IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Heme A biosynthesis in Trypanosoma cruzi, the essential cofactor for the complex Cytochrome c Oxidase
Autor/es:
MARCELO L. MERLI, LUCÍA V. FERRERO, JOSEFINA HERNÁNDEZ AND JULIA A. CRICCO
Lugar:
Ciudad autonoma de Buenos Aires
Reunión:
Simposio; 3° Encuentro Nacional sobre Enfermedades Olvidadas y XIV Simposio Internacional sobre Control Epidemiológico de Enfermedades Transmitidas por Vectores; 2012
Institución organizadora:
Mundo Sano
Resumen:
Heme is an essential cofactor for aerobic organisms. Its biosynthesis is conserved throughout evolution requiring for the action of eight different enzymes. Trypanosoma cruzi, as well as other trypanosomatids, has nutritional requirements for different cofactors where heme is included. Because this organism is unable to synthesize heme, it must be acquired from the environment (the different hosts). Once heme is imported, it is distributed inside the cell and inserted into the target heme-proteins. Heme-proteins form part of the respiratory chain complexes in the parasite mitochondrion, cytochromes involved in polyunsaturated fatty acid metabolism and sterol biosynthesis in the endoplasmic reticulum. As heme is a highly toxic molecule, it is well accepted that heme carriers involved in its distribution might exist. In eukaryotic cells, these types of proteins were not reported yet and the heme transport and distribution processes in trypanosomatids remain unknown. We are interested into elucidate how heme is imported and distributed in T. cruzi, specially we are focused in its traffic to the mitochondrion and its conversion into heme A, the essential cofactor only for the respiratory complex IV, the cytochrome c oxidase (CcO). In our lab, we identified the codifying sequences for the T. cruzi enzymes involved in heme A biosynthesis, TcCox10 (heme O synthase) and TcCox15 (heme A synthase) and characterized their function in the yeast S. cerevisiae. The mRNA levels of these genes (TcCOX10 and TcCOX15) were analyzed by qRT-PCR and we observed the presence of these mRNA along different parasite life stages . In order to have a direct protein measurement, we design strategies to determine the presence and amount of them by western blot analysis. We cloned a small fragment of TcCox10 or TcCox15 (both are integral membrane proteins) as GST-fusion proteins to get specific antibodies. The obtained polyclonal antibodies were purified and used to evaluate TcCox10 and TcCox15 levels in T. cruzi by western blot analysis and their localization by indirect immunofluorescence assays. The protein TcCox15 was observed in different life stages of T. cruzi and was localized in the mitochondrion of the parasite (epimastigote stage). We postulated that the observed changes in mRNA levels could be a form of regulation reflecting differences in respiratory requirements at different life stages. This observation was corroborated by the presence of TcCox15 protein at the same life stages. As an approach to evaluate the relevance of the heme A biosynthesis, we designed strategies to study the effect of shutdown heme A synthesis. Preliminary results indicate that this pathway is relevant, at least for the epimastigote stage of T. cruzi, suggesting this parasite requires heme A for an active respiratory chain.