Cytochrome P450 reductases of Trypanosoma cruzi: Subcellular localization and functional analysis

BLANCO OBREGÓN DALMIRO; PORTAL PATRICIO; FLAWIÁ MIRTHA; PAVETO CRISTINA

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2012

Cytochrome P450 (CYPs) are an extensively described superfamily of enzymes involved in the synthesis of endogenous compounds such as steroids, as well as in the detoxification of foreign compounds, including therapeutic drugs. A single Cytochrome P450 Reductase (CPR) is known to interact with the CYPs in most organisms. We have identified and characterized a gene family consisting of three CPRs in T. cruzi , named TcCPR-A, TcCPR-B and TcCPR-C. Although recombinant TcCPR-A, -B and -C were able to complement CYP activities in an reconstituted system, only TcCPR-B and -C overexpression in the parasite increased ergosterol membrane content, while TcCPR-A resulted lethal. We present here the results of complementation in yeast: expression of TcCPR-B and -C in the CPR knock-out strain WR∆ can partially restore yeast normal growth and susceptibility to the antifungal ketoconazole, while expression of TcCPR-A cannot. This suggests a different role for TcCPR-A, in agreement with our previous observations. Since TcCPR-B and TcCPR-C presented different localization in previous immunofluorescence studies, their putative transmembrane domains were fused to GFP. Chimeric proteins presented similar localizations to the wild type, in the reservosome and ER respectively. This would indicate that the N-terminal transmembrane domain fragments cloned contain sequences that commit protein localization.