Бесплатный автореферат и диссертация по биологии на тему

Изучение влияния концевых структурных модификаций на функции цитохромов Р450 микросом млекопитающих

ВАК РФ 03.00.03, Молекулярная биология

Содержание диссертации, кандидата биологических наук, Жгун, Александр Александрович

I. Введение

II. Обзор литературы

2.1. Общая характеристика цитохромов Р

2.1.1. Краткая история открытия цитохромов Р

2.1.2. Общие свойства Р

2.2. Надсемейства Р450: номенклатура, эволюция, редокс партнеры

2.2.1. Протеомный индекс Р

2.2.2. Эволюция надсемейства Р

2.2.3. Компоненты Р450-зависимых систем и их эволюция

2.2.4. Р450 1А2 и 2В4: характеристики и взаимодействие

2.3. Каталитический цикл цитохромов Р

2.3.1. Организация простетической группы

2.3.2. Каталитический цикл

2.3.3. Типы реакций, катализируемые цитохромами Р

2.4. Пространственная организация цитохромов Р

2.4.1. Пространственная структура Р450саш

2.4.2. Пространственная структура CYP2C

2.4.3. Консервативные мотивы Р

2.4.4. Гипервариабельные районы Р450 (модель Гото и SRS)

2.5. Мембранная топология микросомальных Р

2.5.1. N-концевой якорный сегмент эукариотических Р

2.5.2. Пролин богатый район цитохромов Р

2.5.3. Взаимодействие Р450 с редокс партнерами

2.5.4. Транслокация и удержание Р450 в специфических компартментах

2.5.5. Олигомеризация цитохромов Р

2.6. Гетерологичная экспрессия микросомальных Р450 в клетках E.coli

2.6.1. Общие принципы прокариотической экспрессии Р

2.6.2. Гетерологичная экспрессия CYP1A2 и CYP2B4 в клетках E.coli

2.7. Цитохромы Р450 человека и их значение для организма

2.7.1. Индукция Р450 человека

2.7.2. Цитохромы Р450 лекарственного метаболизма человека

2.7.3. Полиморфизм цитохромов Р450 человека

2.7.4. Цитохромы Р450 и эндогенный метаболизм человека

2.8. Применение флуоресцентных методов для изучения белок-белковых 60 взаимодействий

III. Материалы и методы

3.1 Компьютерное моделирование модифицированных форм CYP1A

3.2 Материалы

3.2.1. Плазмидные вектора и штаммы микроорганизмов

3.2.2. Среды и реактивы

3.2.3. Синтетические олигинуклеотиды

3.3. Создание генно-инженерных конструкций

3.3.1. Получение конструкций для экспрессии СYP 2В4 в клетках Е. coli

3.3.2. Получение конструкций для экспрессии CYP1A2 в клетках E.coli и 68 S. cerevisiae

3.4. Методы исследования

3.4.1. Гетерологичная экспрессия цитохрома Р450 в клетках Е. coli

3.4.2. Фракционирование и очистка CYP2B4 и CYP1А2 из клеток E.coli

3.4.3. Экспрессия рекомбинантных белков в клетках S.cerevisiae и 72 получение микросом

3.4.4. Определение концентрации и подготовка к реакции компонентов

3.4.5. Определение О-деалкилирующей (-дебензилирующей) активности 75 рекомбинантных цитохромов Р450 в реакциях с 07-производными резоруфина

3.4.6. Определение взаимодействия компонентов МОС методом тушения 77 флуоресценции

3.4.7. Аналитические методы

IV. Результаты исследования

4.1. Выбор района для введения протеолитического сайта в молекулу CYP1A

4.2. Конструирование векторов и экспрессия модифицированных форм 86 CYP1A2 и 2В4 в клетках E.coli и S.cerevisiae

4.2.1. Плазмиды и "кассеты экспрессии"

4.2.2. Условия культивирования и уровни экспрессии

4.2.3. Анализ экспрессии рекомбинантных цитохромов методом 91 иммуноблоттинга

4.3. Очистка модифицированных форм CYP1А2 и 2В

4.5. Энзиматические свойства модифицированных форм CYP1A2 и 2В

4.5.1. Энзиматические свойства вариантов CYP2B

4.5.2. Энзиматические свойства вариантов CYP1А

4.5.3. Определение взаимодействия модифицированных форм Р450 1А2 с BMR методом тушения флуоресценции V. Обсуждение результатов

5.1. Концевые модификации

5.1.1. Концевые модификации для аффинного выделения

5.1.2. Концевая модификация для создания водорастворимого аналога Р

5.1.3. Гетерологичная экспрессия и очистка модифицированных микросомальных Р

5.2. Сопряжение микросомальных Р450 с BMR

5.3. Иммунологическое родство цитохромов Р450 1А2 человека и кролика

5.4. Сигмоидальная кинетика мутанта СYP1A2 Xa52(ALV) (AXA)

5.5. Эффективная экспрессия CYP2B4 под контролем Т7 промотора в клетках Е. coli

Выводы

Заключение Диссертация по теме "Молекулярная биология", Жгун, Александр Александрович

Выводы:

1. Получены вектора для экспрессии вариантов цитохромов Р450 1А2 человека и 2В4 кролика в штаммах Е. coli и S. cerevisiae. Модифицированные цитохромы несли: на N-конце - введенный сайт протеазы Ха на границе пролин богатого района (ПБР) и А-спирали либо домен GST, на С-конце -олигогистидиновые а.о. либо домен интеина. Оптимизированы условия экспрессии и очистки мутантных белков.

2. CYP1A2 и CYP2B4 с С-концевыми олигогистидиновыми остатками не отличаются по своим энзиматическим свойствам от природных белков. В то же время рекомбинантный фермент 2В4(Д2-27) с удаленным N-концевым сегментом обладает пониженной каталитической активностью. Показана принципиальная невозможность применения метода аффинной очистки С-концевых гибридов Р450 с интеином вследствие инактивации гемопротеи-на при индукции автопротеолиза гибрида.

3. Рекомбинантный аналог CYP1A2 с введенным сайтом узнавания фактора Ха на границе ПБР и А-спирали обладает измененными энзиматическими свойствами по сравнению с немодифицированным ферментом. При этом возникает новая субстратная специфичность в отношении 7-ПОРФ и 7-БОРФ и негиперболическая зависимость между скоростью реакции О-деметилирования и концентрацией субстрата 7-МОРФ.

4. Прокариотическая НАДФН-цитохром Р450 редуктаза BMR из B.megaterium способна выступать в качестве эффективного редокс-партнера CYP1A2 человека.

Работа поддержана грантом INTAS 96-1549 и ИНКО-КОПЕРНИКУС №

ERB3512PL965070 (контракт № ERBIC15CT960810).

Библиография Диссертация по биологии, кандидата биологических наук, Жгун, Александр Александрович, Москва

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232. Я крайне благодарен моему научному руководителю К.Г. Скрябину, принявшему самое непосредственное участие в моей научной судьбе.

233. Необходимо отдельно выразить самую глубокую благодарность H.A. Пе-тушковой, научившей меня работать с препаратами Р450, посвятившей в многочисленные тонкости их физико-химических измерений.

234. Настоящая работа посвящается моим дорогим родителям, без которых эта работа не была бы выполнена, Г. М. Щукиной и A.A. Жгуну старшему.