Identification and study of a putative catalytic subunit AMPKa in Trypanosoma brucei and Trypanosoma cruzi

PATRICIO GENTA; ALEJANDRA C. SCHOIJET; GUILLERMO D. ALONSO; TAMARA STERNLIEB

Santa Fé

Congreso; XXVIII Reunión Anual de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias; 2016

Sociedad Argentina de Protozoología y CURSO - SIMPOSIO Internacional de Biología Celular y Molecular de la Enfermedad de Chagas

AMPK is an heterotrimer evolutionarily conserved enzyme that typically functions as a metabolic sensor and generates changes in energy homeostasis, through transcriptional and metabolic reprograming. This energy sensing is crucial in protozoa such as Trypanosoma cruzi, which must go through sudden changes in the environment during the passage through its different stages. AMPK can also either directly or indirectly influence other cellular processes such as regulating mitochondrial function, autophagy, endoplasmic reticulum stress, and apoptosis. This protein kinase is composed of the catalytic subunit α and two regulatory subunits β and γ. In Trypanosoma brucei AMPK β and γ subunits have been identified and it was shown that they are involved in surface protein expression changes in response to nutritional stress. Nevertheless, the α subunit couldn?t yet be found in this parasite. Using conserved domains and secondary structure analysis, we have been able to identify candidate genes for the catalytic subunit of AMPKα in T. brucei and T. cruzi, named TbAMPKα and TcAMPKα respectively. In the present work we report the cloning and partial characterization of TbAMPα. The amino acid sequence of TbAMPα shows a high identity with the putative TcAMPα of T. cruzi and TvAMPα of T. vivax, but is not well conserved in other AMPKα orthologues such as human, S. cerevisiae and rat. We analyzed the sequence of T. brucei through Conserved Domain tool and found that it has the same type of catalytic domain of the AMPK protein family. It is a protein domain with triple catalytic action: serine, threonine and tyrosine kinase. In addition, the analysis using the SAS FASTA tool revealed a high similarity between the kinase catalytic domains of TbAMPKα and the alpha subunit of human AMPK, but throughout the rest of the sequence, the similarity is poor. The TbAMPKα sequence was amplified by PCR, its identity confirmed by sequencing and subcloned into the pDEST15 expression vector fused to GST at its N-terminus. The expression of the recombinant protein was confirmed by Western blot assays and afterward the protein was glutathione-based affinity purified. On the other hand, we have also amplified and subcloned into the pRIBOTEX vector the putative orthologous gene of T. cruzi (TcAMPKα). Future studies using transgenic parasites will allow us to evaluate its subcellular localization as well as its biological role in the parasite.