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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.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments., This study was funded by Institute of Health Carlos III (ISCiii) (PI16/00090, PI19/00838 and PI19/01266), Spanish Ministry of Economy and Competitiveness (BFU2016-80006-P), Andalusian Ministry of Economy, Innovation, Science and Employment (BIO-216 and CTS-6264), Andalusian Ministry of Equality, Health and Social Policies (PI-0198-2016) and Valencian Ministry of Education, Culture and Sports (PROMETEO/2019/027). P de la C-O was supported by FPU predoctoral fellowship (FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E N-V was supported by the the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii. We thank the Biomedical Research Network Center for Cardiovascular Diseases (CIBERcv), and the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and co-financed by European Regional Development Fund (ERDF) "A way to achieve Europe" for their financial support.

Nitric oxide (NO) is a physiopathological messenger generating different reactive nitrogen species (RNS) according to hypoxic, acidic and redox conditions. Recent Advances: RNS and reactive oxygen species (ROS) promote relevant post-translational modifications, such as nitrosation, nitration, and oxidation, in critical components of cell proliferation and death, epithelial-to-mesenchymal transition, and metastasis.The pro- or antitumoral properties of NO are dependent on local concentration, redox state, cellular status, duration of exposure, and compartmentalization of NO generation. The increased expression of NO synthase has been associated with cancer progression. However, the experimental strategies leading to high intratumoral NO generation have been shown to exert antitumoral properties. The effect of NO and ROS on cell signaling is critically altered by factors modulating tumor progression such as oxygen content, metabolism, and inflammatory response. The review describes the alteration of key components involved in cell survival and death, metabolism, and metastasis induced by RNS- and ROS-related post-translational modifications.The identification of the molecular targets affected by nitrosation, nitration, and oxidation, as well as their interactions with other post-translational modifications, will improve the understanding on the complex signaling and cell fate decision in cancer. The therapeutic NO-based strategies have to address the complex crosstalk among NO and ROS with regard to critical components affecting tumor cell survival, metabolism, and metastasis in the progression of cancer, as well as close interaction with ionizing radiation and chemotherapy.

Nitric oxide (NO) plays relevant roles in signal transduction in physiopathology and its effects are dependent on several environmental factors. NO has both pro-apoptotic and anti-apoptotic functions but the molecular mechanisms responsible for these opposite effects are not fully understood. The action of NO occurs mainly through redox changes in target proteins, particularly by S-nitrosylation of reactive cysteine residues. Thioredoxin (Trx) and glutaredoxin (Grx) systems are the main cellular controllers of the thiolic redox state of proteins exerting controversial effects on apoptosis with consequences for the resistance to or the development of cancer. The aim of this study was to ascertain whether Trx and/or Grx systems mediate the antiproliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO. Enzyme levels and cysteine redox state at several metabolic checkpoints were consistent with prominence of the pentose phosphate pathway to direct the metabolic flux toward NADPH for antioxidant defense and lowering of nucleotide biosynthesis and hence proliferation. Proteins involved in cell survival pathways, proteins of the redoxin systems and phosphorylation of MAPK were all significantly increased accompanied by a shift of the thiolic redox state of Akt1, Trx1 and Grx1 to more oxidized. Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and -8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells. These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new antitumoral therapies., Graphical abstract, Highlights • Endogenous NO induces NADPH and reduces nucleotide biosynthesis in HepG2 cells. • Trx1 and Grx1 have a pro-oxidant action on key proteins under nitrosative conditions. • Trx1 and Grx1 hamper the antiproliferative action of NO in tumoral cells. • Weakening of thiolic antioxidant defenses could help in the design of anti-tumoral therapies.

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met(35), the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single A to G [corrected] mutation converts Grx2 into a typical protein of the mitochondrial matrix. The "A" denotes adenine, rather than alanine, and the "G" refers to guanine, not glycine [corrected]

Glutaredoxins (Grx) are small (approximately 12kDa) proteins which catalyze thiol disulfide oxidoreductions involving glutathione (GSH) and disulfides in proteins or small molecules. Here, we present data which demonstrate the ability of glutaredoxins to catalyze the reduction of oxidized glutathione (GSSG) by dihydrolipoamide (DHL), an important biological redox catalyst and synthetic antioxidant. We have designed a new assay method to quantify the rate of reduction of GSSG and other disulfides by reduced lipoamide and have tested a set of eight recombinant Grx from human, rat, yeast, and E. coli. Lipoamide dependent activity is highest with the large atypical E. coli Grx2 (k(cat)=3.235 min(-1)) and lowest for human mitochondrial Grx2a (k(cat)=96 min(-1)) covering a wider range than k(cat) for the standard reduction of hydroxyethyldisulfide (HED) by GSH (290-2.851 min(-1)). The lipoamide/HED activity ratio was highest for yeast Grx2 (1.25) and E. coli Grx2 and lowest for E. coli Grx1 (0.13). These results suggest a new role for Grxs as ancillary proteins that could shunt reducing equivalents from main catabolic pathways to recycling of GSSG via a lipoyl group, thus serving biochemical functions which involve GSH but without NAD(P)H consumption.

Specific polyclonal antibodies towards the oxidized form of bovine thioredoxin reductase (TR) have been obtained in rabbits, and purified. The antigenicity was lost upon reduction of TR by NADPH indicating a large conformational change upon reduction of the redox-active disulfide in the enzyme. The antibodies did not cross-react with other bovine NADPH-dependent dehydrogenases. No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with TR from horse. Immunoafinity purified anti-thioredoxin and anti-glutaredoxin antibodies were used to develop competitive indirect ELISA assays that were validated giving very good linearity, reproducibility, sensitivity and parallelism. The glutaredoxin (Grx) immunoassay is the first quantitative method described to measure the protein. When applied to a battery of calf tissues the contents of Grx varied from 7 to 120 μg per gram of fresh tissue. Skeletal and heart muscles gave the lowest values and spleen and salivary glands the highest. However, skeletal muscle showed the highest gluthathione-hydroxyethyl disulfide oxidoreductase specific activity.

1. 1. Purification of horse-liver glutathione reductase was obtained by affinity chromatography on N 6 -(6-aminohexyl)-adenosine-1′5′-bisphosphate Sepharose ( N 6 -2′5′-ADP-Sepharose) and Reactive Red-120-Agarose, and chromatography on DEAE-Sephadex and Sephacryl S-300. 2. 2. The final preparation had 248 U/mg specific activity after 11,174-fold purification with 47% final recovery, and was homogeneous by SDS-electrophoresis. It showed charge heterogeneity in non-denaturing electrophoresis and chromatofocusing, with several peaks of pI between 5.7 and 6.7. 3. 3. The enzyme was homodimeric (107,000 native MW), with S 20 w = 6.31 S, and 41.22 A of hydrodynamic radius. It showed absorption peaks at 270, 370 and 462 nm, a characteristic of flavoproteins. 4. 4. When NADPH was substituted by deamino-NADPH or NADH the enzyme showed 69 and 8.5% activity, respectively, while with glutathione-CoA mixed disulfide the enzyme had 23% of the activity shown with GSSG. Apparent K m values of 8.8, 680, 59, and 560 μ M were measured for NADPH, NADH, GSSG and ferrycianide, respectively.

Streptococcus suis serotype 2 is a major Gram-positive swine pathogen, causing also zoonoses. We describe here the immunoprotective activity in an in vivo animal model of a serotype-2 cell wall protein, designated Sat, which was identified by a previously validated proteomics approach consisting of the protease digestion of live bacteria and the selective recovery of exposed domains, followed by LC/MS/MS analysis. Increased survival rate (80%) and decreased bacterial burden were observed in mice immunized with a recombinant Sat fragment, suggesting that this protein is a potential vaccine candidate against serotype-2 infection.

28 páginas, 5 figuras, 1 tabla., 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 11.9kDa 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 99bp 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., This research has been supported by grants from the Spanish Ministerio de Educación y Cultura (PM98-0166) and from Plan Andaluz de Investigación, Junta de Andalucía (CVI-216) (J.A.B.) and Swedish Medical Research Council (03P-14096-01A; 03X-14041-01A) and the Åke Wibergs Stiftelse (A.M.-V.).

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30 degrees C. Lower but significant protection and reactivation was also observed with NADP+ and NAD+. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.

1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations. 2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective. 3. GSSG fully reactivated the inactive enzyme at 38 degrees C and neutral to acidic pH (5.5-7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.