Annotation of the results obtained in 2020
The sensitivity of the ∆lpcA mutant to a wide range of antibiotics of various chemical nature and various mechanisms of action was determined. It was shown that ∆lpcA mutants exhibit supersensitivity to antibiotics of the gyrase inhibitor group (nalidixic acid, moxifloxacin), aminoglycosides (kanamycin, gentamicin), protein synthesis inhibitors (tetracycline, chloramphenicol), as well as rifampicin, which inhibits RNA synthesis. The results obtained allow us to conclude that the activity of sedoheptulose-7 phosphate isomerase determines the resistance of E. coli cells to a wide range of antibacterial drugs. Modulating the activity of this enzyme can help solve the problem of multidrug resistance. The lpcA gene was cloned into the pET15b expression vector. Thus the 5’ prime end of lpcA gene was fused with the sequence encoding the His-Tag. The His-Tag-LpcA protein was expressed in E. coli cells of the BL21 strain, and preparative amounts of this protein were purified by affinity chromatography on Hi-Trap GE Healthcare columns using the AKTA pure 150 GE chromatographic system. The effect of potential inhibitors of the LpcA (GmhA) protein - inh 35, inh 38, inh 39 on the enzyme activity has been characterized. It was shown that compounds inh38 and inh39 inhibit the enzyme with inhibition constants of about 10-20 μM. For the first time, the role of zinc ions in the formation of the active enzyme GmhA was demonstrated. The parameters of binding of adenine nucleotides and substrate to the enzyme were estimated. It was found that binding of the substrate, sedoheptulose-7 phosphate, occurs only in the presence of adenine nucleotides. It was shown that compounds inh38 and inh39, which are derivatives of the reaction product, bind to the GmhA enzyme. In the presence of these compounds, the binding of the substrate is disrupted, which indicates the competitive nature of inhibition. In vivo experiments demonstrated that inh38 and inh39 compounds enhance the toxic effect of the antibiotics nalidixic acid, gentamicin and rifampicin on the growth of wild-type E. coli. The negative effect of the antioxidants: glutathione and N-acetylcysteine, on the viability of the nematodes Caenorhabditis elegans has been demonstrated. It was shown that the addition of glutathione and N-acetylcysteine to the bacteria lawn on which nematodes are cultivated reduces their lifespan by 10-15%. A model of the pretermination complex that includes the termination factor Rho, the common elongation factors NusA, NusG, and the RNA polymerase elongation complex was obtained. It was shown that in strains with deletion of regions that form protein-protein interactions with Rho in the beta-subunit of RNA polymerase, the termination efficiency is reduced by 2.5 times. It was concluded that the direct interaction of RNA polymerase with Rho is critical in Rho-dependent transcription termination. In vitro experiments have shown that NusA enhances termination and causes a shift in the termination sites further down the course of transcription. Structural information, supported by genetic and biochemical data, formed the basis for a new molecular model of Rho-dependent transcription termination. Specifically obtained lines of D. melanogaster were used to establish that the ubiquitous constitutive overexpression of the genes controlling the biosynthesis of hydrogen sulfide CBS and CSE in combination with the pharmacological action of the precursors of the H2S N-acetyl-L-cysteine (NAC) biosynthesis and cysteine leads to a geroprotective effect which is expressed in males with overexpression of CBS maintained on culture medium supplemented with NAC, as well as in males and females with overexpression of CSE, maintained on the culture medium supplemented with NAC and additional cysteine. For the first time it was demonstrated that the joint conditioned activation of the expression of CBS and CSE genes in the cells of the D. melanogaster nervous system has a geroprotective effect on males and females. Using Illumina technology extensive transcriptome studies were conducted to investigate the role of genes involved in the production of reactive forms of sulfur (hydrogen sulfide-H2S), both at the cellular level and at the level of an entire organism. The sodium hydrosulfide, characterized by a fast release of hydrogen sulfide ("fast donor"), and an artificial compound GYY4137, widely used in research as a "slow donor" of hydrogen sulfide, as well as two human cell lines were used in the study. Cells of neuronal origin (neuroblastoma SH-SY-5Y) and human promonocytic cell line THP-1 were selected to conduct experiments. Transcriptome libraries were obtained after exposure of cells to LPS, GYY4137 and combined exposure to the slow hydrogen sulfide donor GYY4137 and LPS. It is noteworthy that hydrogen sulfide donor was added both before and after the administration of LPS. The analysis of sequenced libraries was carried out using bioinformatic methods, allowing elucidation of signaling pathways and genes that respond to the administration of GYY4137 and LPS, as well as to the combined effect of these two agents in THP-1 cells (Yurinskaya et al., 2020). To study the role of hydrogen sulfide at the organism level, we used obtained lines of D. melanogaster with deletions of three main genes involved in the metabolism of cysteine and responsible for the production of hydrogen sulfide in both higher organisms and bacteria (deletions for CBS, CSE, and MST). The transcriptome analysis revealed that in flies with deletions, the expression of genes involved in redox processes, stress genes, genes encoding enzymes involved in the glutathione synthesis, TNF-a effectors, genes involved in reproduction and smell, as well as other housekeeping genes is disrupted. Analysis of transcriptomes revealed that comparing to the control line the most significant differences in gene expression are observed in lines carrying deletions of the cbs gene. Significant gender differences were found in the expression of the studied genes, as well as characteristic differences between the expression in the whole body and heads of flies. The differences in the expression of certain candidate genes that were shown to change significantly the expression levels in lines with deletions, were confirmed by RT-PCR (Zatsepina et al., 2020). The performed analysis allowed identification of adaptive systems interacting with the system of genes responsible for the production of hydrogen sulfide. The effect of GYY4137 on the total chymotrypsin-like activity, as well as the specific activity of the β5i immunoproteasome subunit was characterized using THP-1 cells. It was shown that prolonged incubation with GYY4137 stimulates the activity of proteasomes. However, when cells were incubated with both GYY4137 and LPS, the total chymotrypsin-like activity of proteasomes and the chymotrypsin-like activity of immunoproteasomes, were decreased. These results clarify the data obtained at the previous stage of the project and indicate that the additional cytoprotective mechanism of GYY4137 is based on a decrease in the expression of immunoproteasomes which affects the development of LPS-induced inflammatory reactions. In addition to the stated plan, we have studied the effect of adenosine triphosphate (ATP), both free and in complex with Mg2+ ions, on the activity of purified preparations of constitutive and immune 20S proteasomes. The obtained results indicate that free ATP and Mg2+ have reciprocal effect on the degradation of various substrates by 20S proteasomes, which is probably dependent on the substrate’s size and charge. In addition, obtained results indicate that the mechanism of proteasome activity regulation based on the balance between free ATP and Mg2+ might exist in cells. It was demonstrated that overexpression of the MST gene in mouse mesenchymal stem cells (MSCs) has a small positive effect on the proliferation rate and the number of passed population doublings under hypoxic conditions. At the same time, the overexpression of CBS gene increased the number of passed population doublings more significantly and led to an increase in the time of the MSCs existence in culture, which can be considered as inhibition of aging in these cells in vitro. Under normoxia the retardation of MSCs aging in conditions of MST and CBS genes overexpression was noticeably more pronounced, indicating that increased expression of CBS and MST promotes an increase in the resistance of mouse MSCs to oxidative stress.

 

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Annotation of the results obtained in 2017
To study the biochemistry of endogenous H2S in E. coli, we generated E. coli strains either lacking a chromosomal copy of the 3MST-encoding gene mstA or carrying it under a strong Ptet promoter (Ptet-mstA). Interaction between H2S production and the expression of the genes involved in cysteine metabolism was investigated. For this purpose E.coli strains with altered expression of genes responsible for cysteine synthesis (cysB, cysK, cysE) and transport (tcyP, tcyJ, ydeD) were generated. We used quantitative RT-PCR to measure the expression of cysB, cysK, cysE, tcyP, tcyJ, ydeD in wild type strain, ΔmstA and Ptet-mstA cells. We found that Ptet-mstA mutant that overproduced H2S exhibited increased level of CysB-regulated genes transcription, while in ΔmstA mutant lacking the ability to produce H2S the expression of these genes was reduced to the basal level and is not subjected to positive CysB-regulation. Reduction of H2S production by Ptet-mstA caused by deletion of cysB gene encoding a master transcriptional regulator of cysteine metabolism or by deletion of cysE gene encoding O-acetylserine autoinducer of CysB was demonstrated. Inactivation of CysB-regulated tcyP and tcyJ genes, encoding L-cystine importers also leads to reduction of H2S production by Ptet-mstA, while in cysK и ydeD deletion mutants production of H2S by Ptet-mstA was not altered. We demonstrated that high level of TcyP expression under the control of Ptet promoter leads to increase of H2S production. In contrast, constitutive expression of YdeD transporter responsible for cysteine export from the cytoplasm to the periplasm leads to detectable reduction of H2S production by Ptet-mstA. The accumulated data allow us to conclude that medium-derived cystine/cysteine is the principle substrate for mstA dependant H2S production. Several Drosophila melanogaster transgenic strains containing two (from the existing four) additional genes (CBS и CSE) involved in generation of H2S synthesis in the cells of this species have been developed. The obtained strains (“binary system”) enable to investigate the protective effects of constitutive and inducible (by mifepristone) superexpression of these genes in the whole body in stress response. A possible role of such superexpression in the life span and other vital characteristics will be also monitored. Preliminary small scale experiments demonstrated positive effect of these two genes superexpression in the whole body on life span and quality of the flies. We have developed experimental approach to generate deletions for all four genes responsible for H2S production in D.melanogaster exploring CRISPR/Cas9 system and pCFD vector. In the process of developing this technique we received a deletion of HSR gene recently described in Prof. Nudler’s laboratory. According to the data accumulated in Nudler’s lab this gene represents an essential component of cellular response to various form of stress in flies and humans. In addition to the planned studies we began the investigation of the effect of H2S on the activity of ubiquitin-proteasome (UPS) system operating in mammalian cell lines and responsible for the degradation of various cellular proteins and, hence, providing protein homeostasis in the eukaryotic cells. Several complementary techniques and general design of the experiments have been developed to monitor the effects of various concentration of GYY4137 reagent (H2S donor molecule) on the activity and content of proteasomes in the cells of neural origin in the presence of substances inducing oxidative stress. The genetically encoded Avitag peptide, which is biotinylated in vivo by biotin ligase BirA, has been inserted in different parts of the transcription termination factor Rho of Escherichia coli. We have shown that expression of the obtained hybrid constructs, encoding Avitag inserted in the solvent-exposed loop or added at the C-terminus of Rho, leads to the same phenotype as in the case with wild type Rho overexpression. An efficient one-step affinity purification of Rho has been demonstrated. We have established a role of Rho-dependent transcription termination in the attenuation of cysE gene that is involved in the first step in the pathway of cysteine biosynthesis. We have constructed E.coli strains with the deletion of rybB and fnrS small RNA genes as well as RNA-binding protein Hfq gene. Analysis of expression of genes Cbs, Cth и Mst responsible for H2S production, in young and aged mouse mesenchymal stem cells was performed. The obtained data indicate modulation of expression of these genes upon transfer from hypoxia to normoxia as well as upon aging of these cells in culture. Activation or inhibition of AMPK signaling in young cells does not affect expression of the above genes, however, a certain modulation of their expression was observed upon AMPK stimulation in aged cells.

 

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Annotation of the results obtained in 2018
A collection of E.coli strains with altered expression of the cyoB, cydB, cydD genes controlling the synthesis of terminal oxidases was obtained and their ability to generate H2S was studied. Increased expression of the cydD gene has been shown to lead to a marked increase in the production of H2S and at the same time causes an increased sensitivity of bacteria to oxidative stress. An additional pathway for the generation of H2S was found in E.coli cells, the activity of which is under the control of the transcriptional regulator CysB. A mutation has been identified in the lpcA gene, which leads to bacterial supersensitivity to a number of antibiotics (nalidixic acid, rifampicin). The results of RT-PCR experiments revealed a different response of terminal oxidases to oxidative stress and the level of H2S generation in the bacterial cell. The data obtained indicate the involvement of terminal oxidases and genes involved in the generation of H2S in maintaining the redox status of the bacterial cell. The results of the proteomic analysis showed that an increase in the fractions corresponding to the Ivy and OsmY proteins involved in protection against osmotic stress and stabilization of the cell wall peptidoglycan is observed in cells of the strain with a deletion of the lpcA gene. A significant change in the cell redox status and a decrease in the viability of E.coli as a result of a point mutation or deletion of the lpcA gene encoding the protein sedoheptulose-7 phosphate isomerase, which catalyzes the first step in the biosynthesis of the main component of lipopolysaccharides, was demonstrated. The deletion of lpcA leads to an increase in the reduced thiols, including glutathione and cysteine, by a factor of 3–4, causes an increase in ROS, and more than 10 times increases the percentage of dead cells in the population. Thus, disruption of lpcA, leading to a significant change in intracellular redox status, significantly reduces the viability of bacterial cells, which is important for creating inhibitors of lpcA for antibiotic therapy. The covalent chemical crosslinking of the factor Rho with partner proteins using disuccinimidyl substrate and formaldehyde was carried out. In addition to the subunits of RNA polymerase, NusA and NusG elongation factors, srkA kinase, and a number of proteins with unknown function were established as Rho protein partners. The factors Rho, NusA and the core of E.coli RNA polymerase were superproduced and purified. It was found that to stabilize the complex of the Rho hexamer with the RNA polymerase, the presence of factor NusA is necessary. Pilot experiments were conducted to select the optimal method for studying the intermolecular interactions of these proteins using a biosensor based on surface plasmon resonance and microscale thermophoresis. The effects of constitutive ubiquitous overexpression of CBS and CSE (hydrogen sulfide control biosynthesis genes) on maximal lifespan, fertility, resistance to stress, and locomotor activity of Drosophila melanogaster males were studied. The geroprotective effect was revealed only with the universal overexpression of CBS. Both favorable and negative effects of overexpression of CSE and CBS on locomotor activity and resistance to various adverse effects (hyperthermia, oxidative stress, a combination of dehydration and fasting) were noted. The results of the study can be used to search for potential targets for pharmacological correction of life expectancy and an increase in healthy lifespan duration. Using 3 species of the genus Drosophila, differing in the maximum lifespan, we investigated the mechanisms of the geroprotective effects of N-acetyl-L-cysteine (NAC) and evaluated the compliance of the NAC with the criteria of geroprotectors. It has been established that NAC does not fully meet the criteria of geroprotectors, since its effects on life expectancy depend on the gender and species of the individual. The results of the analysis of the expression levels of antioxidant protection control genes (Cat / CG6871, Sod1 / CG11793) and the genes involved in the control of hydrogen sulfide biosynthesis (Cbs / CG1753, Eip55E / CG5345, Nfs1 / CG12264) suggest that the geroprotective properties of NAC are interrelated with the activation of the protection against NACs, the biosynthesis of hydrogen sulfide and due to the mechanisms of hormesis. The results can be used to better understand the mechanisms of geroprotectors effects, as well as to identify the geroprotective effects of known drugs. Constructs for CRISPR experiments were developed and synthesized in order to obtain deletions of the main genes controlling sulfur metabolism in Drosophila. Using these constructs, 3-4 lines were obtained with a deletion of each gene. Crossing experiments were started to produce double homozygotes for pairs of genes we studied (for example, CBS and CSE). Experiments are being carried out to clarify the role of the studied genes under various forms of stress, as well as their role in the aging process. It has been established that in SH-SY5Y neuroblastoma cells, under the action of GYY4137, proteasome activity decreases, but there is no change in the content of both structural and catalytic subunits of proteasomes. At the same time, the immune subunits in these cells are practically not detected. Under the action of GYY4137, SH-SY5Y cells do not change the expression levels of the catalytic subunits of proteasomes. Thus, the observed changes in proteasome activity are probably associated with post-translational modifications of proteasome subunits and their cofactors. Studies have been completed to investigate the role of tHSR gene, which according to the lab. of Prof. Nudler, obtained using Drosophila culture cells (S2), produces a long non-coding RNA, which is a thermo-sensor. It has been shown that flies homozygous for the HSR gene do not significantly differ in thermoresistance from control wild type flies. The effect of NaHS hydrogen sulfide donor and AMPK activator (AICAR) on human neuroblastoma cells of the SH-SY-5Y line was characterized. It was established that these substances enhance the antioxidant protection of the cells, leading to an increase in the level of intracellular reduced glutathione. The joint use of the hydrogen sulfide donor and AICAR leads to a significantly greater effect on the redox status of cells than their use separately. Thus, the data obtained indicate the benefits of simultaneous application of hydrogen sulfide donors and AICAR to enhance the antioxidant protection of neuronal cells. Using the line of human neuroblastoma and several other cell cultures, we investigated the protective effect of donors of active forms of sulfur (slow-GYY4137 and fast-NaHS) against the action of bacterial endotoxin LPS. Experiments have shown that the compounds used effectively protect all cell types used in the work from the action of LPS. It is remarkable that the use of these donors proved to be effective and lowered the level of TNFα, reactive oxygen species (ROS) and nitric oxide (NO), both in experiments with prophylactic administration (before toxin) and with donors administration after toxin application. Experiments on the long-term cultivation of mouse mesenchymal stem cells (MSCs) under hypoxia (5% O2) and normoxia (21% O2) in the presence of the hydrogen sulfide donor NaHS were carried out. It was shown that optimal concentrations of hydrogen sulphide donor NaHS slow down the aging of MSCs in culture and stimulate the proliferation of older MSCs both during hypoxia and, especially, with normoxia. At the same time, high concentrations of NaHS, on the contrary, are capable of inhibiting the proliferation of MSCs in a dose-dependent manner. It has been shown that the hydrogen sulphide donor NaHS protects melanoma cells and MSCs from the negative effect of infrared laser radiation. Experiments exploring the effect of NaHS and AICAR (AMPK activator) on mouse MSC were carried out. The data obtained indicate the existence of an interaction between the signal pathways in which AMPK and hydrogen sulfide are involved. Retro-and lentiviral constructs were created for over-expression of the mouse CBS gene in the MSC. Lentiviral constructs were designed to inactivate the mouse CBS gene in MSCs using CRISPR technology.

 

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Annotation of the results obtained in 2019
The relationship between the activity of genes responsible for cysteine transport (tcyP, eamA), H2S production (∆mstA and Ptet-mstA mutants), and the functioning of CydDC terminal oxidase was studied. Experimental evidence is presented that the CydDC complex does not participate in the export of cysteine from cytoplasm to periplasm, as previously assumed. It was shown that the presence of ΔcydD deletion in the bacterial genome leads to an increase in cell resistance to oxidative stress caused by hydrogen peroxide and is accompanied by an increase in the level of transcription of sodA, sodB, and katG genes. It follows that inactivation of cydDC leads to oxidative stress and an increase in the level of ROS generation in the cell. The presented results allow us to conclude that the CydDC complex promotes the restoration of cystine to cysteine and this leads to the promotion of the Fenton reaction and genotoxic stress. Thus, CydDC terminal oxidase plays an important role in maintaining the redox balance of the bacterial cell. The effect of E. coli cyoA and cydA mutations, which disrupt the activity of terminal oxidases bo'and bd-I, respectively, on the lifespan of C. elegans nematodes, was studied. It was shown that the average life span of nematodes when grown on cyoA mutants increased by 15.5%, and on cydA mutants - by 12.8%. It is known that the cyoA and cydA mutants are characterized by an increased level of generation of reactive oxygen species (ROS). We suggest that an increase in the life span of C. elegans is due to moderate oxidative stress that occurs in the nematode organism when cultured on these mutant bacteria. In the present work, we confirmed the previously obtained data that the deletion of the lpcA gene (gmhA) in E. coli cells leads to a change in the normal metabolism of low molecular weight thiols and, as a consequence, to a disruption of the cellular redox balance. The ∆lpcA mutant has been shown to be highly sensitive to oxidative stress caused by paraquat, but not hydrogen peroxide. In addition, the ∆lpcA mutant is characterized by increased sensitivity to the action of quinolones, such as nalidixic acid and rifampicin, which inhibits DNA-dependent RNA polymerase, which may be associated with genotoxic stress caused by increased ROS generation. Based on the analysis of data obtained using the ro-GFP redox sensor and intracellular dye on ROS, it was found that deletion of the lpcA gene leads to a decrease in the content of polysulfides in bacterial cells with deletion of the mstA and lpcA genes compared to the control, with the largest decrease observed in case of lpcA deletion. Using NMR, it was found that cellular non-protein extracts contain mainly reduced glutathione, the amount of other non-protein thiols, cysteine and mercaptopiruvate, are significantly lower and practically do not contribute to the pool of determined non-protein thiols. The proportion of oxidized glutathione is less than 5% of the amount recovered. It was found that a deletion of lpcA leads to a decrease in the level of intracellular reduced glutathione by more than 2 times. Thus, the previously observed increase in intracellular thiols during inactivation or deletion of lpcA was observed due to a decrease in polysulfides and an increase in the pool of reduced protein thiols, while the level of reduced glutathione is significantly reduced. It can be concluded that these changes were one of the reasons for the decrease in cell viability. The role of the mstA and lpcA genes in the process of redox-modifications of proteins (sulfhydration and glutathionylation) in E. coli cells was determined. It was shown that the ΔmstA deletion and, to a lesser extent, the ΔlpcA deletion leads to a decrease in the total level of protein sulfhydration. It was found that the greatest changes in the pattern of glutathionylation were observed in cells with the ΔlpcA gene deletion. It was shown that the major glutathionylation band corresponds to the GAPDH protein, which is necessary for energy metabolism and the production of ATP and pyruvate by glycolysis. In cells with a ΔlpcA deletion and a double ΔmstA ΔlpcA deletion, a significant increase in the level of GAPDH occurs, while its glutathionylation decreases. Thus, a decrease in the level of glutathionylation and sulfhydration of proteins in cells with ΔlpcA deletion correlates with the decrease in the level of persulfides in cells and an increase in the reduced protein groups described in the previous section. The observed decrease in the level of glutathionylation of GAPDH in the lpcA deletion mutant contributes to the activation of this protein. Using the Autodock Vina software a virtual screening was performed in the structure of sedoheptulose 7-phosphate isomerase GmhA from E. coli 2i22 (PDB code) at the sites of binding to sedoheptulose-7-phosphate, including residues 52-NGGS-55, 120T, 168-AD-169, 172Q of the chain B and residues 61H, 65E, 176I, 180H of the chain C of structure 2i22. Ten potential ligands were obtained from the ZINC base: 00640220, 03873988, 01714434, 03954011, 01630310, 03954010, 00056857, 04366948, 05546109, 16942956 for further experimental verification. Using soft chemical fixation in the density gradient of glycerol (GraFix), the elongation complex of RNA polymerase with termination factor Rho was stabilized. Based on the new structural data, using the lambda-Red recombination system and CRISPR / Cas9, precision chromosomal mutations were constructed in E. coli strain MDS42 encoding deletions of polypeptide loops b (105-116) and b (483-491) and domain i9 b- subunits of RNA polymerase. Real-time PCR demonstrated an in vivo decrease in the effectiveness of Rho-dependent termination of transcription in strains with the deletions in the b-subunit of RNA polymerase: b (483-491) and i9 domain which are involved in the interaction with Rho factor. The transcriptome of long-lived Drosophila contained in various contrasting conditions was sequenced. Comparing long-lived flies of different genotypes (E (z) / w VS w / w), subjected to a combination of geroprotective factors, we found a strong difference (Fold change> 8) in the expression of a number of genes, some of which were described previously in connection with their possible role in ensuring longevity in higher organisms, including mammals. The D. melanogaster lines containing single and double deletions of all genes involved in the production of active sulfur (H2S) were obtained using the CRISPR technology. The effect of these deletions on the expression of the genome of flies was studied (transcriptome studies). The effect of the obtained deletions, as well as the overexpression of these genes on various vital parameters of Drosophila, including resistance to various forms of stress, including oxidative stress, and also an increase in temperature (HS), was studied. The effect of the obtained mutations on the life expectancy of males and females separately was investigated. It was shown, in particular, that although double deletions in the genes of sulfur metabolism lead to a reduction in life expectancy, flies with double deletions are characterized by a higher resistance to oxidative stress (paraquat). Of particular interest are the results of experiments in which the effect of hydrogen sulfide donors (GYY4137) on various parameters of flies with deletions of sulfur metabolism genes were investigated. In particular, it was shown that in males and females of the double deletion line (BE (1-2), a positive effect of GYY4137 on life expectancy (according to censored data) and survival under hyperthermia was noted. The effects of hydrogen sulfide donors on various indicators of cultured human cells, in particular, on the inflammatory response caused by bacterial toxin (LPS) were studied. It turned out that the use of hydrogen sulfide donors has a protective effect, reducing inflammation (ROS, TNFa, etc.). It was shown that a decrease in LPS-induced expression of proteasome immune subunits under the action of a hydrogen sulfide donor (GYY4137) can be an important molecular mechanism providing cytoprotective functions of hydrogen sulfide donors. А comparative analysis of mRNA expression of the catalytic proteasome subunits genes in MSC cells cultured for various periods of time was performed. It has been demonstrated that MSC aging is accompanied by a decrease in the expression of proteasome immune subunits, which, apparently, affects the functional state of cells. Lentiviral constructs have been created to inhibit CBS gene expression in mammalian cells. In addition, retro- and lentiviral constructs were created for over-expression of the mouse MST gene (MPST) in MSCs. Lentiviral constructs for inactivation of the mouse MST gene (MPST) in MSCs using CRISPR technology were created and their effectiveness was demonstrated.

 

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