Avaliação do fator de início de tradução de eucariotos 5A (eIF5A) na tradução das proteínas S6Ks
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Data
2017-03-24
Autores
Meneguello, Letícia [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O fator de início de tradução de eucariotos 5A (eIF5A) caracteriza-se por ser a única proteína conhecida que apresenta o aminoácido hipusina, formado a partir de uma modificação pós-traducional chamada hipusinação. Apesar de originalmente eIF5A ter sido denominado como fator de início de tradução, a função de eIF5A têm sido relacionada principalmente com a etapa de elongação da tradução. Desde 2013, trabalhos importantes apresentaram evidências de que eIF5A hipusinado possui um papel fundamental na tradução de mRNAs específicos de proteínas que contém consecutivos resíduos de aminoácidos prolina (PPP ou PPG). Tendo em vista estas informações, o objetivo principal deste trabalho consiste no estudo da dependência de eIF5A na tradução da proteína ribosomal S6 kinase 2 (S6K2), pois a mesma apresenta em sua extremidade C-terminal uma região contendo cinco resíduos consecutivos de prolinas. Neste contexto, por meio da produção de uma proteína mutante (S6K2∆Pro), substituindo a região de poli-prolina de S6K2 pela sequência correspondente da proteína homóloga ribosomal S6 kinase 1 (S6K1), foram avaliadas a produção e habilidade fosforilativa da proteína mutante em comparação com S6K2 selvagem. Observou-se que S6K2ΔPro é produzida e sua superexpressão aumenta o conteúdo da proteína ribossomal S6 fosforilada, principal alvo de fosforilação das proteínas S6Ks, assim como a superexpressão das proteínas S6K1 e S6K2. Análises de silenciamento gênico em células HeLa, utilizando moléculas de siRNA para eIF5A, mostraram que a proteína S6K2 endógena não tem seu conteúdo alterado em função da depleção do conteúdo de eIF5A nas condições avaliadas. Além disso, regulando a expressão gênica de TIF51A (homológo do gene EIF5A humano) em Saccharomyces cerevisiae foi observado que a tradução de S6K2 foi apenas levemente afetada pela redução do conteúdo de eIF5A quando comparado à proteína LDB17, uma proteína com conhecida dependência de eIF5A para sua tradução. Ademais, ensaios de imunoprecipitação de S6K2 e S6K2ΔPro seguido de identificação dos peptídeos por espectrômetria de massas mostraram que a região de poli-prolina influencia as interações proteína-proteina de S6K2, visto que, com a mudança da sequência dessa região, observou-se uma interferência no perfil de proteínas associadas, com destaque para as proteínas com atividade transportadora. Dentre as proteínas identificadas, os resultados indicam que a interação de S6K2 com o fator de elongação 2 (eEF2) é influenciada pela região de prolinas presentes no C-terminal de S6K2.
The eukaryotic translation initiation factor 5A (eIF5A), which is highly conserved among eukaryotes, contain the unusual amino acid hypusine, synthesized by a posttranslational modification called hypusination. Even though eIF5A was first denominated as an initiation factor, it has been related to elongation step of the translation process. Since 2013, many studies proposed that eIF5A acts on rescue the ribosome stalling promoted by synthesis of poly-proline motifs containing PPP or PPG sequences. The aim of this study is to evaluate the eIF5A dependence on Ribosomal S6 kinase 2 protein (S6K2) translation, once S6K2 contains a poly-proline stretch on its C-terminus with five consecutive prolines (PPPPP). By producing a mutant protein (S6K2Pro), replacing the polyproline motif on S6K2 for the same region found on homologous protein Ribosomal S6 kinase 1 (S6K1), the content and phosphorylation activity were evaluated. It was observed that S6K2ΔPro is produced by HeLa cells and its RPS6 phosphorylation ability, which is the mainly target of S6Ks phosphorylation, is maintained. Analyzes using siRNA molecules for eIF5A gene silencing in HeLa cells have shown that the endogenous S6K2 protein does not have its content altered as a function of the depletion of the eIF5A content, under the conditions evaluated. Furthermore, by regulating the TIF51A gene expression (homologous of the human EIF5A gene) in Saccharomyces cerevisiae it was observed that the translation of S6K2 was only slightly affected by the reduction of the content of eIF5A when compared to the poly-proline LDB17 protein, a protein with known dependence of eIF5A for its translation. In addition, immunoprecipitation assays of S6K2 and S6K2ΔPro followed by identification of the peptides by mass spectrometry showed that the poly-proline region influences the protein-protein interactions of S6K2, since it was observed an interference in the profile of associated proteins, especially proteins classified as transport activity, in function of poly-proline replacement. Among the identified proteins, the results indicate that the interaction between S6K2 and the elongation factor 2 (eEF2) is influenced by the poly-proline region present at the C-terminal of S6K2.
The eukaryotic translation initiation factor 5A (eIF5A), which is highly conserved among eukaryotes, contain the unusual amino acid hypusine, synthesized by a posttranslational modification called hypusination. Even though eIF5A was first denominated as an initiation factor, it has been related to elongation step of the translation process. Since 2013, many studies proposed that eIF5A acts on rescue the ribosome stalling promoted by synthesis of poly-proline motifs containing PPP or PPG sequences. The aim of this study is to evaluate the eIF5A dependence on Ribosomal S6 kinase 2 protein (S6K2) translation, once S6K2 contains a poly-proline stretch on its C-terminus with five consecutive prolines (PPPPP). By producing a mutant protein (S6K2Pro), replacing the polyproline motif on S6K2 for the same region found on homologous protein Ribosomal S6 kinase 1 (S6K1), the content and phosphorylation activity were evaluated. It was observed that S6K2ΔPro is produced by HeLa cells and its RPS6 phosphorylation ability, which is the mainly target of S6Ks phosphorylation, is maintained. Analyzes using siRNA molecules for eIF5A gene silencing in HeLa cells have shown that the endogenous S6K2 protein does not have its content altered as a function of the depletion of the eIF5A content, under the conditions evaluated. Furthermore, by regulating the TIF51A gene expression (homologous of the human EIF5A gene) in Saccharomyces cerevisiae it was observed that the translation of S6K2 was only slightly affected by the reduction of the content of eIF5A when compared to the poly-proline LDB17 protein, a protein with known dependence of eIF5A for its translation. In addition, immunoprecipitation assays of S6K2 and S6K2ΔPro followed by identification of the peptides by mass spectrometry showed that the poly-proline region influences the protein-protein interactions of S6K2, since it was observed an interference in the profile of associated proteins, especially proteins classified as transport activity, in function of poly-proline replacement. Among the identified proteins, the results indicate that the interaction between S6K2 and the elongation factor 2 (eEF2) is influenced by the poly-proline region present at the C-terminal of S6K2.
Descrição
Palavras-chave
S6K2, eIF5A, Poli-prolina, S6Ks