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Evaluation of eukaryotic translation initiation factor 5A (eIF5A) in the translation of S6Ks proteins

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Author(s):
Letícia Meneguello
Total Authors: 1
Document type: Master's Dissertation
Press: Rio Claro. 2017-05-16.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
Defense date:
Advisor: Augusto Ducati Luchessi
Abstract

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. (AU)

FAPESP's process: 14/27154-0 - EVALUATION OF EUKARYOTIC TRANSLATION INITIATION FACTOR 5A (EIF5A) IN THE TRANSLATION OF S6KS PROTEINS
Grantee:Letícia Meneguello
Support Opportunities: Scholarships in Brazil - Master