Diego G. Arias

Entamoeba histolytica thioredoxin reductase: molecular and functional characterization of its atypical properties.

BACKGROUND Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance. METHODS The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR). RESULTS Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S-nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR. CONCLUSIONS AND GENERAL SIGNIFICANCE Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.

Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.

Redox metabolism in Trypanosoma cruzi: Functional characterization of tryparedoxins revisited

Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various thiol proteins. In Trypanosoma cruzi, two genes coding for TXN-like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.

Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin II

UNLABELLED Trypanosoma cruzi, the causative agent of Chagas disease, possesses two tryparedoxins (TcTXNI and TcTXNII), belonging to the thioredoxin superfamily. TXNs are oxidoreductases which mediate electron transfer between trypanothione and peroxiredoxins. This constitutes a difference with the host cells, in which these activities are mediated by thioredoxins. These differences make TXNs an attractive target for drug development. In a previous work we characterized TcTXNI, including the redox interactome. In this work we extend the study to TcTXNII. We demonstrate that TcTXNII is a transmembrane protein anchored to the surface of the mitochondria and endoplasmic reticulum, with a cytoplasmatic orientation of the redox domain. It would be expressed during the metacyclogenesis process. In order to continue with the characterization of the redox interactome of T. cruzi, we designed an active site mutant TcTXNII lacking the resolving cysteine, and through the expression of this mutant protein and incubation with T. cruzi proteins, heterodisulfide complexes were isolated by affinity chromatography and identified by mass spectrometry. This allowed us to identify sixteen TcTXNII interacting proteins, which are involved in a wide range of cellular processes, indicating the relevance of TcTXNII, and contributing to our understanding of the redox interactome of T. cruzi. BIOLOGICAL SIGNIFICANCE T. cruzi, the causative agent of Chagas disease, constitutes a major sanitary problem in Latin America. The number of estimated infected persons is ca. 8 million, 28 million people are at risk of infection and ~20,000 deaths occur per year in endemic regions. No vaccines are available at present, and most drugs currently in use were developed decades ago and show variable efficacy with undesirable side effects. The parasite is able to live and prolipherate inside macrophage phagosomes, where it is exposed to cytotoxic reactive oxygen and nitrogen species, derived from macrophage activation. Therefore, T. cruzi antioxidant mechanisms constitute an active field of investigation, since they could provide the basis for a rational drug development. Peroxide detoxification in this parasite is achieved by ascorbate peroxidase and different thiol-dependent peroxidases. Among them, both mitochondrial and cytosolic tryparedoxin peroxidases, typical two-cysteine peroxiredoxins, were found to be important for hydrogen peroxide and peroxynitrite detoxification and their expression levels correlated with parasite infectivity and virulence. In trypanosomes tryparedoxins and not thioredoxins act as peroxiredoxin reductases, suggesting that these enzymes substitute thioredoxins in these parasites. T. cruzi possesses two tryparedoxin genes, TcTXNI and TcTXN II. Since thioredoxins are proteins with several targets actively participating of complex redox networks, we have previously investigated if this is the case also for TcTXNI, for which we described relevant partners (J Proteomics. 2011;74(9):1683-92). In this manuscript we investigated the interactions of TcTXNII. We have designed an active site mutant tryparedoxin II lacking the resolving cysteine and, through the expression of this mutant protein and its incubation with T. cruzi proteins, hetero disulfide complexes were isolated by affinity chromatography purification and identified by electrophoresis separation and MS identification. This allowed us to identify sixteen TcTXNII interacting proteins which are involved in different and relevant cellular processes. Moreover, we demonstrate that TcTXNII is a transmembrane protein anchored to the surface of the mitochondria and endoplasmic reticulum.

Immunolocalization and enzymatic functional characterization of the thioredoxin system in Entamoeba histolytica.

The components of the redox metabolism in Entamoeba histolytica have been recently revisited by Arias et al. (Free Radic. Biol. Med. 42:1496-1505; 2007), after the identification and characterization of a thioredoxin-linked system. The present work deals with studies performed for a better understanding of the localization and identification of different components of the redox machinery present in the parasite. The gene encoding for amoebic thioredoxin 8 was cloned and the recombinant protein typified as having properties similar to those of thioredoxin 41. The ability of these thioredoxins and the specific reductase to assemble a system utilizing NADPH to metabolize hydroperoxides in association with a peroxiredoxin has been kinetically characterized. The peroxiredoxin behaved as a typical 2 cysteine enzyme, exhibiting a ping-pong mechanism with hyperbolic saturation kinetics for thioredoxin 8 (K(m)=3.8 microM), thioredoxin 41 (K(m)=3.1 microM), and tert-butyl hydroperoxide (K(m) about 35 microM). Moreover, the tandem system involving thioredoxin reductase and either thioredoxin proved to be operative for reducing low molecular weight disulfides, including putative physiological substrates as cystine and oxidized trypanothione. Thioredoxin reductase and thioredoxin 41 (by association also the functional redox system) have been immunolocalized underlying the plasma membrane in Entamoeba histolytica cells. These findings suggest an important role for the metabolic pathway involving thioredoxin as a redox interchanger, which could be critical for the maintenance and virulence of the parasite when exposed to highly toxic reactive oxygen species.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis.

Background Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.

Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol glutaredoxin dependent cellular pathways

In Trypanosoma cruzi, the modification of thiols by glutathionylation-deglutathionylation and its potential relation to protective, regulatory or signaling functions have been scarcely explored. Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating activity, having potential functionality to control intracellular homeostasis of protein and non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the contribution of the protein to increase the general resistance against oxidative damage and intracellular replication of the amastigote stage. Also, studies performed with epimastigotes overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests novel insights about the protein involvement in redox signaling.

New enzymatic pathways for the reduction of reactive oxygen species in Entamoeba histolytica

BACKGROUND Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. METHODS The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. RESULTS We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. CONCLUSIONS The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. GENERAL SIGNIFICANCE To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.

Kinetic and structural characterization of a typical two-cysteine peroxiredoxin from Leptospira interrogans exhibiting redox sensitivity

Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H2O2 (Chyp 1% ~30 µM at pH 7.0 and 30°C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.

Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.