Caracterização funcional e estrutural das proteínas RUV-1 e RUV-2 do fungo Neurospora crassa
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Data
2016-02-18
Autores
Mateos, Pablo Acera [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho foi realizado um estudo de caracterização das proteínas RUV-1 e RUV-2 de Neurospora crassa, as quais são proteínas ubiquamente encontradas e descritas estar envolvidas em diferentes processos celulares. Resultados anteriores obtidos pelo nosso grupo identificaram, através de espectrometria de massas, a proteína RUV-1 como uma proteína capaz de se ligar ao promotor do gene da glicogênio sintase (gsn) durante a resposta ao choque térmico. Mais tarde, foi demonstrado que a proteína foi capaz de se ligar in vitro, e de maneira especifica, ao motivo de DNA STRE, também presente no promotor gsn. Considerando que a proteína RUV-1 interage com a proteína parceira RUV-2 e, juntas, participam de grandes complexos proteicos que atuam na regulação de diferentes processos celulares, este trabalho teve como objetivo realizar estudos de caracterização das proteínas RUV-1 e RUV-2. Os resultados de expressão gênica mostraram que o gene ruv-1 foi superexpresso na condição de choque térmico e que o gene ruv-2 não mostrou alteração na expressão na mesma condição. Além disso, foi demonstrado que o transcrito do gene ruv-2 é parcialmente processado na situação de choque térmico, e não em outra condição indutora de estresse, através do processo conhecido como intron retention levando à síntese de uma proteína truncada. No entanto, os resultados mostraram que a proteína RUV-2 foi detectada em extratos celulares do fungo obtidos até 4 h de choque térmico. Os cDNAs (ruv-1 e ruv-2) foram inseridos no vetor pET28a e as proteínas expressas em E. coli. Entretanto, ainda não foi finalizada a expressão de ambas utilizando o plasmídeo bicistrônico pETDUET-1, para a análise de interação de ambas proteínas. Neste trabalho também foi construída uma linhagem do fungo modificada geneticamente, a qual produz a proteína RUV-1 fusionada ao tag V5 (RUV-1-V5) e será posteriormente utilizada em ensaio de imunoprecipitação para identificação de proteínas parceiras. Ensaios de imunoprecipatação de cromatina (ChIP) com esta linhagem foram realizados com o objetivo de analisar se a proteína RUV-1 se liga in vivo ao mesmo fragmento de DNA. Entretanto, os experimentos ainda não permitiram identificar a ligação proteína-DNA. Análises de modelagem e dinâmica molecular das proteínas RUV-1 e RUV-2 de N. crassa sugeriram interação entre elas e homologia estrutural a outras proteínas RUV conhecidas
In this work, we performed characterization studies of Neurospora crassa RUV-1 and RUV-2, which are ubiquitous protein and have been described to be involved in different cellular processes. Previous results obtained by our group identified, by mass spectrometry, RUV-1 as a protein able of binding to the glycogen synthase gene promoter (gsn) during heat shock response. Later, it was demonstrated that the protein was able to bind in vitro, and in a specific manner, to the STRE DNA motif, also present in gsn promoter. Since RUV-1 interacts with RUV- 2 protein, and together participate in large protein complexes that act in the regulation of different cellular processes, this study aimed to conduct characterization studies of RUV-1 and RUV- 2 proteins. Gene expression results showed that ruv-1 gene, but not ruv-2 gene was overexpressed under heat shock. Furthermore, it was shown that ruv-2 transcript was incompletely processed under heat shock through a process known as intron retention that leads to the production of a truncated protein. No other stress inducing conditions led to the same phenomenon. However, RUV-2 protein was detected in cell extracts of the fungus exposed to heat shock up to 4 h. The cDNAc (ruv-1 and ruv-2) were inserted into the pET28a vector and the proteins were expressed in E. coli. However, it has not yet been completed the molecular cloning in the bicistronic plasmid pETDUET-1, which allows the production of both proteins in the same cell, what is required for protein-protein interaction analysis. In this work, was also constructed a genetically modified strain, which produces the V5- tagged RUV-1 protein (V5-RUV-1), which that will be later used in immunoprecipitation assay for the identification of partner proteins. Chromatin immunoprecipitation (ChIP) assay using this strain was performed in order to investigate whether RUV-1 protein binds in vivo the DNA fragment from the gsn promoter. Experiments have not allowed the identification of protein-DNA binding yet. Analysis of molecular modelling and dynamics of RUV-1 and RUV-2 proteins suggested the existence of interaction between them and structural homology to other known RUV proteins.
In this work, we performed characterization studies of Neurospora crassa RUV-1 and RUV-2, which are ubiquitous protein and have been described to be involved in different cellular processes. Previous results obtained by our group identified, by mass spectrometry, RUV-1 as a protein able of binding to the glycogen synthase gene promoter (gsn) during heat shock response. Later, it was demonstrated that the protein was able to bind in vitro, and in a specific manner, to the STRE DNA motif, also present in gsn promoter. Since RUV-1 interacts with RUV- 2 protein, and together participate in large protein complexes that act in the regulation of different cellular processes, this study aimed to conduct characterization studies of RUV-1 and RUV- 2 proteins. Gene expression results showed that ruv-1 gene, but not ruv-2 gene was overexpressed under heat shock. Furthermore, it was shown that ruv-2 transcript was incompletely processed under heat shock through a process known as intron retention that leads to the production of a truncated protein. No other stress inducing conditions led to the same phenomenon. However, RUV-2 protein was detected in cell extracts of the fungus exposed to heat shock up to 4 h. The cDNAc (ruv-1 and ruv-2) were inserted into the pET28a vector and the proteins were expressed in E. coli. However, it has not yet been completed the molecular cloning in the bicistronic plasmid pETDUET-1, which allows the production of both proteins in the same cell, what is required for protein-protein interaction analysis. In this work, was also constructed a genetically modified strain, which produces the V5- tagged RUV-1 protein (V5-RUV-1), which that will be later used in immunoprecipitation assay for the identification of partner proteins. Chromatin immunoprecipitation (ChIP) assay using this strain was performed in order to investigate whether RUV-1 protein binds in vivo the DNA fragment from the gsn promoter. Experiments have not allowed the identification of protein-DNA binding yet. Analysis of molecular modelling and dynamics of RUV-1 and RUV-2 proteins suggested the existence of interaction between them and structural homology to other known RUV proteins.
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Palavras-chave
Genética, Bioquimíca, Biología Molecular, Proteínas de choque térmico, Bioinformática, Expressão génica, ChIP, RT-qPCR