On the functionality of the N-terminal domain in xylanase 10A from Ruminococcus albus 8

We analyzed the structure to function relationships in Ruminococcus albus 8 xylanase 10A (RalXyn10A) finding that the N-terminus 34-amino acids sequence (N34) in the protein is particularly functional. We performed the recombinant wild type enzyme’s characterization and that of the truncated mutant lacking the N34 extreme (RalΔN34Xyn10A). The truncated enzyme exhibited about half of the activity and reduced affinity for binding to insoluble saccharides. These suggest a (CBM)-like function for the N34 motif. Besides, RalXyn10A activity was diminished by redox agent dithiothreitol, a characteristic absent in RalΔN34Xyn10A. The N34 sequence exhibited a significant similarity with protein components of the ABC transporter of the bacterial membrane, and this motif is present in other proteins of R. albus 8. Data suggest that N34 would confer RalXyn10A the capacity to interact with polysaccharides and components of the cell membrane, enhancing the degradation of the substrate and uptake of the products by the bacterium.Fil: Storani, Alem. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

A fluorometric method for the assay of protein kinase activity

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0–10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.Fil: Rojas, Bruno Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Santin, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

A Differential Redox Regulation or the Pathways Metabolizing Glyceraldehyde-3-phosphate Tunes the Production of Reducing Power in the Cytosol of Plant Cells

Adaptation to aerobic life leads organisms to sense reactive oxygen species and use the signal for coordination of the entire metabolism. Glycolysis in plants is a particular network where specific steps, like oxidation of glyceraldehydes-3-phosphate (Ga3P), are critical in order for it to function. The triose-phosphate can be converted into 3-phosphoglycerate through the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12) producing ATP and NADH, or via the non-phosphorylating enzyme (np-Ga3PDHase; EC 1.2.1.9) generating NADPH. In this work we found redox regulation to be a posttranslational mechanism allowing the fine-tuning of the triose-phosphate fate. Both enzymes were inactivated after oxidation by reactive oxygen and nitrogen species. Kinetic studies determined that Ga3PDHase is marked (63-fold) more sensitive to oxidants than np-Ga3PDHase. Thioredoxin-h reverted the oxidation of both enzymes (although with differences between them), suggesting a physiological redox regulation. The results support a metabolic scenario where the cytosolic triose-phosphate dehydrogenases are regulated under changeable redox conditions. This would allow coordinate production of NADPH or ATP through glycolysis, with oxidative signals triggering reducing power synthesis in the cytosol. The NADPH increment would favor antioxidant responses to cope with the oxidative situation, while the thioredoxin system would positively feedback NADPH production by maintaining np-Ga3PDHase at its reduced active state.Fil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

On the kinetic and allosteric regulatory properties of the ADP-glucose pyrophosphorylase from Rhodococcus jostii: An approach to evaluate glycogen metabolism in oleaginous bacteria

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.Fil: Cereijo, Antonela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Dávila Costa, José Sebastián. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Proteolytic cleavage of Arabidopsis thaliana phosphoenolpyruvate carboxykinase-1 modifies its allosteric regulation

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.Fil: Rojas, Bruno Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Hartman, Matias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Redox Regulation of UDP-glucose Pyrophosphorylase from Entamoeba histolytica.

Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V(max) values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Garay, Alberto. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Rodrigues, Daniel Enrique. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentin

Structure, function, and evolution of plant ADP-glucose pyrophosphorylase

Key message: This review outlines research performed in the last two decades on the structural, kinetic,regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzymefor starch biosynthesis. Abstract: ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Maryland (lum);Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis

Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.Fil: Calloni, Rodrigo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Muchut, Robertino José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Reconquista; ArgentinaFil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans

Background: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. Methods: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. Results: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. Conclusions and General significance: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.Fil: Sasoni, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Hartman, Matias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin II

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.Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Piñeyro, María Dolores. Instituto Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Robello, Carlos. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Urugua