Regulatory characterization of leucine zipper EF-containing transmembrane protein 1 (Letm1) in cell bioenergetic of Trypanosoma cruzi

• Grant number: 18/04559-6
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): July 01, 2018
• Effective date (End): June 30, 2020
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Aníbal Eugênio Vercesi
• Grantee: Guilherme Rodrigo Reis Monteiro dos Santos
• Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

The parasite Trypanosoma cruzi is the etiological agent of Chagas Disease. The existing drugs have low effectiveness and high toxicity. Structural and functional studies of several proteins are underway, and they are a powerful tool in the identification and development of new chemotherapy targets. Recognized molecular targets for new chemotherapies are the proteins involved in the regulation of mitochondrial functions. Calcium ions have important signaling functions in the life and death processes in the mitochondria matrix. Although extremely important, the regulation mechanism of calcium transport in T. cruzi is poorly understood. Much of the research has shown the importance of the Mitochondrial Calcium Uniporter (MCU) and the transporter responsible for Ca2+ extrusion from the mitochondrial matrix, the leucine zipper EF-containing transmembrane protein 1 (Letm1). While experiments of gene knockout in Drosophila showed the role of Letm1 in Ca2+ transport from the mitochondria matrix in exchange with H+, in Trypanosoma brucei, the function of Letm1 has been proposed to be a K+/H+ exchanger. In the T. cruzi parasite, the function of Letm1 protein is unknown. Therefore, the goal of this project is to study the regulation of the mitochondrial bioenergetics by Letm1 in T. cruzi, evaluating mitochondrial Ca2+ levels, reactive oxygen species generation and cell death induction in T. cruzi by apoptosis or autophagy across decreasing of ATP levels. To carry out the study we will use molecular interventions in Letm1, such as overexpression, site direct mutation using CRISPR/Cas9 system and gene silencing by expression of glmS ribozyme. In addition, we will perform protein expression and purification of Letm1 in order to reconstruct the liposome transport system. (AU)