Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi.

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.

Safety assessment of nonbrowning potatoes: opening the discussion about the relevance of substantial equivalence on next generation biotech crops.

It is expected that the next generation of biotech crops displaying enhanced quality traits with benefits to both farmers and consumers will have a better acceptance than first generation biotech crops and will improve public perception of genetic engineering. This will only be true if they are proven to be as safe as traditionally bred crops. In contrast with the first generation of biotech crops where only a single trait is modified, the next generation of biotech crops will add a new level of complexity inherent to the mechanisms underlying their output traits. In this study, a comprehensive evaluation of the comparative safety approach on a quality-improved biotech crop with metabolic modifications is presented. Three genetically engineered potato lines with silenced polyphenol oxidase (Ppo) transcripts and reduced tuber browning were characterized at both physiological and molecular levels and showed to be equivalent to wild-type (WT) plants when yield-associated traits and photosynthesis were evaluated. Analysis of the primary metabolism revealed several unintended metabolic modifications in the engineered tubers, providing evidence for potential compositional inequivalence between transgenic lines and WT controls. The silencing construct sequence was in silico analysed for potential allergenic cross-reactivity, and no similarities to known allergenic proteins were identified. Moreover, in vivo intake safety evaluation showed no adverse effects in physiological parameters. Taken together, these results provide the first evidence supporting that the safety of next generation biotech crops can be properly assessed following the current evaluation criterion, even if the transgenic and WT crops are not substantially equivalent.

Multiple NADPH-cytochrome P450 reductases from Trypanosoma cruzi suggested role on drug resistance.

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. Cytochrome P450 reductase (CPR, E.C.1.6.2.4) transfers electrons from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. This work presents the complete sequences of three non-allelic CPR genes from Trypanosoma cruzi. The encoded proteins named TcCPR-A, TcCPR-B and TcCPR-C have calculated molecular masses of 68.6kDa, 78.4kDa and 71.3kDa, respectively. Deduced amino acid sequences share 11% amino acid 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. Every T. cruzi CPRs, 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 K(m) 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 stages, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole. We suggest a participation of TcCPR-B in the detoxification metabolism of the parasite.

TcPARP: A DNA damage-dependent poly(ADP-ribose) polymerase from Trypanosoma cruzi.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme present in most eukaryotes and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catabolised by poly(ADP-ribose) glycohydrolase. Here, we describe the cloning and characterisation of a PARP from Trypanosoma cruzi (TcPARP). The recombinant enzyme (Mr=65) required DNA for catalytic activity and it was strongly enhanced by nicked DNA. Histones purified from T. cruzi increased TcPARP activity and the covalent attachment of [32P]ADP-ribose moieties to histones was demonstrated. TcPARP required no magnesium or any other metal ion cofactor for its activity. The enzyme was inhibited by 3-aminobenzamide, nicotinamide, theophylline and thymidine but not by menadione. We demonstrated an automodification reaction of TcPARP, and that the removal of attached PAR from this protein resulted in an increase of its activity. The enzyme was expressed in all parasite stages (amastigotes, epimastigotes and trypomastigotes). When T. cruzi epimastigotes were exposed to DNA-damaging agents such as hydrogen peroxide or beta-lapachone, PAR drastically increased in the nucleus, thus confirming PAR synthesis in vivo and suggesting a physiological role for PARP in trypanosomatid DNA repair signalling.

TcrPDEA1, a cAMP-specific phosphodiesterase with atypical pharmacological properties from Trypanosoma cruzi.

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of cAMP and cGMP, and regulate a variety of cellular processes by controlling the levels of these second messengers. We have previously described the presence of both a calcium-stimulated adenylyl cyclase and two membrane-bound cAMP-specific PDEs (one of them strongly associated to the flagellum and the other one with a possible vesicular localization) in Trypanosoma cruzi. Here we report the identification and characterization of TcrPDEA1, a singular phosphodiesterase of T. cruzi which is resistant to the typical phosphodiesterase inhibitors, such as IBMX, papaverine and theofylline. TcrPDEA1 is a single copy gene that encodes a 620-amino acid protein, which is grouped with PDE1 family members, mainly with its kinetoplastid orthologs. TcrPDEA1 was able to complement a mutant yeast strain deficient in PDE genes, demonstrating that this enzyme is a functional phosphodiesterase. TcrPDEA1 is specific for cAMP with a high K(m) value (191.1+/-6.5 microM). Cyclic GMP neither activates the enzyme nor competes as a substrate. In addition, calcium-calmodulin did not affect the kinetic parameters and, as its counterpart in T. brucei, magnesium showed to be crucial for its activity and stability. Although TcrPDEA1 function remains unclear, its presence points out the high complexity of the cAMP signaling in trypanosomatids and the possible compartmentalization of the enzymes involved in the cAMP pathway.

TcPDE4, a novel membrane-associated cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Cyclic nucleotide phosphodiesterases constitute the only known mechanism to inactivate regulatory signals involving cAMP or cGMP. In our laboratory a cAMP-specific phosphodiesterase associated to the flagellar apparatus, named TcPDE1, was identified in Trypanosoma cruzi. By using the catalytic domain sequence of TcPDE1 to screen a Trypanosoma cruzi genomic data base, a novel T. cruzi phosphodiesterase sequence was found and characterized. TcPDE4 encodes a 924-amino acid protein and shows homology with the PDE4 vertebrate subfamily. The sequence shows three conserved domains, FYVE, phosphohydrolase and PDEaseI. The FYVE zinc-finger domain is characteristic of proteins recruited to phosphatidylinosytol 3-phosphate-containing membranes, whereas the two others are characteristic of phosphohydrolases and members of the cyclic nucleotide phosphodiesterases. Sequence analysis shows all characteristic domains present at the type-4 phosphodiesterases specific for cAMP. Moreover, TcPDE4 shows the inhibition profile characteristic for PDE4 subfamily, with an IC50 of 10.46 microM for rolipram and 1.3 microM for etazolate. TcPDE4 is able to complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. The enzyme is specific for cAMP, Mg(2+)-dependent and its activity is not affected by cGMP or Ca(2+). The association of TcPDE4 with membranes was studied by subcellular fractionation of recombinant yeast and extraction in several conditions. Most of the enzyme remained associated to the membrane fraction after treatment with high salt concentration, detergent, or chaotropic agents. This support previous hypotheses that in this parasite cAMP phosphodiesterases, and consequently cAMP levels, are compartmentalized.

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution.

The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution.

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei.

BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. METHODS: The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H2O2, was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. RESULTS: Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H2O2 treatment when compared to their wild type counterparts. PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. CONCLUSIONS: PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems.

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes.

Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

Arginine metabolism in Trypanosoma cruzi is coupled to parasite stage and replication.

L-Arginine plays an essential role in the energetic metabolism of Trypanosoma cruzi. In this work we propose a relationship between L-arginine uptake, arginine kinase activity and the parasite replication ability. In epimastigote cultures L-arginine uptake decreases continuously accompanying a cell replication rate reduction. The use of conditioned or fresh medium mimics uptake variations. Interestingly, in non-replicative trypomastigote cells, L-arginine uptake was undetectable. The association between L-arginine uptake and cell replication was demonstrated using the antimitotic agent hydroxyurea. Arginine kinase, the enzyme responsible for phosphoarginine and ATP synthesis, also shows a differential activity in epimastigote and trypomastigote parasite stages.

Arginine kinase overexpression improves Trypanosoma cruzi survival capability.

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5-fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.

Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases.

A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.

Trypanosoma cruzi poly-zinc finger protein: a novel DNA/RNA-binding CCHC-zinc finger protein.

A poly-zinc finger protein, designated PZFP1 was identified in Trypanosoma cruzi for the first time. The protein has 191 amino acids, contains seven motifs Cys(X)(2)Cys(X)(4)His(X)(4)Cys. A recombinant PZFP1 was generated in E. coli and the expected 21kDa polypeptide co-purified with two other inducible products of about 42 and 63kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41kDa. Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins. The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis. PZFP1 interacted specifically with a T. cruzi serine-arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.

An early ancestor in the evolution of splicing: a Trypanosoma cruzi serine-arginine-rich protein (TcSR) is functional in cis-splicing.

A novel serine-arginine-rich protein designated TcSR was identified in Trypanosoma cruzi. The deduced amino acid sequence reveals that TcSR is a member of the SR protein family of splicing factors that contains two RNA-binding domains at the N-terminal side and several serine-arginine repeats at the COOH-terminus. Over expression of either TcSR or the human SR-protein associated splicing factor/splicing factor 2 (ASF/SF2) in wild-type Schizosaccharomyces pombe, provoked an elongated phenotype similar to that of fission yeast over expressing the SR-containing splicing factor Prp2, a U2AF(65) orthologue. When a double mutant strain lacking two SR protein-specific protein kinases was used, expression of TcSR or human SR ASF/SF2 splicing factor reverted the mutant to a wild-type phenotype. Transient expression of TcSR in HeLa cells stimulated the inclusion of the EDI exon of human fibronectin in an in vivo functional alternative cis-splicing assay. Inclusion was dependent on a splicing enhancer sequence present in the EDI exon. In addition, TcSR and peptides carrying TcSR-RS domain sequences were phosphorylated by a human SR protein kinase. These results indicate that TcSR is a member of the SR splicing network and that some components common to the trans- and cis-splicing machineries evolved from the early origins of the eukaryotic lineage.

Host cell poly(ADP-ribose) glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H(2)O(2) or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways.

The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway.

Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. 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. 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.