Homology Modeling and Molecular Dynamics Simulations of Trypanosoma cruzi Phosphodiesterase b1.

Cyclic nucleotide phosphodiesterases have been implicated in the proliferation, differentiation and osmotic regulation of trypanosomatids; in some trypanosomatid species, they have been validated as molecular targets for the development of new therapeutic agents. Because the experimental structure of Trypanosoma cruzi PDEb1 (TcrPDEb1) has not been solved so far, an homology model of the target was created using the structure of Trypanosoma brucei PDEb1 (TbrPDEb1) as a template. The model was refined by extensive enhanced sampling molecular dynamics simulations, and representative snapshots were extracted from the trajectory by combined clustering analysis. This structural ensemble was used to develop a structure-based docking model of the target. The docking accuracy of the model was validated by redocking and cross-docking experiments using all available crystal structures of TbrPDEb1, whereas the scoring accuracy was validated through a retrospective screen, using a carefully curated dataset of compounds assayed against TbrPDEb1 and/or TcrPDEb1. Considering the results from in silico validations, the model may be applied in prospective virtual screening campaigns to identify novel hits, as well as to guide the rational design of potent and selective inhibitors targeting this enzyme.

The Phosphatidylinositol 3-kinase Class III Complex Containing TcVps15 and TcVps34 Participates in Autophagy in Trypanosoma cruzi.

Autophagy is a degradative process by which eukaryotic cells digest their own components to provide aminoacids that may function as energy source under nutritional stress conditions. There is experimental evidence for autophagy in parasitic protists belonging to the family Trypanosomatidae. However, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzi differentiation and thus might have a role in pathogenicity. Here, we report the cloning and biochemical characterization of TcVps15. In addition, we demonstrate that TcVps15 interact with the PI3K TcVps34 and that both proteins associate with cellular membranes. Under nutritional stress conditions, TcVps15 and TcVps34 modify their subcellular distribution showing a partial co-localization in autophagosomes with TcAtg8.1 and using an active site TcVps15-mutated version (TcVps15-K219D-HA) we demonstrated that this relocalization depends on the TcVps15 catalytic activity. Overexpression of TcVps15-HA and TcVps15-K219D-HA also leads to increased accumulation of monodansylcadaverine (MDC) in autophagic vacuoles under nutritional stress conditions compared to wild-type cells. In addition, the MDC-specific activity shows to be significantly higher in TcVps15-HA overexpressing cells when compared with TcVps15-K219D-HA. Our results reveal for the first time a role of TcVps15 as a key regulator of TcVps34 enzymatic activity and implicate the TcVps15-Vps34 complex in autophagy in T. cruzi, exposing a new key pathway to explore novel chemotherapeutic targets.

Improving genome-wide mapping of nucleosomes in Trypanosome cruzi.

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.

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.

An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Methods to Investigate Signal Transduction Pathways in Trypanosoma cruzi: Cyclic Nucleotide Phosphodiesterases Assay Protocols.

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways.In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs.In this chapter, we describe a simple and sensitive radioisotope-based method to measure cyclic 3',5'-nucleotide phosphodiesterase using [3H]cAMP.

Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi.

Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomine's midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while in trypomastigotes an increase in intracellular cAMP doesn't seem to aid in this response, although it does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.

Venom alkaloids against Chagas disease parasite: search for effective therapies.

Chagas disease is an important disease affecting millions of patients in the New World and is caused by a protozoan transmitted by haematophagous kissing bugs. It can be treated with drugs during the early acute phase; however, effective therapy against the chronic form of Chagas disease has yet to be discovered and developed. We herein tested the activity of solenopsin alkaloids extracted from two species of fire ants against the protozoan parasite Trypanosoma cruzi, the aetiologic agent of Chagas disease. Although IC50 determinations showed that solenopsins are more toxic to the parasite than benznidazole, the drug of choice for Chagas disease treatment, the ant alkaloids presented a lower selectivity index. As a result of exposure to the alkaloids, the parasites became swollen and rounded in shape, with hypertrophied contractile vacuoles and intense cytoplasmic vacuolization, possibly resulting in osmotic stress; no accumulation of multiple kinetoplasts and/or nuclei was detected. Overexpressing phosphatidylinositol 3-kinase-an enzyme essential for osmoregulation that is a known target of solenopsins in mammalian cells-did not prevent swelling and vacuolization, nor did it counteract the toxic effects of alkaloids on the parasites. Additional experimental results suggested that solenopsins induced a type of autophagic and programmed cell death in T. cruzi. Solenopsins also reduced the intracellular proliferation of T. cruzi amastigotes in infected macrophages in a concentration-dependent manner and demonstrated activity against Trypanosoma brucei rhodesiense bloodstream forms, which is another important aetiological kinetoplastid parasite. The results suggest the potential of solenopsins as novel natural drugs against neglected parasitic diseases caused by kinetoplastids.

Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication.

Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein.

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.

TbVps15 is required for vesicular transport and cytokinesis in Trypanosoma brucei.

The class III phosphatidylinositol 3-kinase (PI3K) Vps34 is an important regulator of key cellular functions, including cell growth, survival, intracellular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with the putative serine/threonine protein kinase Vps15, however, its role in signaling has not been deeply evaluated. Here, we have identified the Vps15 orthologue in Trypanosoma brucei, named TbVps15. Knockdown of TbVps15 expression by interference RNA resulted in inhibition of cell growth and blockage of cytokinesis. Scanning electron microcopy revealed a variety of morphological abnormalities, with enlarged parasites and dividing cells that often exhibited a detached flagellum. Transmission electron microscopy analysis of TbVps15 RNAi cells showed an increase in intracellular vacuoles of the endomembrane system and some cells displayed an enlargement of the flagellar pocket, a common feature of cells defective in endocytosis. Moreover, uptake of dextran, transferrin and Concanavalin A was impaired. Finally, TbVps15 downregulation affected the PI3K activity, supporting the hypothesis that TbVps15 and TbVps34 form a complex as occurs in other organisms. In summary, we propose that TbVps15 has a role in the maintenance of cytokinesis, endocytosis and intracellular trafficking in T. brucei.

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.

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.