Elucidating the Cytochrome P450 Reductase functions in Trypanosoma cruzi

PORTAL P; ALONSO G D; FLAWIA MIRTHA M; FERNANDEZ VILLAMIL S H; PAVETO C

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

Cytochrome P450 hemoproteins (CYPs) are involved in the synthesis of endogenous compounds such as steroids, fatty acids and prostaglandins as well as in the activation and detoxification of foreign compounds including therapeutic drugs. The membrane-bound form of cytochrome P450 reductase (CPR) orchestrates the stepwise electron transfer from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. Although there are numerous functional CYP genes in mammals, there is only one CPR gene in each species. The main sterol found in a variety of membranes, is cholesterol however other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. The production of a special class of sterols, which includes ergosterol and other 24-methyl sterols required for cell growth and viability, is an important metabolic pathway in these organisms that is absent in mammalian cells. The member CYP51, is a key enzyme in sterol synthesis present in Trypanosomatids (Lepesheva et al, Biochem Soc Trans. 2006, 34(Pt 6):1161-4). We have identified a gene family consisting of three putative CPRs in T. cruzi. The encoded proteins, named TcCPR-A, TcCPR-B and TcCPR-C, have calculated molecular masses of 68.6 kDa, 78.4 kDa and 71.3 kDa, respectively. Deduced amino acid sequences share 11% identity, possess the conserved binding domains for FMN, FAD and NADPH and differ in the hydrophobic 27-amino acid residues of the N-terminal extension, which is absent in TcCPR-A. Interestingly, phylogenetic tree analysis revealed that TcCPR-A and other related CPRs clustered with human NR1, a cytoplasmic flavoprotein oxidoreductase which appears widely expressed in human cancer cell lines. A common feature of all CPRs belonging to this cluster is the absence of the characteristic N-terminal transmembrane domain. Every T. cruzi CPR, TcCPR-A, TcCPR-B and TcCPR-C, were cloned and expressed in Escherichia coli. All of the recombinant enzymes reduced cytochrome c in a NADPH absolutely dependent manner with low Km values for this cofactor. They all were also strongly inhibited by diphenyleneiodonium, a classical flavoenzyme inhibitor. In addition, TcCPRs could support CYP activities when assayed in reconstituted systems containing rat liver microsomes. Polyclonal antiserum rose against the recombinant enzymes TcCPR-A and TcCPR-B demonstrated its presence in every T. cruzi developmental stage, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP+/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole also suggenting a participation of TcCPR-B in the detoxification metabolism of the parasite (Portal et al. Mol. Biochem. Parasitol. 2008, 160: 42?51). In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations. (De Vas et al. International Journal for Parasitol. 2011, 41:99?108).