New clues to metabolic regulation through changes in the thiol redox proteome

Comunicaciones a congreso

Cis- and trans-regulatory mechanisms of gene expression in the ASJ sensory neuron of Caenorhabditis elegans

© 2015 by the Genetics Society of America. The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons.Some C. elegans strains were provided by the CGC, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), and by the Japanese National Bioresource Project, which is funded by the Japanese Ministry of Education, Culture, Sport, Science and Technology. We thank Nuria Flames for advice and support and María Jesús Rodríguez-Palero and Francisco José Naranjo-Galindo for excellent technical assistance. This work was financed by grants to A.M.-V. from the Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). Work in the laboratory of P.S. was supported by grants from the Swedish Research Council and the Torsten Söderberg Foundation. E.K. was supported by a grant from the European Union FP6 Marie Curie Research Training Network “EUrythron” MRTN-CT-2004-005499.Peer Reviewe

Role of glutathione reductase in proteostasis regulation

Trabajo presentado en la V Spanish Worm Meeting, celebrada en Salamanca el 5 y 6 de marzo de 2015.Peer Reviewe

Estudio de las especies de nitrógeno reactivo en plantas durante el proceso de estrés biótico en la interacción girasol-mildiu

En hipocótilos de plántulas de girasol sensibles y resistentes a la infección por el hongo parásito Plasmopara halstedii, responsable del mildiu, los análisis mediante quimioluminiscencia de ozono revelaron una mayor producción de óxido nítrico en la variedad sensible frente a la resistente, tanto en plantas controles como en plantas inoculadas.La inmunolocalización mediante microscopía de fluorescencia y microscopía confocal láser mostró la localización extensiva de NOS (Óxido nítrico sintasa) y Snitrosoglutation (GSNO) en células parenquimáticas. La lozalización tisular preferente del GSNO en la zona de entrada del patógeno en el hipocótilo, evidencia la posible participación del óxido nítrico en los mecanismos de defensa celulares de respuesta inmediata frente a la invasión por patógenos y antes de la inducción de la producción de óxido nítrico por las proteínas responsables de su generación.El análisis de estos resultados evidencia la presencia en células de hipocótilos de girasol de proteínas tipo NOS y sugiere la participación de la NOS, del óxido nítrico (NO·) y del GSNO en la respuesta de la planta frente al estrés biótico por el hongo Plasmopara halstedii

Proctective role of glutathione reductase in Caenorhabditis elegans models of polyQ diseases

Póster presentado en la Thiol Based Redox Regulation & Signaling. Gordon Research Conference, celebrada en Girona del 20 al 25 de julio de 2014.Protein aggregation is a major hallmark of many neurodegenerative disorders such as Alzheimer, Parkinson and Huntington Diseases. However, the signaling events and molecular pathways governing this aggregation process are not completely understood. Huntington Disease and other polyQ diseases are characterized by an abnormal CAG codon expansion (that encodes the amino acid glutamine) inspecific genes. When these CAG expansions increase above a threshold of 35 to 40 glutamine residues, the resulting protein shows toxic gain-of-function features, associated to protein misfolding and aggregation. Using Caenorhabditis elegans models of polyQ diseases we have identified a novel protective role of glutathione reductase (GSR-1) against polyQ aggregation and toxicity. First, we found that gsr-1 RNAi downregulation inworms expressing the fusion protein Q40::YFP caused a strong developmental arrest while Q35::YFP animals developed normally. This phenotype was phenocopied by treatment with buthionine sulphoximine (an inhibitor of glutathione synthesis) and diethyl maleate (a glutathione depletory), confirming that 40 glutamine residues set the pathological threshold of polyQ proteins alsoinworms. Consistently, Q40::YFP animals carrying a gsr-1 loss of function mutation showed a remarkable increase of polyQ aggregation in muscle cells. Interestingly, embryos from Q40::YFP worms lacking gsr-1 display a striking phenotype consisting incell membrane blebbing and explosion, a phenotype that is much milder inQ35::YFP; gsr-1 embryos. Finally, our results demonstrate that, inC. elegans, the gsr-1 gene encodes two isoforms that localize to cytoplasm and mitochondria, respectively. Importantly, the cytoplasmic variant is shown to be essential for C. elegans viability while animals devoid of the mitochondrial isoform have no phenotype. Our future plans will focus on validating the results obtained with C. elegans in mammalian cell culture and mice models of Huntington s Disease and other polyQ Diseases. In addition, we will explore the role of glutaredoxins and other glutathione-dependentenzymesinworm models of polyQ and other aggregation-dependent neuro degenerative diseases.Peer Reviewe

Mitochondria of Saccharomyces cerevisiae Contain One-conserved Cysteine Type Peroxiredoxin with Thioredoxin Peroxidase Activity

6 páginas, 7 figuras, 1 tabla. Este artículo esta dedicado a la memoria del Dr. Antonio López Ruiz.Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.This work was supported by Swedish Medical Research Council Project Grant 13X-10370, by funds from the Karolinska Institutet, the Svenska Institutet, and Åke Wibergs Stiftelse, and by Training and Mobility of Researchers Marie Curie Research Training Grant ERBFMBICT972824.The costs of publication of this article were defrayed in part by the payment of page charges.Peer reviewe

Redox-dependent and redox-independent functions of Caenorhabditis elegans thioredoxin 1

Thioredoxins (TRX) are traditionally considered as enzymes catalyzing redox reactions. However, redox-independent functions of thioredoxins have been described in different organisms, although the underlying molecular mechanisms are yet unknown. We report here the characterization of the first generated endogenous redox-inactive thioredoxin in an animal model, the TRX-1 in the nematode Caenorhabditis elegans. We find that TRX-1 dually regulates the formation of an endurance larval stage (dauer) by interacting with the insulin pathway in a redox-independent manner and the cGMP pathway in a redox-dependent manner. Moreover, the requirement of TRX-1 for the extended longevity of worms with compromised insulin signalling or under calorie restriction relies on TRX-1 redox activity. In contrast, the nuclear translocation of the SKN-1 transcription factor and increased LIPS-6 protein levels in the intestine upon trx-1 deficiency are strictly redox-independent. Finally, we identify a novel function of C. elegans TRX-1 in male food-leaving behaviour that is redox-dependent. Taken together, our results position C. elegans as an ideal model to gain mechanistic insight into the redox-independent functions of metazoan thioredoxins, overcoming the limitations imposed by the embryonic lethal phenotypes of thioredoxin mutants in higher organisms.We thank the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for providing worm strains and SunyBiotech Corporation for the generation of the trx-1(sgps) strain. We thank Prof. Stefan Taubert for providing the lips-6::gfp reporter strain. AMV was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BFU2015-64408-P), cofinanced by the Fondo Social Europeo (FEDER). DAG was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI076406. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. AMV is a member of the GENIE and EU-ROS Cost Actions of the European Union

Peroxiredoxin 2 and Peroxidase Enzymatic Activity of Mammalian Spermatozoa

10 páginas, 6 figuras, 2 tablas.Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.Supported in part by the Food for the 21st Century Program of the University of Missouri and by National Research Initiative Competitive Grant No. 2007-01319 from the USDA CSREES to P.S.Peer reviewe

Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments.

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the “cadherin switch”. These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies