TcAMPK: Identi cation and characterization of a cellular energy homeostasis hub regulator in Trypanosoma cruzi

STERNLIEB T; SCHOIJET AC; GENTA PD; ALONSO GD

Resistencia, Chaco

Congreso; XXX Reunión Anual de la Sociedad Argentina de Protozoología; 2018

Sociedad Argentina de Protozoología

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to dierent stresses in many organisms. Trypanosoma cruzi is the causative agent of Chagas disease, which affects between 6 and 7 million people. During the transition between the mammalhost and the insect vector, T. cruzi faces nutritional, oxidative, osmotic and other types of stress, all of which can prompt the parasite to remodel its metabolism and force it to re-establish energy homeostasis.The ability to respond to stress, allows the parasite to differentiate and survive. Recently, it was shown that Trypanosoma brucei AMPK is involved in the differentiation from the slender to stumpy stages and in surface protein expression changes in response to nutritional stress. This underscores the relevance of AMPK in critical responses for parasite life cycle progression. We identied four candidate genes for the AMPK subunits of T. cruzi(alfa1 and alfa2 catalytic subunits, beta and gamma regulatory subunits). The alpha subunits showsignicant sequence and structure differences from the human orthologs. The presence of these subunits in T. cruzi epimastigotes was confirmed by RT-PCR, Western blot with a phospho-AMPKalfa specic antibody, mass spectrometry and by incorporation of 32P to the specic AMPK substrate SAMS in a kinase activityassay. This last assay also allowed us to observe the upregulation of AMPK activity under epimastigote starvation, and the inhibition of this activity with dorsomorphin, a specic inhibitor. Also, each of these subunits was capable of reverting the "glucose dependent" phenotype of S. cerevisiae conditional mutantsalternatively lacking one subunit of the AMPK ortholog SNF1. Dansylcadaverine incorporation assays show that the over-expression of the alfa1 subunit could increase autophagy between 20% and 40% in starvation assays. Our results show, for the first time, the presence of a functional AMPK ortholog in Trypanosomacruzi, and its involvement in one relevant metabolic pathway. In the future, we aim to dig deeper in its role through the life cycle and stress responses of this parasite.