Participação de eIF2α e sua fosforilação durante o ciclo de vida e sobrevivência de Trypanosoma

• Ano de defesa: 2018
• Autor(a) principal: Machado, Fabricio Castro [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Trypanosomatids; Trypanosome; Tripanossomatídeos; Trypanosoma Cruzi
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=6734885; https://repositorio.unifesp.br/handle/11600/52985

Trypanosomatids regulate their gene expression mainly at the posttranscriptional level through mRNA processing, exporting, stabilization and by the control of translation. Translation initiation can be regulated by phosphorylation of the eukaryotic initiation factor 2 (eIF2) at serine 51 of its  subunit (eIF2), which stops overall protein synthesis by decreasing the availability of ribosomes coupled with initiator tRNA. The Nterminal of eIF2α is extended in trypanosomatids, so the threonine at position 169 (T169) is the regulatory phosphorylation residue. In this thesis we evaluated eIF2 expression and phosphorylation during the life cycle of trypanosome species, especially Trypanosoma cruzi, protozoa parasite that cause Chagas disease. Initially for T. cruzi, we observed the total levels of eIF2α relatively diminished in infective and nonreplicative forms, called trypomastigotes, in comparison to forms that multiply in the intestine of the insect vector or in the interior of mammalian cells. This result is convergent with a decreased global translation in infective forms. We also found that the phosphorylation of eIF2α occurs in proliferative intracellular forms prior to differentiation into trypomastigotes. To assess the role of eIF2 regulatory phosphorylation, we generated parasites overexpressing eIF2 with substitutions on phosphorylation sites by a nonphosphorylatable alanine residue. The presence of alanine instead of T169 in overexpressing eIF2α parasites led to a deregulation in protein synthesis with constitutive expression of common trypomastigotemetacyclic proteins. The mutated parasites infected less and proliferated more slowly in cultured cells and in mice. In addition, they were more susceptible to benznidazole, suggesting that eIF2 regulatory phosphorylation can be relevant in controlling gene expression during T. cruzi infection and stress response. For this reason, we tested urea derivatives compounds which have already been used to inhibit the proliferation of cancer cells by the induction of eIF2 phosphorylation. Of 25 compounds tested, only compound I17 inhibited 50% of T. cruzi and T. brucei parasite proliferation at low concentrations (1 to 3 μM) also presenting a 10 to 20fold selectivity increase for parasites in relation to mammalian cells. I17 reversibly affected the shape of parasites, stopping its cell cycle stopped in G1 phase and arresting global translation. As this compound was less effective in T. brucei containing only eIF2α with alanine substituting T169, and seemed to be partially depended on the expression of a specific protein kinase (TbK3), we infer that I17 partially acts on the parasites through the phosphorylation of eIF2α. As a conclusion of this thesis, we have revealed that the eIF2 phosphorylation pathway is important during T. cruzi life cycle and can be explored in the understanding and the development of therapies for Chagas disease and sleep sickness.