A functional characterization of AMP-activated protein kinase in Trypanosoma cruzi

Abstract

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to different stresses in many organisms. Sequence and structure of its subunits may change between organisms, but they maintain the same function. The α subunit contains a N-terminal kinase catalytic domain, the β subunit acts as a scaffold and intervenes in the localization of the complex and the γ subunits binds AMP. During the transition from the mammal host to the insect vector, Trypanosoma cruzi suffers nutritional stress from the absence of glucose in the insect?s midgut. The ability to respond to this stress, allows the parasite to differentiate and survive. Recently, it was shown that Trypanosoma brucei AMPK is involved in surface protein expression changes in response to nutritional stress and differentiation. We identified four candidate genes for the AMPK subunits of T. cruzi (α1, α2, β and γ). Each of these subunits was capable of reverting the ?glucose dependent? phenotype of S. cerevisiae conditional mutants alternatively lacking one subunit of the AMPK ortholog SNF1. Also, we overexpressed the α1, β and γ subunits with a hemagglutinin (HA) Tag in CL Brener epimastigotes and evaluated their localization and possible post-translational modifications. Western blots using anti-PhosphoAMPK antibody showed specific stripes corresponding to the expected MW of the alpha subunits. These stripes are reduced in intensity or completely deleted with Lambda phosphatase treatment. Also, epimastigotes treated with an AMPK specific activator show a shift in the intensity pattern of the stripes. Thus, the phosphorylation pattern of the α subunits can be modified in vivo. RT-PCR assays also revealed the endogenous α2 mRNA, but not the α1 mRNA in epimastigotes. Our results show, for the first time, the presence of an AMPK ortholog in Trypanosoma cruzi. In the future, we aim to discover its role in the life cycle and stress responses of this parasite.