In vivo evaluation of the biological function of an AMP-Activated Protein Kinase alfa subunit in Trypanosoma cruzi.

PATRICIO D. GENTA; TAMARA STERNLIEB; NADIA M. BARRERA; MILENA MASSIMINO STEPÑICKA; ALEJANDRA C. SCHOIJET; GUILLERMO D. ALONSO

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB). XXIX Reunión Anual de la Sociedad Argentina de Protozoología (SAP).; 2017

CONJUNTO DE SOCIEDADES DE BIOCIENCIAS

The AMP-activated protein kinase (AMPK) is a heterotrimeric evolutionarily conserved enzyme that typically functions as a metabolic sensor and generates changes in energy homeostasis, through transcriptional and metabolic reprograming. In parasitic protozoa, such as Trypanosoma cruzi, energy sensing is crucial since they must go through sudden environmental changes during the passage through its different stages. AMPK can also influence other cellular processes such as mitochondrial activity, autophagy, endoplasmic reticulum stress, and apoptosis. This protein kinase is composed of the catalytic subunit α and two regulatory subunits β and γ. Using conserved domains and secondary structure analysis, we have been able to identify four candidate genes corresponding to two α subunits, named TcAMPKα1 and TcAMPKα2, and β and γ subunits, named TcAMPKβ and TcAMPKγ respectively. In the present work we have focused on the characterization of TcAMPKα1; for this goal we subcloned into the pRIBOTEX vector the putative orthologous gene of T. cruzi (TcAMPKα1), to which we added an HA tag to perform further experiments.To study its expression, we first evaluated its transcription through RT-PCR and the expression of the HA-fussed protein was confirmed in the soluble fraction by subcellular fractionation and subsequent western blot. After confirming its correct transcription and translation we demonstrated its localization in granules distributed in the cytoplasm by indirect immunofluorescence using an anti-HA antibody.In the present time, we are assessing epimastigote cultures growth of wild type and TcAMPKalfa overexpressing parasites, with or without Compound C (Dorsomorphin) which is a potent and specific inhibitor of the AMPK activity. These studies will allow us to identify the biological function of this protein, which may be essential to maintain the energy homeostasis of the cell and to complete their life cycle.