ИСТИНА

Поиск ионных жидкостей, разобщающих дыхание и фосфорилирование в максимально широком диапазоне нетоксичных концентраций, а также изучение механизма токсического действия ионных жидкостей на митохондрии

Ionic liquids (IL) are traditionally called organic salts, the melting point of which is lower than the boiling point of water. Recently, they have become widely used in industry, biotechnology, environmentally friendly chemistry, in particular, as environmentally relatively harmless solvents. The low melting point of IL is due to the steric features of the structure. As a rule, this is the presence of several side alkyl chains in the IL cation, which complicates crystallization. We assume that the same feature of ILs can predetermine their ability to penetrate through biological membranes. Hydrophobic moieties can reduce the degree of hydration of the charged group, thereby facilitating passage across the membrane. We have previously shown that such a transition is a key electrogenic stage in the uncoupling cycle of a lipophilic uncoupler cation. Thus, it can be assumed that IL based on lipophilic cations can have uncoupling properties, that is, uncouple respiration and oxidative phosphorylation by increasing the electrical permeability of mitochondrial membranes. We assume that ILs with lipophilic cations can be used as non-toxic uncouplers. The action of uncouplers of respiration and oxidative phosphorylation occurs due to a decrease in the electrical resistance of mitochondrial membranes. The increased membrane potential of mitochondria can cause the formation of reactive oxygen species. Therefore, a slight decrease in the electrical resistance of the inner mitochondrial membranes can have an antioxidant effect. In addition, this reduction leads to the dissipation of nutrient energy into heat, which can be useful in the treatment of obesity, and also as a mimetic of calorie restriction. There have been several attempts to use uncouplers for therapeutic purposes. Thus, it has been shown that the lipophilic cation C12TPP (dodecyltriphenylphosphonium) prevents weight gain in mice fed a diet with a high fat content (DOI: 10.1038 / ijo.2016.146). The main problem is the narrow range of action of uncouplers, that is, a decrease in resistance in non-toxic concentrations. We recently developed an experimental system for rapid screening of dozens of substances for non-toxic uncoupling properties. The system is based on stimulating the growth of a yeast strain with a deletion of the tps1 gene. We have previously shown that a slight decrease in the intracellular ATP level in this strain stimulates its growth in the presence of glucose (doi: 10.3390 / biom10091345). According to our preliminary data, the range of non-toxic concentrations of several imidazole-based ILs exceeds that of C12TPP. We plan to test the IL based on imidazolium, phosphonium (partially done), pyridinium piperidinium and morpholinium in our system. The most promising ILs will be further tested for the ability to stimulate respiration (test for uncoupling ability) of yeast cells and, subsequently, mitochondria isolated from rat liver. According to our preliminary data, one of the phosphonium-based ILs stimulates cell growth in our experimental system, as well as the rate of cell respiration, which indicates its uncoupling activity. We also plan to study the mechanism of the toxic effect of IL on cells. It is believed that this effect is mediated by the action of IL on mitochondria, namely, a decrease in the respiration rate, which is confirmed by our preliminary data, and the formation of reactive oxygen species (ROS). It was suggested that imidazolium IL with an octyl alkyl residue is reduced by the respiratory chain of mitochondria, and thus ROS is generated. This seems unlikely to us, since the reduction potential of the respiratory chain, theoretically, is not high enough for electron transfer to the imidazole residue in the IL structure. We plan to evaluate IL-induced ROS production in the wild-type strain and in the mutant with inactivated respiratory chain. According to our preliminary data, a decrease in the respiratory rate with a high concentration of imidazolium IL does not occur if combined with the addition of uncouplers. This indicates that the decrease is mediated not by the inhibition of the respiratory chain, but by the inhibition of ATP synthetase. We plan to test this assumption experimentally by combining IL with known inhibitors of the respiratory chain or ATP synthetase. As a result of the work, we expect to obtain a general picture of the influence of IL on the cell energetics dependent on mitochondria.

Наша группа имеет многолетний опыт работы с использованием дрожжей S. cerevisiae как модельной системы. В последние годы мы опубликовали несколько работ по изучению эффектов разобщителей на дрожжи S. cerevisiae. Для данного проекта мы имеем обширные наработки по всем необходимым для успешного выполнения методам: микробиологическим, молекулярно-биологическим, микроскопическим, биохимическими. Имеем все необходимое оборудование (термостатируемые качалки, стерильные боксы, флуоресцентный микроскоп, охлаждаемые центрифуги, микропланшетный ридер и многое другое). Обладаем доступом к большой коллекцией химических веществ необходимых для выполнения проекта. Ранее нами на основе штамма дельта tps1 дрожжей Saccharomyces cerevisiae была разработана уникальная методика поиска нетоксичных веществ обладающих свойствами разобщителей дыхания и окислительного-фосфорилирования. Принцип метода состоит в том, что белок Tps1 нужен для регулирования скорости входа глюкозы в гликолиз, поэтому в штамме дельта tps1 происходит истощение клеточного фосфата, который, через АТФ тратится на фосфорилирование входящей в гликолиз глюкозы. Вещества разобщители помогают снизить синтез АТФ митохондриями и тем самым сохранить фосфат, который тогда будет потрачен в нижних этапах гликолиза и тем самым “метаболическая пробка” верхнего гликолиза будет просачиваться и исчезнет, когда вся глюкоза добавленная к клеткам будет истрачена. В результате, если мы наблюдаем, что добавление вещества в некоторой концентрации восстанавливает рост дельта tps1, остановленный добавлением глюкозы, то мы знаем, что вещество в такой концентрации одновременно и снижает эффективность митохондрий, и не является токсичным.По нашим предварительным данным, как минимум две из этих ИЖ ингибируют работу АТФ-синтетазы. Это наблюдение является новым в рамках исследования механизма токсичности ИЖ.