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Article: Redox metabolism in trypanosoma cruzi: functional characterization of tryparedoxins revisited.
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ABSTRACT: Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to different 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 transmenbrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione or dihydrolipoamide to different acceptors, including methionine sulfoxide reductases and peroxiredoxins. Results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in redox scenario in T. cruzi.Free radical biology & medicine 05/2013; · 5.42 Impact Factor -
Article: REDOX METABOLISM IN TRYPANOSOMA CRUZI: FUNCTIONAL CHARACTERIZATION OF TRYPAREDOXINS REVISITED
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ABSTRACT: REDOX METABOLISM IN TRYPANOSOMA CRUZI: FUNCTIONAL CHARACTERIZATION OF TRYPAREDOXINS REVISITED Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to different 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 transmenbrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione or dihydrolipoamide to different acceptors, including methionine sulfoxide reductases and peroxiredoxins. Results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in redox scenario in T. cruzi. Article Type: Original Contribution Corresponding Author: Dr. Sergio A. Guerrero All Authors:Free Radical Biology and Medicine 04/2013; · 5.42 Impact Factor -
Article: A chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators
International Journal of Molecular Sciences 04/2013; · 2.60 Impact Factor -
Chapter: Capítulo XVI: Glutathione metabolism in protozoan parasites. Making the difference, in Glutathione: Biochemistry, Mechanisms of Action and Clinical Implications.
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ABSTRACT: Generation of reactive oxygen species (ROS) is a common feature of abiotic and biotic stress reactions. ROS need to be detoxified to avoid the beginning of deleterious reactions. Glutathione is the most abundant low-molecular weight thiol acting in cellular redox system. It plays a central role in thiol-disulfide redox homeostasis in many organisms by providing electrons to essential enzymes, defense against oxidative stress and transmission of redox signals. Parasitic diseases such as sleeping sickness, Chagas disease, and malaria, between others, are major health problems in poverty-stricken areas. There is priority urgency in public health, which is the development of a new, specific, economic and sure chemotherapeutic drug against these diseases. The design of efficacious and safe drugs is expected from, for example, inhibition of metabolic pathways that are unique and essential to the parasite, being absent in the host. Many of the known glutathione-dependent processes are directly related to specific life-style from different parasites. Thus, Malaria parasites have a dual antioxidative system based on glutathione and thioredoxin. Proteins involved in GSH-dependent metabolic pathways include glutathione reductase, glutaredoxins, glyoxalases, glutathione Stransferases, and thioredoxins. These proteins, as well as ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. In contrast, in trypanosomes and leishmanias, the redox network is centered around the trypanothione (N1,N8-bis(glutathionyl)spermidine) instead of the ubiquitous glutathione. Trypanothione is an “exclusive” glutathione-derived dithiol, an unusual thiol containing two glutathione molecules linked by a spermidine molecule. This dithiol is the main non-protein thiol in trypanosomatids, which is kept reduced by a unique flavoenzyme, the trypanothione reductase. In these parasites, trypanothione participates in essential thiol–disulfide exchange reactions as electron donor to different trypanothione-dependent enzymes such as tryparedoxin, glutaredoxin and peroxiredoxins. Since trypanosomatids lack glutathione reductase and thioredoxin reductase, trypanothione is the central node electrons take from NADPH to achieve antioxidant enzymes. Anaerobic parasites such as Entamoeba histolytica or Giardia lamblia are human pathogen that lacks the capacity to synthesize glutathione. Cysteine is the major low-molecular weight thiol, instead of glutathione. However, these parasites could incorporate glutathione from growth media or presumably from the human host. Here, we describe the glutathione dependent metabolism in parasitic cells, pointing out its relevance in vital functions of the parasite. We analyze comparatively differences and similarities between glutathione metabolisms in different protozoa. Particular attention is given to the role of glutathione in redox regulation and adaptation to stresses, highlighting the importance of the enzyme belonging to this metabolism, many of them proposed as target of antiparasitic drugs.02/2013: pages 295-326; , ISBN: 978-1-62417-460-5 -
Article: The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms.
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ABSTRACT: ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate. The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP. After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.BMC Evolutionary Biology 01/2013; 13:51. · 3.52 Impact Factor
