Caracterização de proteínas importantes para a manutenção da função mitocondrial durante o estresse genotóxico em Saccharomyces cerevisiae
Data
2023-02-14
Autores
Silva, Aline Montenegro 
Orientadores
Cussiol, José Renato Rosa
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Mitocôndrias são organelas responsáveis por desempenhar processos celulares vitais, tais como a produção de ATP, grupos ferro-enxofre, heme e regulação do cálcio. Possui DNA próprio, e sua replicação ocorre de maneira independente do DNA nuclear. A instabilidade genômica do DNA mitocondrial (mtDNA) está associada a processos patológicos, como doenças neurodegenerativas, envelhecimento precoce e câncer. Portanto, a manutenção e reparo do mtDNA são de suma importância para os organismos. Foi observado anteriormente que regiões deletadas do mtDNA são flanqueadas por sequências repetidas (DRMD do inglês, Direct Repeat Mediated Deletions) sugerindo que mecanismos de reparo por recombinação poderiam explicar a existência dessas deleções. Entretanto, pouco se sabe ainda sobre a contribuição de mecanismos de recombinação homóloga para o reparo do mtDNA. Temos como objetivo investigar e caracterizar o papel de proteínas potencialmente envolvidas na manutenção e reparo do mtDNA assim como na função mitocondrial utilizando a levedura Saccharomyces cerevisiae como organismo modelo. Resultados preliminares obtidos pelo nosso grupo de pesquisa mostraram através de proteômica quantitativa que as proteínas Rad54 e Yhb1 estavam enriquecidas em frações mitocondriais de leveduras tratadas com a genotoxina MMS, indicando um possível papel mitocondrial dessas proteínas durante o estresse genotóxico. Dessa forma, produzimos linhagens nocautes rad54 e yhb1. Utilizando um sistema repórter capaz de quantificar DRMDs, mostramos que linhagem rad54 apresentou uma redução nos eventos de recombinação sugerindo que poderia atuar diretamente no reparo do mtDNA. Embora não tenhamos observado mudanças na frequência de reparo por recombinação mitocondrial, a linhagem yhb1 apresentou uma maior resistência ao tratamento com a genotoxina MMS. Além disso, ensaio de western blot mostrou que o tratamento com MMS leva a um aumento dos níveis proteicos de Yhb1 e o aparecimento de uma isoforma com maior mobilidade eletroforética sugerindo um possível endereçamento de Yhb1 para a mitocôndria durante o estresse genotóxico.
Mitochondria are organelles responsible for performing vital cellular processes, such as the production of ATP, ironsulfur groups, heme and calcium regulation. It has its own DNA and its replication occurs independently of the nuclear DNA. The genomic ins tability of mitochondrial DNA (mtDNA) is associated with pathological processes, such as neurodegenerative diseases, premature aging and cancer. Therefore, maintenance and repair of mtDNA are of paramount importance for organisms. It has been previously ob served that deleted regions of the mtDNA are flanked by Repeat Mediated Deletions Direct (DRMD) suggesting that recombination repair mechanisms could explain the existence of these deletions. However, little is known yet about the contribution of homologou s recombination mechanisms to mtDNA repair. We aim to investigate and characterize the role of proteins potentially involved in mtDNA maintenance and repair as well as mitochondrial function using the yeast Saccharomyces cerevisiae as a model organism. Pre liminary results obtained by our research group showed through quantitative proteomics that Rad54 and Yhb1 proteins were enriched in mitochondrial fractions of yeast treated with the genotoxin MMS, indicating a possible mitochondrial role of these proteins during genotoxic stress. Thus, we produced rad54 and yhb1 knockout strains. Using a reporter system capable of quantifying DRMDs, we showed that strain rad54 showed a reduction in recombination events suggesting that it could act directly on mtDNA repa ir. Although we did not observe changes in the frequency of repair by mitochondrial recombination, cells lacking Yhb1 showed increased resistance to treatment with the genotoxin MMS. Furthermore, western blot assay showed that MMS treatment leads to an inc rease in Yhb1 protein levels and the appearance of an isoform with higher electrophoretic mobility suggesting a possible addressing of Yhb1 to mitochondria during genotoxic stress.
Mitochondria are organelles responsible for performing vital cellular processes, such as the production of ATP, ironsulfur groups, heme and calcium regulation. It has its own DNA and its replication occurs independently of the nuclear DNA. The genomic ins tability of mitochondrial DNA (mtDNA) is associated with pathological processes, such as neurodegenerative diseases, premature aging and cancer. Therefore, maintenance and repair of mtDNA are of paramount importance for organisms. It has been previously ob served that deleted regions of the mtDNA are flanked by Repeat Mediated Deletions Direct (DRMD) suggesting that recombination repair mechanisms could explain the existence of these deletions. However, little is known yet about the contribution of homologou s recombination mechanisms to mtDNA repair. We aim to investigate and characterize the role of proteins potentially involved in mtDNA maintenance and repair as well as mitochondrial function using the yeast Saccharomyces cerevisiae as a model organism. Pre liminary results obtained by our research group showed through quantitative proteomics that Rad54 and Yhb1 proteins were enriched in mitochondrial fractions of yeast treated with the genotoxin MMS, indicating a possible mitochondrial role of these proteins during genotoxic stress. Thus, we produced rad54 and yhb1 knockout strains. Using a reporter system capable of quantifying DRMDs, we showed that strain rad54 showed a reduction in recombination events suggesting that it could act directly on mtDNA repa ir. Although we did not observe changes in the frequency of repair by mitochondrial recombination, cells lacking Yhb1 showed increased resistance to treatment with the genotoxin MMS. Furthermore, western blot assay showed that MMS treatment leads to an inc rease in Yhb1 protein levels and the appearance of an isoform with higher electrophoretic mobility suggesting a possible addressing of Yhb1 to mitochondria during genotoxic stress.