danДоклады Национальной академии наук БеларусиDoklady of the National Academy of Sciences of Belarus1561-83232524-2431The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8323-2022-66-2-176-186dan-1052Research ArticleХИМИЯCHEMISTRYФизико-химические свойства Δ3−12 цистеин-обедненного цитохрома P450 3A4 с аминокислотной заменой S291CPhysical and chemical properties of Δ3−12 cysteine-depleted cytocrome P450 3A4 with amino acid substitution of S291CБритиковВ. В.BritikovV. V.

Бритиков Владимир Владимирович – научный сотрудник

ул. Купревича, 5/2, 220141, Минск

Britikov Vladimir V. – Researcher

5/2, Kuprevich Str., 220141, Minsk

britikov@iboch.byБритиковаЕ. В.BritikovaE. V.

Бритикова Елена Вячеславовна – научный сотрудник

ул. Купревича, 5/2, 220141, Минск

Britikova Elena V. – Researcher

5/2, Kuprevich Str., 220141, Minsk

britikova@iboch.byБочаровЭ. В.BocharovE. V.

Бочаров Эдуард Валерьевич – кандидат химических наук, старший научный сотрудник

ул. Миклухо-Маклая, 16/10, 117997, Москва

Bocharov Eduard V. – Ph. D. (Chemistry), Senior Researcher

16/10, Miklukho-Maklai Str., 117997, Moscow

edvbon@mail.ruБершацкийЯ. В.BershatskyYa. V.

Бершацкий Ярослав Витальевич – младший научный сотрудник

ул. Миклухо-Маклая, 16/10, 117997, Москва

Bershatsky Yaroslav V. – Junior Researcher

16/10, Miklukho-Maklai Str., 117997, Moscow

bershackyjaroslav@gmail.comКузьминаН. В.KuzminaN. V.

Кузьмина Наталья Викторовна – кандидат физико-математических наук, научный сотрудник

Ленинский пр., 33/2, 119071, Москва

Kuzmina Natalya V. – Ph. D. (Physics and Mathematics), Researcher

33/2, Leninsky Ave., 119071, Moscow

kuzmina-natsha@inbox.ruБойкоК. М.BoykoK. M.

Бойко Константин Михайлович – кандидат биологических наук, старший научный сотрудник

Ленинский пр., 31/4, 119071, Москва

Boyko Konstantin M. – Ph. D. (Вiology), Senior Researcher

31/4, Leninsky Ave., 119071, Moscow

boiko_konstantin@inbi.ras.ruУсановС. А.UsanovS. A.

Усанов Сергей Александрович – член-корреспондент, доктор химических наук, профессор

ул. Купревича, 5/2, 220141, Минск

Usanov Sergey A. – Corresponding Member, D. Sc. (Chemistry), Professor

5/2, Kuprevich Str., 220141, Minsk

usanov@iboch.byИнститут биоорганической химии Национальной академии наук БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of BelarusИнститут биоорганической химии имени академиков М. М. Шемякина и Ю. А. Овчинникова Российской академии наукShemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesФедеральный исследовательский центр «Фундаментальные основы биотехнологии» Российской академии наукResearch Center of Biotechnology of the Russian Academy of ScienceИнститут физической химии и электрохимии имени А. Н. Фрумкина Российской академии наукFrumkin Institute of Physical Chemistry and Electrochemistry of the Russian Academy of Sciences202206052022662176186Copyright © Бритиков В.В., Бритикова Е.В., Бочаров Э.В., Бершацкий Я.В., Кузьмина Н.В., Бойко К.М., Усанов С.А., 20222022Бритиков В.В., Бритикова Е.В., Бочаров Э.В., Бершацкий Я.В., Кузьмина Н.В., Бойко К.М., Усанов С.А.Britikov V.V., Britikova E.V., Bocharov E.V., Bershatsky Y.V., Kuzmina N.V., Boyko K.M., Usanov S.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://doklady.belnauka.by/jour/article/view/1052

Цитохром P450 3A4 (3A4) экспрессируется в клетках печени человека и играет ключевую роль в метаболизме ксенобиотиков, в том числе и более 50 % лекарственных препаратов. Регуляция активности данного фермента может происходить на уровне экспрессии генов, а также на уровне конформационного состояния структуры самого белка, за счет изменения молекулярного окружения, в том числе за счет взаимодействия с высокомолекулярными эффекторами. Понимание изменения структуры и динамики 3A4 в ответ на изменение условий среды необходимо для предсказания изменения уровня его активности, который в значительной степени обуславливает гомеостаз организма. Для проведения in vitro экспериментов по исследованию структуры, динамики и белоклигандных/белковых взаимодействий ферментов современными спектральными методами используется подход, в котором в целевой белок селективно вводятся методами белковой инженерии цистеиновые остатки в заданные локусы полипептидной цепи для последующего мечения специализированными молекулярными метками. Для этих целей в данной работе была получена мутантная форма мембрансвязанного (полноразмерного) рекомбинантного цитохрома P450 3A4 человека C58A/C64M/C98A/C239T/C377A/C468S/S291C. По данным спектроскопии кругового дихроизма нами было установлено, что введенные мутации не вызывают значимых изменений во вторичной структуре полученной формы 3A4, что свидетельствует о сохранении свернутости полипептидной цепи. Проведены спектрофотометрические измерения для сравнительного анализа изменения сродства к лигандам активного центра. Более того, нами было показано, что тестостерон гидроксилирующая активность в in vitro реконструированной системе для данной мутантной формы 3A4 многократно увеличивается относительно дикой формы фермента.

Cytochrome P450 3A4 (3A4) is highly expressed in the human liver cells and plays a decisive role in the metabolism of xenobiotics, including more than 50 % of medical products. The activity of this enzyme can be regulated at the expression level of genes, as well as at the conformation level of the structure of the protein itself, due to changes in the molecular environment, including due to the interaction with high-molecular effectors. The understanding of the structure changes and the 3A4 dynamics in response to the environmental changes is necessary to predict the changes in the level of its activity that to a considerable extent regulates the body’s homeostasis. To perform in vitro experiments on the structure, dynamics, and protein-ligand/protein interactions of the enzymes by the modern spectral methods, the approach is used, in which the target protein is selectively added with cysteine residues in the given polypeptide chain loci by the protein engineering methods for subsequent labeling with specialized molecular labels. To do this, the human mutant form of membrane-bound (full length) recombinant cytochrome P450 3A4 C58A/C64M/C98A/C239T/C377A/C468S/S291C was obtained. According to the circular dichroism spectroscopy data we established that the introduced mutations do not cause significant changes in the secondary structure of the obtained form 3A4, which shows the preservation of the folding of the peptide chain. The spectral photometric measurements were made to comparatively analyze the changes in the affinity to the ligands of the active center. Moreover, we showed that the testosterone hydroxylase activity in the in vitro reconstructed system for a given mutation form of 3A4 increases many times with respect to the wild form of the enzyme.

 

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The authors declare that there are no conflicts of interest present.