| Abstract [eng] |
Prions are proteinaceous particles that are able to replicate and spread as an infection. Prions cause transmissible spongiform encephalopathies (TSEs), a fatal neurodegenerative disease in mammals. Neurodegenerative diseases also include amyloidoses, better known as Alzheimer's, Parkinson's or tauopathy. The sequence of the yeast protein Sup35 GNNQQNY is sufficient for the formation of amyloid fibrils. Molecular dynamics studies have shown that several amino acids in this sequence are important in prion aggregation. On the basis of these results, it was chosen to develop such mutant variants of the GNNQQNY sequence in an in vivo system. Four mutations were chosen for the study, N8A, N9A, Q10A and Q11A. The yeast Saccharomyces cerevisiae is a widespread model system suitable for the study of prions and exhibits many conserved mechanisms with higher eukaryotes. Prion formation, aggregation, proliferation and elimination and interactions between prion proteins have been identified in this system. The aim of the present work was to create mutant variants of the Sup35 protein GNNQQNY sequence and to induce the synthesis of target proteins by copper ions in yeast S. cerevisiae cells. To achieve this goal, targeted mutations (N8A, N9A, Q10A and Q11A) were introduced into the SUP35-GFP gene. These mutations were generated by PCR and cloned into vectors to amplify the genes in Escherichia coli cells. The mutated genes were subsequently transferred into a yeast expression vector and introduced into S. cerevisiae 74-D694 [psi-][PIN+] strain cells. Protein synthesis in yeast cells was induced using 15-100 μM copper sulphate solution and cell samples were collected after 0 h, 4 h, 18 h and 24 h. The results showed that the target genes were successfully amplified by PCR. Sequencing results confirmed that site-specific mutagenesis by PCR was successful in introducing selected mutations into the SUP35-GFP gene and that these mutations were not lost during all steps of the study. The constructed expression vectors pRSCupSup35-GFP containing the targeted mutations were successfully amplified in E. coli cells and transferred to S. cerevisiae cells. Unfortunately, the expression of the target proteins could not be detected by induction of the target proteins with copper ions in yeast cells. |
