[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.

Hsp70 affects memory formation and behaviorally relevant gene expression in Drosophila melanogaster.

Heat shock proteins, in particular Hsp70, play a central role in proteostasis in eukaryotic cells. Due to its chaperone properties, Hsp70 is involved in various processes after stress and under normal physiological conditions. In contrast to mammals and many Diptera species, inducible members of the Hsp70 family in Drosophila are constitutively synthesized at a low level and undergo dramatic induction after temperature elevation or other forms of stress. In the courtship suppression paradigm used in this study, Drosophila males that have been repeatedly rejected by mated females during courtship are less likely than naive males to court other females. Although numerous genes with known function were identified to play important roles in long-term memory, there is, to the best of our knowledge, no direct evidence implicating Hsp70 in this process. To elucidate a possible role of Hsp70 in memory formation, we used D. melanogaster strains containing different hsp70 copy numbers, including strains carrying a deletion of all six hsp70 genes. Our investigations exploring the memory of courtship rejection paradigm demonstrated that a low constitutive level of Hsp70 is apparently required for learning and the formation of short and long-term memories in males. The performed transcriptomic studies demonstrate that males with different hsp70 copy numbers differ significantly in the expression of a few definite groups of genes involved in mating, reproduction, and immunity in response to rejection. Specifically, our analysis reveals several major pathways that depend on the presence of hsp70 in the genome and participate in memory formation and consolidation, including the cAMP signaling cascade.

Heat shock protein 70 from a thermotolerant Diptera species provides higher thermoresistance to Drosophila larvae than correspondent endogenous gene.

Heat shock proteins (Hsp70s) from two Diptera species that drastically differ in their heat shock response and longevity were investigated. Drosophila melanogaster is characterized by the absence of Hsp70 and other hsps under normal conditions and the dramatic induction of hsp synthesis after temperature elevation. The other Diptera species examined belongs to the Stratiomyidae family (Stratiomys singularior) and exhibits high levels of inducible Hsp70 under normal conditions coupled with a thermotolerant phenotype and much longer lifespan. To evaluate the impact of hsp70 genes on thermotolerance and longevity, we made use of a D. melanogaster strain that lacks all hsp70 genes. We introduced single copies of either S. singularior or D. melanogaster hsp70 into this strain and monitored the obtained transgenic flies in terms of thermotolerance and longevity. We developed transgenic strains containing the S. singularior hsp70 gene under control of a D. melanogaster hsp70 promoter. Although these adult flies did synthesize the corresponding mRNA after heat shock, they were not superior to the flies containing a single copy of D. melanogaster hsp70 in thermotolerance and longevity. By contrast, Stratiomyidae Hsp70 provided significantly higher thermotolerance at the larval stage in comparison with endogenous Hsp70.

Molecular mechanisms underlying thermal adaptation of xeric animals.

For many years,we and our collaborators have investigated the adaptive role of heat shock proteins in different animals,including the representatives of homothermic and poikilothermic species that inhabit regions with contrasting thermal conditions. Adaptive evolution of the response to hyperthermia has led to different results depending upon the species. The thermal threshold of induction of heat shock proteins in desert thermophylic species is, as a rule, higher than in the species from less extreme climates. In addition,thermoresistant poikilothermic species often exhibit a certain level of heat shock proteins in cells even at a physiologically normal temperature. Furthermore,there is often a positive correlation between the characteristic temperature of the ecological niche of a given species and the amount of Hsp70-like proteins in the cells at normal temperature. Although in most cases adaptation to hyperthermia occurs without changes in the number of heat shock genes, these genes can be amplified in some xeric species. It was shown that mobile genetic elements may play an important role in the evolution and fine-tuning of the heat shock response system,and can be used for direct introduction of mutations in the promoter regions of these genes.