IDENTIFICATION AND CHARACTERIZATION OF AMP-ACTIVATED PROTEIN KINASE IN TRYPANOSOMA CRUZI

STERNLIEB T; GENTA PD; FIGUERAS MJ; FLAWIÁ MM; ALONSO GD

CABA

Congreso; XXVII Reunión anual de la Sociedad Argentina de Protozoología; 2015

Sociedad Argentina de Protozoología

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to nutrient stress in many organisms. Sequence and structure of its subunits may change between organisms, but they maintain the same function. The α subunit contains a kinase domain as well as a regulatory domain that inhibits the enzyme in the absence of AMP. The β subunit acts as a scaffold for the other components, while the γ subunit is thought to be involved in AMP binding. 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, AMPK β and γ subunits have been identified in Trypanosoma brucei, and it was shown that they are involved in surface protein expression changes in response to nutritional stress. Nevertheless, the α subunit couldn?t be found. Using conserved domains and secondary structure analysis, we have been able to identify candidate genes for the catalytic subunit of both organisms, named TbAMPKα and TcAMPKα. On the other hand, we have identified the genes corresponding to AMPK β and γ subunits in T. cruzi (TcAMPKβ and γ), which present significant sequence differences from human AMPK. These three gene sequences were all successfully expressed in E. coli attached to a GST tag. Subsequently, this will allow us to purify the recombinant proteins and perform different binding assays. Also, we generated transgenic epimastigotes lines overexpressing TcAMPKγ along with a hemagglutinin tag, which allowed us to determine its co-localization with glycosomes through immunofluorescence assays. Among other objectives, we are trying to elucidate the effects of nutritional stress on the subunits localization and function by starvation assays.