Oxidative stress response mechanisms in phosphorylation of eIF2± and AMPK in T. cruzi

Abstract

In this project we will evaluate in Trypanosoma cruzi, the protozoan causing Chagas Disease, the role of phosphorylation of eIF2± in the response to different stresses and the activation of AMPK, sensor of the energy state of eukaryotic cells. During nutritional stress eIF2± is phosphorylated, decreasing protein synthesis, and leading to differentiation to the infective form of the parasite. Oxidative stress and low heme levels also cause phosphorylation of eIF2± by TcK2 kinase. In our group we obtained T. cruzi with eIF2± in which threonine T169 was replaced by alanine using CRISPR-Cas9. These parasites were not able to phosphorylate eIF2, making them more susceptible to hydrogen-peroxide induced stress. We have also verified that the protein kinase AMPK, which is a sensor of nutrient levels and oxidative stresses, is phosphorylated both after oxidative stresses or in the lack of nutrients. After phosphorylation, AMPK components of higher molecular mass are detected, which could correspond to the ubiquitination of the AMPK chains. This enzyme in other eukaryotes can be activated by the protein kinase CamKII through calcium accumulation in cells, in the presence of ROS and heme derivative, and it has been verified that these processes are related to the proliferation of parasites in the epimastigote form. In our group we also obtained parasites with mNeonGreen tags in the N-terminal in AMPK±1 and ±2 by the CRISPR-Cas9 system. Thus, we intend to verify how phosphorylation of eIF2±, and AMPK occurs under oxidative stress conditions and the importance of these processes for the survival and differentiation of the parasite. For this we intend to: 1) to determine the susceptibility to oxidative stress-induced by oxidative compounds or nitrous agents, in the parasites modified in eIF2± and knockout parasites for TcK2, both already obtained in the laboratory; 2) to determine the effect of the same agents on phosphorylation of the chains of AMPK ±1 and ±2 and changes in the ATP/ADP/AMP ratio in each situation; 3) identify enzymes involved in AMPK phosphorylation using CamKII pharmacological inhibitors or altering intracellular Ca2+ levels of the parasite; 4) check whether the signaling pathways for phosphorylation of eIF2± are related to AMPK activation using mutant parasites eIF2±/T169A. This study will allow us to understand how the parasite responds to situations faced during the life cycle. (AU)