Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 55014 10.29039/rusjbpc.2022.0514 МЕДИЦИНСКАЯ БИОФИЗИКА И БИОФИЗИЧЕСКАЯ ХИМИЯ MEDICAL BIOPHYSICS AND BIOPHYSICAL CHEMISTRY МЕДИЦИНСКАЯ БИОФИЗИКА И БИОФИЗИЧЕСКАЯ ХИМИЯ STUDY ON THE AMYLOIDOIGENIC POTENTIAL OF NON-SPECIFIC YERSINIA PSEUDOTUBERCULOSIS PORINS Исследование амилоидоигенного потенциала неспецифических поринов Yersinia pseudotuberculosis Рыбинская Т. В. Rybinskaya T. V. tati2099.k@gmail.com Портнягина О. Ю. Portnyagina O. Yu. Зелепуга Е. А. Zelepuga E. A. Хоменко В. А. Khomenko V. A. Ким Н. Ю. Kim N. Yu. Чингизова Е. А. Chingizova E. A. Менчинская Е. С. Menchinskaya E. S. Глазунов В. П. Glazunov V. P. Чистюлин Д. К. Chistyulin D. K. cdk27@mail.ru Новикова О. Д. Novikova O. D. Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the RAS Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the RAS Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation Тихоокеанский институт биоорганической химии им. Г.Б. Елякова ДВО РАН Владивосток Россия G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences Vladivostok Russian Federation 25 06 2022 25 06 2022 7 2 273 279 20 06 2022 20 06 2022 https://rusjbpc.ru/en/nauka/article/55014/view

В работе рассматривается процесс образования и свойства амилоидоподобных агрегатов неспецифических поринов (OmpC и OmpF) наружной мембраны грамотрицательной бактерии Yersinia рseudotuberculos в кислой среде (рН 4,5) при повышенной температуре. Динамику формирования амилоидоподобных агрегатов поринов контролировали через две и четыре недели инкубации (при 42 ºС) и через 3-5 ч (при 90 ºС) с помощью окраски образцов амилоид-специфическим красителем тиофлавином Т, анализа спектров кругового дихроизма в дальней УФ-области, ИК-спектроскопии и конфокальной микроскопии. Обнаружено, что в случае OmpC порина инкубация в мягких условиях (42 ºС) приводит к обратимому накоплению α-спиральных участков в полипептидной цепи белка. В пространственной структуре OmpF порина в этих условиях существенных изменений не наблюдается, однако в жестких условиях (95 ºС) происходит формирование амилоидоподобных агрегатов, которые отличаются повышенным содержанием структуры β-листа. С помощью ИК-спектроскопии показано, что конформационная перестройка в молекуле OmpF порина связана с изменением количества и качества элементов β-структуры. По данным конфокальной микроскопии агрегаты исследованных неспецифических поринов можно рассматривать как промежуточные продукты амилоидогенного пути – олигомеры. Согласно литературным данным эти олигомеры, предшествующие образованию зрелых фибрилл, обладают мембранолитическими и цитотоксическими свойствами. Для прогретых образцов исследованных поринов при реконструкции в бислойные липидные мембраны не было обнаружено ни порообразующей, ни мембранолитической активности. По отношению к клеткам нейробластомы мыши Neuro-2a CCL-131™ полученные после инкубации агрегаты OmpF и OmpC поринов обладали более высокой токсичностью по сравнению с исходными образцами белков.

The paper considers the process of formation and properties of amyloid-like aggregates of outer membrane non-specific porins (OmpC and OmpF) of the gram-negative bacterium Yersinia pseudotuberculos in an acidic medium (pH 4.5) at elevated temperature. The dynamics of the formation of amyloid-like aggregates of porins was monitored after two and four weeks of incubation (at 42 °C) and after 3-5 hours (at 90 °C) by staining the samples with amyloid-specific dye thioflavin T, analyzing the spectra of circular dichroism in the far UV region, IR -spectroscopy and confocal microscopy. It was found that in the case of porin OmpC, incubation under mild conditions (42°C) leads to a reversible accumulation of α-helical regions in the protein polypeptide chain. No significant changes are observed in the spatial structure of OmpF porin under these conditions, however, under harsh conditions (95 ºC) amyloid-like aggregates are formed, which are characterized by an increased content of the β-sheet structure. Using IR spectroscopy, it was shown that the conformational rearrangement in the molecule of OmpF porin is associated with a change in the quantity and quality of elements of the β-structure. According to confocal microscopy, the aggregates of the studied non-specific porins can be considered as intermediate products of the amyloidogenic pathway - oligomers. According to the literature data, these oligomers, which precede the formation of mature fibrils, have membranolytic and cytotoxic properties. For heated samples of the studied porins during reconstitution into bilayer lipid membranes, neither pore-forming nor membranolytic activity was detected. With respect to Neuro-2a CCL-131™ mouse neuroblastoma cells, the aggregates of OmpF and OmpC porins obtained after incubation had a higher toxicity compared to the initial protein samples.

 порины грамотрицательных бактерий олигомеры амилоидные фибриллы взаимодействие с тиофлавином Т цитотоксическая активность porins of gram-negative bacteria oligomers amyloid fibrils interaction with thioflavin T cytotoxic activity

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