Análise comparativa da expressão de homólogos do fator de iniciação da tradução eIF4G ao longo do ciclo de vida de Leishmania amazonensis

The genus Leishmania comprises 30 species of flagellated protozoa from the family Tripanosomatidae, of which 20 are pathogenic to humans. These organisms have complex life cycles and molecular particularities not observed in most eukaryotes, like absence of transcriptional regulation. Thus, the regulation of gene expression occurs in post-transcriptional steps, with the initiation of mRNA translation representing the most important event. Different factors called eIFs (eukariotic initiation factors) are involved, with emphasis in eIF4Fcomplex, which promotes mRNA recognition and its ribosome interaction. This eIF4F complex consists of three subunits: eIF4A (RNA helicase), eIF4E (cap binding protein) and eIF4G (scaffold protein, for eIF4F complex maintenance). In trypanosomatids, five eIF4G homologous (EIF4G1 to G5) was described, however, little is known about the specific cellular functions of these homologous. In this manner, we evaluated the expression and characterized post-translational modifications of the eIF4G homologous during the Leishmania amazonensis life cycle, especially phosphorylation, considering the translational regulation by phosphorylation of eIF s via MAP kinases observed in higher eukaryote. For this reason, cultures were performed with the L. amazonensis promastigote and amastigote axenic forms, and the protein extract, collected from different growth phases, were analyzed by Western blotting. These results demonstrated the detection of all eIF4G homologues during the life cycle of L. amazonensis, while more than one protein isoform were observed for the EIF4G1, G4 and G5 homologues, suggesting possible post-translational modifications. Posteriorly, the EIF4G3 and EIF4G4 gene expression were investigated under differential growth conditions using six distinct inhibitors, however no change was observed in the expression pattern, which suggests that these proteins are quite stable and have long half-life extending. Finally, In silico analysis shows specifics MAP kinase-dependent phosphorylation sites presents in all eIFG homologues and further analysis with EIF4G3 and EIF4G4 confirmed the existence of phosphorylation mechanisms acting on these factors.These present data help to clarify the molecular mechanisms involved in post-transcriptional regulation of gene expression characteristic of these organisms.