Publicação
Heterologous expression of human KRASwt cDNA in Saccharomyces cerevisiae and its mutants from the Ras signalling pathway and phenotype screening
| Resumo: | Most of our knowledge about basic cellular processes has originated from model organisms. Saccharomyces cerevisiae is consider a model system, representing the simplest eukaryotic organism, whose genome can be easily manipulated allowing suitable analysis and efficient assessment of gene products from humans. RAS genes encode low molecular weight, GTP-binding, GTP-hydrolysing proteins that are highly conserved throughout all eukaryotic species. RAS pathway has attracted attention because of its importance in malignant transformation of human cells. KRAS is a human RAS isoform, expressed in almost all cell types and essential for the normal cellular development, in addition it is the isoform most frequently mutated in many cancer types. The S. cerevisiae has two RAS genes, RAS1 and RAS2 that are essential, once their double deletion renders yeast unviable. These genes are structurally and functionally homolog of the mammalian RAS proto-oncogenes. This conservation allows the use of yeast genetics to the study of KRAS. Considering this, the first aim of this work was to build a set of S. cerevisiae strains (using haploid BY4741 wild type, ras1Δ, and ras2Δ as a basis) expressing the human KRASwt cDNA, using a plasmid-based expression. The second aim was the phenotype screening of these humanized yeasts, as well as of the correspondent recipient strains. The effect of KRASwt expression was evaluate on the cell stress response, growth, chronological aging, cell cycle progression and haploid invasive growth. According to the results, the KRASwt heterologous expression in yeast (1) had a negative or null effect on the resistance to the temperature, pH, osmotic and oxidative stresses; (2) decreased growth on non-fermentable carbon sources; (3) increased the adhesion capacity; (4) stimulated the haploid invasive growth in the ras2Δ strain; (5) modified the budding pattern of the wild type cells; (6) changed the cellular proliferation in a strain-dependent way; and (7) decreased the chronological lifespan. The results indicated that the expression of KRASwt in the wild type strain, possibly leads to a hyperactivaction of the RAS/cAMP/PKA signalling pathway, which in turn triggers a decrease of stress resistance and longevity. This study also highlighted the relevance of the yeast background, to the possible achievement of different results, regardless the functional conditions used. The present work contributed to gain insight into KRAS mechanism of action using yeast as a model organism. Furthermore, all obtained results will constitute part of the development of a yeast-based high throughput phenotype platform for future pharmacological testing. |
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| Autores principais: | Carneiro, Eliana Raquel Dias |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Most of our knowledge about basic cellular processes has originated from model organisms. Saccharomyces cerevisiae is consider a model system, representing the simplest eukaryotic organism, whose genome can be easily manipulated allowing suitable analysis and efficient assessment of gene products from humans. RAS genes encode low molecular weight, GTP-binding, GTP-hydrolysing proteins that are highly conserved throughout all eukaryotic species. RAS pathway has attracted attention because of its importance in malignant transformation of human cells. KRAS is a human RAS isoform, expressed in almost all cell types and essential for the normal cellular development, in addition it is the isoform most frequently mutated in many cancer types. The S. cerevisiae has two RAS genes, RAS1 and RAS2 that are essential, once their double deletion renders yeast unviable. These genes are structurally and functionally homolog of the mammalian RAS proto-oncogenes. This conservation allows the use of yeast genetics to the study of KRAS. Considering this, the first aim of this work was to build a set of S. cerevisiae strains (using haploid BY4741 wild type, ras1Δ, and ras2Δ as a basis) expressing the human KRASwt cDNA, using a plasmid-based expression. The second aim was the phenotype screening of these humanized yeasts, as well as of the correspondent recipient strains. The effect of KRASwt expression was evaluate on the cell stress response, growth, chronological aging, cell cycle progression and haploid invasive growth. According to the results, the KRASwt heterologous expression in yeast (1) had a negative or null effect on the resistance to the temperature, pH, osmotic and oxidative stresses; (2) decreased growth on non-fermentable carbon sources; (3) increased the adhesion capacity; (4) stimulated the haploid invasive growth in the ras2Δ strain; (5) modified the budding pattern of the wild type cells; (6) changed the cellular proliferation in a strain-dependent way; and (7) decreased the chronological lifespan. The results indicated that the expression of KRASwt in the wild type strain, possibly leads to a hyperactivaction of the RAS/cAMP/PKA signalling pathway, which in turn triggers a decrease of stress resistance and longevity. This study also highlighted the relevance of the yeast background, to the possible achievement of different results, regardless the functional conditions used. The present work contributed to gain insight into KRAS mechanism of action using yeast as a model organism. Furthermore, all obtained results will constitute part of the development of a yeast-based high throughput phenotype platform for future pharmacological testing. |
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