удаленным геном Каизо приобретают устойчивость к возникновению рака кишечника.

Исходя из выше сказанного, возрастает важность изучения взаимодействия Каизо и катенина р120, особенно если предположить существование некоторого сигнального пути, ответственного за передачу сигналов с поверхности клетки в ядро, приводящего к выключению некоторых метилированных генов. Предположительно, не только Каизо с р 120, но и Е-кадхерин могут являться компонентами такого сигнального каскада. Таким образом, становиться понятно, что изучение подобного взаимодействия способно внести существенный вклад в понимание процессов как опухолеобразования, так и функционирования нормальной соматической/ эмбриональной клетки при транслировании эпигенетической информации.

1. Обзор литературы

Заключение диссертации по теме «Молекулярная генетика», Айтхожина, Дана Сабировна

4. Выводы

1. Определено, что связывание с катенином р120 препятствует взаимодействию Каизо с метилированной ДНК, и подавляет активацию транскрипции с метилированных матриц конструкцией Каизо УР16 без ВТВ.

2. Установлено, что для взаимодействия Каизо с р120 необходимы второй и третий домены цинковые пальцы С2Н2 типа.

3. Охарактеризован функциональный сигнал ядерной локализации, показано, что р120 катенин может маскировать этот сигнал.

4. Показано, что при трансфекции мутированной формы 1кВа в 61% клеток белок Каизо локализован в цитоплазме.

5. Определено, что Каизо является фосфобелком, и фосфорилируется по 2-м или 3-м остаткам серина, расположенным в ВТВ домене.

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