项目来源

俄罗斯科学基金(RSF)

项目主持人

Zhivotovsky Boris

项目受资助机构

Federal State Budgetary Educational Institution of Higher Education Lomonosov Moscow State University

项目编号

19-15-00125

立项年度

2019

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

未知

学科

未公开

学科代码

未公开

基金类别

未公开

关键词

Cell death ; caspases ; mitotic catastrophe ; evolution ; cancer

参与者

未公开

参与机构

未公开

项目标书摘要：nnotationThe phenomenon of programmed cell death (PCD) is characteristic of all living organisms. It is compulsory for the development of embryos and in an adult organism to maintain the homeostasis of all tissues and organs. It is obvious that the mechanism responsible for the regulation of cell death is equally important for the organism, as are the mechanisms responsible for cell division and differentiation. Disturbances in the functioning of any of these mechanisms lead to the emergence of a large number of diseases. One of the causes of the emergence and course of one of the most important and most terrible diseases - cancer - is the inability of the organism to destroy pathological tumor cells. It is also important to note that if the PCD phenomenon is one of several physiological alterations leading to the onset and development of a tumor, then this phenomenon is the only one responsible for the elimination of tumor cells. The investigation of various aspects of cell death is the fastest growing area of biomedicine and, as a result, the only area of biomedicine that awarded two Nobel Prizes in Physiology or Medicine in the 21st century (2002 and 2016).The most studied form of PCD is apoptosis. The caspase family of cysteine proteases plays an important role in the initiation and development of apoptosis. Currently, 13 proteins of this family have been identified, of which 11 are in humans. Proteins of this family have also been found in nematodes (2 proteins), in Drosophila (6 proteins) and other organisms. It is obvious that the number of caspases in different organisms grows in proportion to the complexity of the latter. In addition to participating in the development of apoptosis, caspases are involved in the regulation of the inflammatory response of the cell, as well as in cell differentiation. Unfortunately, the evolutionary role of caspases is not clear. In this project, the evolution of caspases will be analysed for the first time on the basis of the Markov modeling and an analysis of the changes in all known physicochemical properties of their amino acids will be carried out. A unique feature of the system will be the ability to directly correlate the calculated features of molecular evolution with the evolution of given phenotypic traits of organisms and, thus, it will be easy to interpret the obtained results. In addition, with the help of the created caspase evolution system, a correlation analysis will be carried out, which will allow to identify new functional features of the enzymes, as well as to establish the role of previously little-investigated caspases in various PCD processes.The first caspase found in humans was caspase-2, which, however, remains the least studied. We and our foreign colleagues have established that caspase-2, as a member of the caspase family, has both pro-apoptotic and tumor-suppressive functions, as well as functions to maintain genetic stability, and plays a key role in the activation of apoptosis in cells in response to genotoxic stress. Recent studies have demonstrated its role in the elimination of potentially dangerous oncogenic aneuploid cells, resulting from disruption of the cell division process. However, the detailed mechanisms for the activation and functioning of caspase-2 remain unclear and require further investigation. In the course of the previous project, supported by the RSF, we obtained new data concerning both the apoptotic and non-apoptotic functions of this protein, described a new platform for its activation, obtained heterozygotes of mice carrying wild-type genes and genes with deletions in caspase-2. In this project, we are planning to investigate homozygote caspase-2 knockout mice. We will analyze the phenotype of caspase-2 knockout mice, the effect of the lack of this protein on the reproductive, nervous and immune systems.Specific objectives of this project include (see also file in appendix):1. Molecular phylogenetic analysis of caspases and the construction of phylogenetic trees, which will establish not only the relationship between organisms and describe the evolutionary component of the PCD process, but also predict new functions of enzymes, as well as their role in the cellular processes of oncogenesis.2. On the model of mice deficient in the caspase-2 gene, investigation of the phenotype of animals and the physiological role of this protein will be performed, which will give an opportunity to answer the question about the importance of caspase-2 as a regulator of normal processes in the body.3. Establish the role of caspase-2 in the processes of various types of PCD, its role in the detection of DNA damage and its repair. The novelty of the proposed project is to establish the functional characteristics of caspases, which have arisen as a result of evolution and their significance in various PCD processes. For the first time, phenotypic changes depending on the expression of caspase-2 in cells/tissues will be analysed. The physiological role of this enzyme in normal and pathological conditions, its participation in the regulation of various types of PCD, DNA damage detection and repair/replication processes will be studied. All fundamentally new knowledge about the molecular basis of PCD processes will be used as the basis for the development in the future of new approaches to enhance the effectiveness of chemotherapy for malignant tumors. The conditions for the successful implementation of this project are based on the presence of: (a) a team that includes both mature and young researchers with experience in studying various forms of cell death; (b) preliminary obtained interesting and promising results; (c) equipment, essential for conducting experiments (confocal microscope, a unique device for evaluating cell bio-energy — Seahorse Analyzer, equipment for spectral analysis, flow cytometer, availability of many programs for building phylogenetic trees, etc.).
        By the time the project is completed we are planning:1. To establish and understand the evolutionary component of the programmed cell death (PCD) process, construct a phylogenetic caspase tree, and predict new functions of these enzymes, as well as their role in the process of oncogenesis.2. To figure out the phenotype of animals deficient in caspase-2 gene and understand the apoptotic and non-apoptotic functions of this protein.3. To obtain new important information about the role of caspase-2 in the processes of various types of PCD, its role in the detection of DNA damage and its repair. It is known that the machine regulating PCD is constantly expressed in cells, but is under the control of inhibitory factors. It is important to note that many genes and their products involved in the regulation of PCD can perform "normal" functions, not associated with death. Unfortunately, most of these functions to date has not been investigated. Therefore, the identification of genes and their products involved in the regulation of PCD and the relationship between them is an important scientific task. At the same time, the ever-increasing amount of knowledge about the mechanism of their action laid the bases for the development of new therapeutic strategies that can use as targets various links of metabolic pathways of cell death. Caspases, a family of cysteine proteases, play an important role in the initiation and development of apoptosis, the most studied mechanism of PCD. They are found in many organisms, but their role in evolution is completely unknown. As a result of this project, we expect to find out the evolutionary component of the PCD process, to build a phylogenetic caspase tree and, as far as possible, predict new functions of these enzymes. There are currently no such data and it is completely obvious that such results will correspond to the most advanced level of knowledge. These results will also help to establish the role of caspases in the cellular processes of oncogenesis.In this project, we will continue to study the properties of caspase-2. In frame of the previous project, supported by RSF, for the first time we were able to show the existence of an alternative caspase-2 activation platform, different from the PIDDosome platform described earlier. The mechanism of caspase-2 activation in the high-molecular weight complex in response to the treatment of cancer cells with the DNA damaging drug cisplatin was identified; an original method to isolate the high-molecular weight complex of caspase-2 activation was developed (a patent was obtained); cell lines were created using the CRISPR/Cas9 genome editing system, characterized by the complete absence of caspase-2 and heterozygotes of the corresponding mice were generated. In this project, after receiving homozygous mice, we expect to receive new data on the phenotype of animals deficient in the caspase-2 gene and understand both the apoptotic and non-apoptotic functions of this enzyme. Moreover, since caspase-2 can fulfill oncosuppressive functions, our results will help to establish the mechanism of this phenomenon. For the first time, we will be able to describe the role of caspase-2 in the regulation of the immune and reproductive systems of the body, as well as in cognitive function. Fundamentally important and interesting to obtain new information on the role of caspase-2, not only in apoptosis, but also other types of PCD, its role in the detection and repair of DNA damage. All expected results are new, original and correspond to the high international level.
        Annotation of the results obtained in 2021During the current period, the analysis of the representation of functional groups of proteins in various suborders of rodents Sciuromorpha, Hystricomorpha, Castorimorpha, Myomorpha was carried out in order to identify unique molecular cell pathways characteristic of rodents with a long lifespan. The analysis revealed that species of the suborders Sciuromorpha, Hystricomorpha, Castorimorpha, which are characterized by a longer lifespan, are characterized by the presence of a number of proteins of the chemokine-cytokine pathway group, as well as beta-defensin proteins, which are involved in the regulation and migration of immune cells, as well as antimicrobial protection. Based on the previous data and current results, a model was formed that demonstrates significant changes in the immune system of rodents in the suborders Sciuromorpha, Hystricomorpha, Castorimorpha.To analyze the functions of caspase-2 at the level of the whole organism, we created 2 strains of mice knockout for this gene using the CRISPR/Cas9 approach. The investigation of these two strains gave a completely unexpected result. We have shown that knockout females are unable to produce offspring when crossed with any variant of the male genotype – wild-type, heterozygote, or knockout. At the same time, knockout males have the opportunity to produce offspring, both with wild-type females and heterozygous individuals. This phenotypic manifestation could be explained by the caspase-2-dependent development of the female reproductive system. An analysis of the crossing of all possible pairs from WTxWT to KO-casp2xKO-casp2 (WT is a wild-type of mice, KO-casp2 is any of the two knockout strains) showed that genotype splitting occurs according to the classical laws of inheritance. Analysis of body weight and individual organs in order to reveal the difference in the phenotype showed that, apart from visceral fat, females have no statistical differences in the weight of organs between knockout animals as compared to the wild-type. This indicated that, upon knockout of caspase-2, shifts in metabolic homeostasis could occur. Analysis of sperm and testes of males demonstrated that neither testis morphology nor sperm quality differs in knockout animals from the wild-type. Analysis of organs and hormones of the reproductive system showed that only animals with a deletion of 20 nucleotides have any difference from wild-type animals - males were characterized by an increased content of testosterone, and females by an increased weight of the uterus and ovaries. This difference could be caused by individual changes in the mouse strain due to genome editing using the CRISPR/Cas9 system.Analysis of the female sexual cycle showed that some of the knockout animals for an abnormally long time are in the same phase of the cycle, compared with wild-type animals. In this case, the motor activity of females - an indicator correlating with cycling - changed depending on the time of the day. It turned out that knockout females are less active during the day than wild-type animals. This indicated a possible imbalance in hormones. Analysis of progesterone and estradiol noted only a trend towards an increase in estradiol production and a decrease in progesterone, but this change was not statistically significant. We conducted a study on whether the studied animals can tolerate pregnancy after transferring healthy embryos to knockout mice. The results of these experiments showed that more than half of the del7/del7 knockout mice and all del20/del20 mice are unable to tolerate pregnancy. Thus, the infertility of caspase-2 knockout females was caused not only by changes in the ovary but also by the inability to endure pregnancy. Analysis of the male reproductive system showed that knockout animals do not have any pronounced differences in testes morphology or sperm quality. It should be noted that the knockout females were characterized by strong morphological changes in the ovary - at the age of 2-3 months, they were characterized by hyperplasia of the corpus luteum and a reduction or almost complete absence of oocytes in the follicles. Consequently, crossed knockout males may have offspring, unlike females. Thus, it can be concluded that caspase-2 performs a very important and specific function in the body, ensuring the correct functioning of the female reproductive system.In addition to experiments with animals, we analyzed the formation of a mitotic catastrophe and its outcome depending on the presence of the p53 protein, one of the main protectors of genome integrity. Caspase-2 is a regulator of p53 stability because can cleave the negative regulator of this protein - MDM2 ubiquitin ligase. Mitotic catastrophe is an oncosuppressive mechanism and is characterized by the formation of multi- or micronuclei in the cell after DNA damage in the G2/M phase of the cell cycle. The further fate of these cells depends on the balance of a number of proteins, including, as we assumed, the presence of p53. In a set of experiments, we showed that the treatment of HCT116 cancer cells with low doses of doxorubicin leads to the accumulation of cells in a state of mitotic catastrophe, and the absence of p53 reduces this accumulation. Moreover, under conditions of the formation of a mitotic catastrophe, the absence of p53 leads to the stimulation of autophagy, which often contributes to the survival of cells under stress conditions. Thus, it is highly probable that p53 regulates the formation of a mitotic catastrophe and the accompanying autophagy, which determines the outcome of a mitotic catastrophe, the death of a tumor cell, or its survival.During the reporting period, we continued to study the effect of inhibiting the activity of the anti-apoptotic protein Mcl-1 with the aim of elucidating the use of BH3 mimetics in the treatment of solid neoplasms. We have shown for the first time that the Bak and Bcl-xL proteins can regulate the sensitivity of tumor cells to Mcl-1 inhibitors. We also found that BH3 mimetic S63845 induced apoptosis more effectively than another Mcl-1 inhibitor, A1210477, via a Bak-dependent mechanism. We further demonstrated that the sensitivity of cancer cells correlates with low expression of the Bcl-xL protein. Moreover, acquired resistance to inhibition of Mcl-1 may be associated with increased expression of the Bcl-xL protein. In addition, low levels of Bcl-xL and high levels of Bak of both mRNA and proteins were observed in tumor tissues obtained from patients with lung adenocarcinoma. Overall, our results have shown an important role for the Bak and Bcl-xL proteins in regulating the sensitivity and resistance of tumor cells to Mcl-1 inhibition.As a result of our research, during 2021 we published 1 article in domestic and 7 articles in international journals, of which 6 in journals related to Q1. In addition, 5 invited presentations were made at conferences held in Russia. For publications supported by this grant, 4 project participants were recognized as laureates of the Moscow State University Competition in the Outstanding Articles category. One participant (A.V. Zamaraev) was awarded the RAS Gold Medal with a prize for young scientists of Russia for carrying out research included in this project.