[The Effect of the Knockout of Major Transsulfuration Genes on the Pattern of Protein Synthesis in D. melanogaster].

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production-cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila strains with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of strains with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the strains with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control strain. A decrease in the total number of proteasomes and their activity was found in the strains with deletions of the cbs and cse genes.

Genome-wide transcriptional effects of deletions of sulphur metabolism genes in Drosophila melanogaster.

In recent years, the gasotransmitter hydrogen sulphide (H2S), produced by the transsulphuration pathway, has been recognized as a biological mediator playing an important role under normal conditions and in various pathologies in both eukaryotes and prokaryotes. The transsulphuration pathway (TSP) includes the conversion of homocysteine to cysteine following the breakdown of methionine. In Drosophila melanogaster and other eukaryotes, H2S is produced by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (MST). In the experiments performed in this study, we were able to explore the CRISPR/Cas9 technique to obtain single and double deletions in homozygotes of these three major genes responsible for H2S production in Drosophila melanogaster. In most cases, the deletion of one studied gene does not result in the compensatory induction of two other genes responsible for H2S production. Transcriptomic studies demonstrated that the deletions of the above CBS and CSE genes alter genome expression to different degrees, with a more pronounced effect being exerted by deletion of the CBS gene. Furthermore, the double deletion of both CBS and CSE resulted in a cumulative effect on transcription in the resulting strains. Overall, we found that the obtained deletions affect numerous genes involved in various biological pathways. Specifically, genes involved in the oxidative reduction process, stress-response genes, housekeeping genes, and genes participating in olfactory and reproduction are among the most strongly affected. Furthermore, characteristic differences in the response to the deletions of the studied genes are apparently organ-specific and have clear-cut sex-specific characteristics. Single and double deletions of the three genes responsible for the production of H2S helped to elucidate new aspects of the biological significance of this vital physiological mediator.

Age-dependent expression profiles of two adaptogenic systems and thermotolerance in Drosophila melanogaster.

Here, we monitored the expression of three genes (hsp70, hsp22, and hsf1) involved in heat shock response in Drosophila melanogaster in males and females of different age. Also, we investigated age- and sex-dependent expression of three major genes participating in the production of hydrogen sulfide (H2S) (cse, cbs, and mst), implicated in stress resistance and aging. In addition to the control strain, we monitored the expression of all of these genes in a cbs knockout strain (cbs-/-) generated using the CRISPR technique. The tested strains differ in the induction capacities of the studied genes. Relative to the control strain, under normal conditions, the cbs-/- strain expresses all of the studied genes more abundantly, especially hsp22. In the control strain, aging leads to a dramatic increase in hsp22 synthesis, whereas in the cbs-/- strain, hsp22 induction is not pronounced. Furthermore, in 30-day-old cbs-/- flies, the constitutive expression of hsp70 and mst is decreased. Surprisingly, in the cbs-/- strain, we detected an upregulation of hsf1 transcription in the 30-day-old females. After heat shock in the control strain, hsp70 and hsp22 induction decreased with age in males and hsp22 decreased in females, while in the cbs-/- strain, a pronounced drop in the induction capacity of both hsp genes was seen in 30-day-old males and females. However, in most cases, the expression levels of hsf1 and H2S-producing genes do not exhibit pronounced changes depending on sex, age, or heat shock. Flies of control and cbs-/- strain exhibited strong reduction in basal thermotolerance with age. Our data suggest a cross-talk between the two studied ancient and universal adaptive systems.