Characterization of T. cruzi nuclear adenylate kinase

CAMARA, MARIA DE LOS MILAGROS; BOUVIER, LEÓN ALBERTO; MIRANDA, MARIANA; PEREIRA, CLAUDIO

Guaruja

Congreso; 1st International meeting of Cell Biology of Pathogens; 2011

Background: Adenylate kinases (ADKs) are enzymes involved in cellular energetic homeostasis; they are involved in nucleotide interconvertion. In typical eukaryotic cells, there are very few isoforms of ADKs however, in trypanosomatids there have been characterized a large number of isoforms. Nuclear ADKS have recently been found in several organisms. The yeast homologous FAP7 is an essential protein involved in the processing of the ribosome?s small subunit. In this work we study the nuclear ADK isoform (ADKN) of T. cruzi. Methods: First of all we measured the biochemical activity of this enzyme. In order to determine its subcellular localization we generated transgenic parasites which express the protein fused to GFP. Bioinformatic analysis revealed that this protein does not present classical nuclear localization signals (NLS), so we mapped the non-canonical NLS, by segmenting the protein and making fusions to GFP. Furthermore we studied ADKN localization and expression under different media treatments and along the parasites growth curve by Western Blot and immunofluorescence assays. We analyzed its expression by real time PCR along the parasite?s growth curve. Finally we realized complementation assays in yeast, using a strain which has the FAP7 gene under the control of a galactose inducible promoter. Conclusions: Using a bioinformatic approach we identified a possible isoform of nuclear adenylate kinase from T. cruzi. In vitro assays revealed that it presents Adenylate kinase and ATPase activity. We detected higher levels of ADKN in the first days of the growth curve, being almost absent after day seven. Supplementation of media with carbon sources induces higher levels of ADKN in old cultures. By deletion analyses we were able to narrow the atypical nuclear localization signal to the amino terminal of the protein (1-83 aa). Furthermore we detected that the 3?UTR of ADKN could be the responsible of the differential expression levels along the growth curve. Finally complementation assays in yeasts revealed that it could be involved in similar processes as its homolog FAP7.