Papel da proteína quinase ativada por AMP (AMPK) em Trypanosoma cruzi

• Autor(a) principal: Gregório, Nathaly Renata Henrique [UNIFESP]
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: https://repositorio.unifesp.br/xmlui/handle/11600/62139
• topic: AMPK; T. cruzi; fosforilação; ATP; Composto C

Trypanosoma cruzi is a flagellated protozoan that causes Chagas disease. During its biological cycle, it has a high capacity for environmental adaptation, as it lives in different hosts at each stage of its life cycle. However, the mechanisms that control these adaptations are not yet well known and it is postulated that they depend on the availability of nutrients, which results in different signaling mechanisms leading to energy production in states of nutritional deficiency. Adenosine monophosphate kinase (AMPK) is a heterotrimer formed by two subunits (β and γ) and a catalytic subunit (α). It has been shown that AMPK activation of Trypanosoma brucei is capable of inducing differentiation of replicative forms. Genes encoding proteins homologous to AMPK are also present in T. cruzi, and their role is still poorly characterized in this parasite. In this work, we investigated the role of AMPK in T. cruzi by examining its phosphorylation under different situation. We performed in silico tests to estimate the structural conservation and amino acid composition of the TcAMPK subunits with other organisms. T. cruzi were found to contain two catalytic subunits, TcAMPK α1 and α2 with a high degree of conservation in the catalytic loop. Only TcAMPKα1 showed a typical phosphorylatable serine (Ser 172). In contrast, TcAMPKα2 has at the same position a serine residue. In parasites maintained in rich medium both chains were found phosphorylated. We observed that glucose levels did not affect TcAMPKα1 and α2 phosphorylation. A band recognized by the same anti-phospho-172 was recognized with the expected size when the α-1 chain was tagged with mNeonGreen-Myc protein. The presence of Compound C (Dorsomorphin), an inhibitor of AMPK of different organisms, inhibited growth of epimastigotes but did not cause dephosphorylation of TcAMPK. However, we noticed that the parasites in a nutrient-poor environment, in which ATP levels decreased, the presence of this inhibitor, did not recover the phosphorylation of TcAMPKα and the parasites underwent morphological changes, exhibiting a spherical and flagella-free format becoming like intracellular amastigotes. Addition of nutrients in poor medium in the presence of Compound C restored partially the ATP levels favoring the survival of epimastigotes. Several attempts to generated parasites lacking either the α1 or α2 chains were unsuccessful, suggesting it’s essential role for the parasite.