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Introduction and objectives: The heterogenous nature of hepatocellular carcinoma (HCC) motivated this attempt at developing and validating a model based on combined biomarkers for improving early HCC detection. Patients/materials and methods: This study examined 196 patients for an estimation study (104 patients with HCC, 52 with liver cirrhosis and 40 with liver fibrosis) and 122 patients for the validation study (80 patients with HCC, 42 with liver cirrhosis). All patients were positive for hepatitis C virus. Four markers were measured: Midkine and thioredoxin using ELISA, 1-methyladenosine and 1-methylguanosine using a gas chromatography–mass spectrometry (GC–MS). The results were compared with alpha-fetoprotein (AFP). The performance of the model was estimated in BCLC, CLIP and Okuda staging systems of HCC. Results: The model yielded high performance with an area under ROC (AUC) of 0.94 for predicting HCC in patients with liver cirrhosis, compared with AUC of 0.69 for AFP. This model had AUCs of 0.93, 0.94 and 0.94 in patients who had only one single nodule, absent macrovascular invasion and tumor size

Cardiac hypertrophy is a current, major, global health challenge. Oxidative stress is an important mechanism that contributes to the pathogenesis of cardiac hypertrophy. Schisandra chinensis polysaccharides (SCP), the primary active constituent in Schisandra chinensis, have antioxidative properties. Here, we investigated the role played by SCP in a cardiac hypertrophy model mouse induced by transverse aortic constriction (TAC). We found that SCP treatment improved cardiac function by inhibiting myocardial hypertrophy and oxidative stress. Angiotensin II was used to induce cardiomyocyte hypertrophy and oxidative stress in vitro. We discovered that the antioxidant effects of SCP were mediated through the regulation of the thioredoxin-interacting protein (TXNIP)/Thioredoxin-1 (Trx-1) pathway. Using molecular docking, we found that SCP binds to Arg207, Ser169, Lys166, Lys286 and Ser285 in TXNIP through hydrogen bonds. TXNIP is an endogenous inhibitor of Trx-1, and the binding SCP with TXNIP may restrict or interfere with the binding between TXNIP and Trx-1, resulting in Trx-1 activation. In conclusion, our findings demonstrated that the potential use of SCP as a TXNIP inhibitor to attenuate oxidative stress, suggesting that TXNIP might represent a potential therapeutic target for the treatment of cardiac hypertrophy.

Summary: Appropriate regulation of genes that coordinate pancreas progenitor proliferation and differentiation is required for pancreas development. Here, we explore the role of H3K4 methylation and the Trithorax group (TrxG) complexes in mediating gene expression during pancreas development. Disruption of TrxG complex assembly, but not catalytic activity, prevented endocrine cell differentiation in pancreas progenitor spheroids. In vivo loss of TrxG catalytic activity in PDX1+ cells increased apoptosis and the fraction of progenitors in the G1 phase of the cell cycle. Pancreas progenitors were reallocated to the acinar lineage, primarily at the expense of NEUROG3+ endocrine progenitors. Later in development, acinar and endocrine cell numbers were decreased, and increased gene expression variance and reduced terminal marker activation in acinar cells led to their incomplete differentiation. These findings demonstrate that TrxG co-activator activity is required for gene induction, whereas TrxG catalytic activity and H3K4 methylation help maintain transcriptional stability. : Campbell et al. demonstrate that the non-catalytic activity of TrxG complexes is required for endocrine and acinar cell differentiation of pancreas progenitor spheroids. They also find that TrxG catalytic H3K4 methyltransferase activity is critical for pancreas endocrine lineage specification, as well as the transcriptional stability and differentiation of acinar cells. Keywords: H3K4 methylation, TrxG complexes, WDR5, DPY30, pancreas development, transcriptional variation

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells., Graphical abstract Graphical Abstract. Sorafenib reduced ROS/RNS cytoplasmic concentrations which were associated with decreased Nrf2 nuclear translocation, Trx1 expression/activity, and SNO–NOS3 expression and NO intracellular generation in HepG2. The reduction of SNO–NOS3 expression and NO generation might also be influenced by the induction of transient GSNOR expression/activity and reduced NOS3 expression by Sorafenib. The diminution of SNO-CD95 expression by Sorafenib involved reduction of caspase-8 activity and cell proliferation, and increased downstream caspase-3 activity in liver cancer cells.Image 1, Highlights • Sorafenib induces mitochondrial ROS generation, but also acts as nucleophilic scavenger. • Sorafenib reduces Nrf2-depenent Trx1 expression, and SNO–NOS3 and SNO-CD95 ratios. • Sorafenib-related antitumoral in vivo activity involves diminution of Trx1 and SNO-CD95.

The failure of insulin-producing β-cells is the underlying cause of hyperglycemia in diabetes mellitus. β-cell decay has been linked to hypoxia, chronic inflammation, and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We have analyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress using β-cell culture, isolated pancreatic islets, and pancreatic islet transplantation modelling low oxygen supply. Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reoxygenation. Interestingly, Trx1 was secreted by β-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation. Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin. Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 on β-cells were examined. In fact, exogenously added recombinant hTrx1 mitigated apoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects were studied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function. Moreover, hTrx1 inhibited the migration of primary murine macrophages. In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreatic β-cells., Graphical abstract Image 1

Intestinal ischemia-reperfusion (I/R) injury is a life-threatening vascular emergency and has long been a disturbing problem for surgeons. Oxidative stress is considered a vital factor in I/R injury. Metformin has anti-oxidative properties and protects against I/R injury. The present study aimed to investigate whether Metformin protects against intestinal I/R injury and reveal the protective mechanism of Metformin. I/R injury was induced in mice by temporary superior mesenteric artery occlusion, and Caco-2 cells were subjected to OGD/R to establish an in vitro model. Different doses of Metformin were administered in vivo and in vitro. We found that I/R injury led to intestinal barrier disruption and cell death by examining histopathological results and the intestinal barrier index, including TER, tight junction proteins and serum biomarkers. We confirmed the existence of pyroptosis in intestinal I/R injury. Moreover, we confirmed the role of pyroptosis in intestinal I/R injury by silencing the gasdermin D (GSDMD). Then, we confirmed that Metformin treatment protected barrier function against intestinal I/R injury and reduced oxidative stress and the inflammatory response. Importantly, Metformin reduced pyroptosis-related proteins, including NLRP3, cleaved caspase-1, and the N-terminus of GSDMD. Knocking down the GSDMD could reversed the protective effects of Metformin, which showed pyroptosis was one of the major cell death pathways controlled by Metformin treatment in setting of intestinal I/R injury. We also discovered that Metformin suppressed the expression of TXNIP and the interaction between TXNIP and NLRP3. We performed siRNA knockdown and found that the protective effects were abolished, which further confirmed our findings. In conclusion, we believe that Metformin protects against intestinal I/R injury in a TXNIP-NLRP3-GSDMD-dependent manner., Highlights • Pyroptosis plays an important role in intestinal I/R injury. • Metformin protects against intestinal I/R injury in mice. • Metformin protects Caco-2 cells subjected to OGD/R. • Metformin inhibits pyroptosis, inflammation and oxidative stress during I/R injury. • Metformin exerts protective effect through TXNIP-NLRP3-GSDMD pathway.

Quinones can modify biological molecules through both redox-cycling reactions that yield radicals (semiquinone, superoxide and hydroxyl) and via covalent adduction to nucleophiles (e.g. thiols and amines). Kinetic data indicate that Cys residues in GSH and proteins are major targets. In the studies reported here, the interactions of a prototypic quinone compound, p-benzoquinone (BQ), with the key redox protein, thioredoxin-1 (Trx1) were examined. BQ binds covalently with isolated Trx1 forming quinoprotein adducts, resulting in a concentration-dependent loss of enzyme activity and crosslink formation. Mass spectrometry peptide mass mapping data indicate that BQ forms adducts with all of the Trx1 Cys residues. Glutathione (GSH) reacts competitively with BQ, and thereby modulates the loss of activity and crosslink formation. Exposure of macrophage-like (J774A.1) cells to BQ results in a dose-dependent loss of Trx and thioredoxin reductase (TrxR) activities, quinoprotein formation, and a decrease in GSH levels without a concomitant increase in oxidized glutathione. GSH depletion aggravates the loss of Trx and TrxR activity. These data are consistent with adduction of GSH to BQ being a primary protective pathway. Reaction of BQ with Trx in cells resulted in the activation of apoptosis signal-regulating kinase 1 (ASK1), and p38 mitogen-activated protein kinase (MAPK) leading to apoptotic cell death. These data suggest that BQ reacts covalently with Cys residues in Trx, including at the active site, leading to enzyme inactivation and protein cross-linking. Modification of the Cys residues in Trx also results in activation of the ASK1/p38-MAPK signalling pathway and promotion of apoptotic cell death., Graphical abstract Image 1, Highlights • Quinone (e.g. p-benzoquinone, BQ) toxicity is linked to Michael adduction reactions. • Adduction of BQ to Cys residues in proteins are rapid (≤105 M−1 s−1) and selective. • BQ reaction with Cys inactivates thioredoxin (Trx) and yields quinone- and disulfide-linked dimers. • GSH reacts competitively with BQ and modulates damage, without GSSG formation. • BQ activates ASK1 and p38 pathways and induced apoptosis in cells via Trx damage.

Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems – glutathione (GSH) and thioredoxin (Trx) systems – are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5 μM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells., Graphical abstract fx1, Highlights • Trx is functional even when TrxR is inhibited by Hg compounds. • Depletion of GSH with BSO increases oxidation of Trx by Hg compounds. • Mice neurons lacking mitochondrial Grx2 show increased oxidation of Trx2 by Hg. • GSH/Grx act as a backup for TrxR during Hg exposure. • Oxidation of Trx by Hg leads to activation of ASK-1 and apoptotic cell death.

Accumulating mutations may drive cells into the acquisition of abnormal phenotypes that are characteristic of cancer cells. Cancer cells feature profound alterations in proliferation programs that result in a new population of cells that overrides normal tissue construction and maintenance programs. To achieve this goal, cancer cells are endowed with up regulated survival signaling pathways. They also must counteract the cytotoxic effects of high levels of nitric oxide (NO) and of reactive oxygen species (ROS), which are by products of cancer cell growth. Accumulating experimental evidence associates cancer cell survival with their capacity to up-regulate antioxidant systems. Elevated expression of the antioxidant protein thioredoxin-1 (Trx1) has been correlated with cancer development. Trx1 has been characterized as a multifunctional protein, playing different roles in different cell compartments. Trx1 migrates to the nucleus in cells exposed to nitrosative/oxidative stress conditions. Trx1 nuclear migration has been related to the activation of transcription factors associated with cell survival and cell proliferation. There is a direct association between the p21Ras-ERK1/2 MAP Kinases survival signaling pathway and Trx1 nuclear migration under nitrosative stress. The expression of the cytoplasmic protein, the thioredoxin-interacting protein (Txnip), determines the change in Trx1 cellular compartmentalization. The anti-apoptotic actions of Trx1 and its denitrosylase activity occur in the cytoplasm and serve as important regulators of cell survival. Within this context, this review focuses on the participation of Trx1 in cells under nitrosative/oxidative stress in survival signaling pathways associated with cancer development.

SUMMARY Energetic nutrients are oxidized to sustain high intra-cellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity., In Brief All cells require cytosolic disulfide-reducing power fueled by intracellular NADPH or, in mammals, alternatively generated by catabolism of the essential amino acid methionine. Prigge et al. show that liver homeostasis under cytosolic disulfide reductase paucity involves noncanonical trafficking of reducing power and a hyperproliferation-based organ survival strategy.

Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system., Highlights • EGCG increases hepatic activities of TrxR, GR and Grx in selenium-optimal mice. • EGCG fails to manipulate the above-mentioned enzymes in selenium-deficient mice. • EGCG in turn activates hepatic Nrf2 response in selenium-deficient mice. • Selenium deficiency does not increase EGCG toxicity due to potent Nrf2 response., Graphical abstract fx1

Aims The present study aimed to investigate the effect of metformin on diabetes-accelerated atherosclerosis and whether Nod-like receptor protein 3 (NLRP3) inflammasome is a target for metformin. Materials and methods ApoE−/− male mice were divided randomly into control, streptozocin-induced diabetes mellitus and metformin groups. Metabolic parameters, atherosclerotic lesion, activation of NLRP3 inflammasomes and related signaling pathways were detected. THP-1-differentiated macrophages were used in in vitro experiments. Results Compared with control mice, increased plasma lipids and proinflammatory interleukin-1β, aggravated macrophage infiltration into the atherosclerotic lesion, and accelerated development of atherosclerosis were observed in diabetic mice, which were associated with the activation of NLRP3 inflammasomes and dysregulation of thioredoxin-1 and thioredoxin-interacting protein. Treatment with metformin alleviated diabetes-induced metabolic disorders and atherosclerosis, as well as NLRP3 inflammasomes activation and dysregulation of thioredoxin-1/thioredoxin-interacting protein. In vitro experiments showed that high glucose induced the accumulation of reactive oxygen species and activated NLRP3 inflammasomes, which was significantly suppressed by treatment with metformin or antioxidant N-acetyl-L-cysteine. Moreover, Compound C, an inhibitor of adenosine 5′-monophosphate-activated protein kinase (AMPK), blocked the anti-inflammatory effect of metformin, indicating that metformin inhibited high glucose-induced NLRP3 inflammasomes activation through AMPK activation. Moreover, high glucose decreased thioredoxin-1 expression and increased thioredoxin-interacting protein expression, which was also reversed by metformin. Conclusions Metformin inhibited NLRP3 inflammasomes activation and suppressed diabetes-accelerated atherosclerosis in apoE−/− mice, which at least partially through activation of AMPK and regulation of thioredoxin-1/thioredoxin-interacting protein.

Sulfane sulfur species including hydrogen polysulfide and organic persulfide are newly recognized normal cellular components, and they participate in signaling and protect cells from oxidative stress. Their production has been extensively studied, but their removal is less characterized. Herein, we showed that sulfane sulfur at high levels was toxic to Escherichia coli under both anaerobic and aerobic conditions. OxyR, a well-known regulator against H2O2, also sensed sulfane sulfur, as revealed via mutational analysis, constructed gene circuits, and in vitro gene expression. Hydrogen polysulfide modified OxyR at Cys199 to form a persulfide OxyR C199-SSH, and the modified OxyR activated the expression of thioredoxin 2 and glutaredoxin 1. The two enzymes are known to reduce sulfane sulfur to hydrogen sulfide. Bioinformatics analysis indicated that OxyR homologs are widely present in bacteria, including obligate anaerobic bacteria. Thus, the OxyR sensing of sulfane sulfur may represent a preserved mechanism for bacteria to deal with sulfane sulfur stress., Graphical abstract Image 1, Highlights • OxyR also senses sulfane sulfur stress and activates the genes for its removal. • OxyR senses hydrogen polysulfide via persulfidation of OxyR at Cys199. • OxyR responds to sulfane sulfur stress under both aerobic and anaerobic conditions. • OxyR is widely distributed in bacterial genomes, including anaerobic bacteria.

NLRP3/IL-1β activation via thioredoxin (TRX)/thioredoxin-interacting protein (TXNIP) following mitochondria ROS (mtROS) overproduction plays a key role in inflammation. However, the involvement of this process in tubular damage in the kidneys of patients with diabetic nephropathy (DN) is unclear. Here, we demonstrated that mtROS overproduction is accompanied by decreases in TRX expression and TXNIP up-regulation. In addition, we discovered that mtROS overproduction is also associated with increases in NLRP3/IL-1β and TGF-β expression in the kidneys of patients with DN and db/db mice. We reversed these changes in db/db mice by administering a peritoneal injection of MitoQ, an antioxidant targeting mtROS. Similar results were observed in human tubular HK-2 cells subjected to high-glucose (HG) conditions and treated with MitoQ. Treating HK-2 cells with MitoQ suppressed the dissociation of TRX from TXNIP and subsequently blocked the interaction between TXNIP and NLRP3, leading to the inhibition of NLRP3 inflammasome activation and IL-1β maturation. The effects of MitoQ were enhanced by pretreatment with TXNIP siRNA and abolished by pretreatment with monosodium urate (MSU) and TRX siRNA in vitro. These results suggest that mitochondrial ROS-TXNIP/NLRP3/IL-1β axis activation is responsible for tubular oxidative injury, which can be ameliorated by MitoQ via the inhibition of mtROS overproduction.

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes., This work was funded by the Junta de Andalucía (BIO-199); the Plan Nacionalde Investigación Científica, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014) and FEDER; and the Plan Nacional (SAF2016-79957-R). We thank Dr Carlos Robello for kindly providing the anti-TbTRYP1 antibody and Dr David Horn for the anti-TbγH2A antibody.

Aims: Oxidative stress caused by exposure to drugs of abuse such as ethanol or 3, 4 methylenedioxymethamphetamine (MDMA) may derive from direct or indirect effects in many organs including the heart. The aim of the present work was to evaluate cardiac sympathetic activity and the expression and activation of two antioxidant proteins: heat shock protein27 (HSP27) and thioredoxin-1 (Trx-1) after voluntary binge ethanol consumption, alone and in combination with MDMA. Material and methods: Adolescent mice received MDMA, ethanol or both. Drinking in the dark (DID) procedure was used as a model of binge. HSP27 expression and phosphorylation at serine 82 (pHSP27), Trx-1 expression, tyrosine hydroxylase (TH) and TH phosphorylated at serine 31 (pTH) were evaluated in adolescent mice 48 h and 7 days after treatments in the right ventricle. TH, HSP27 expression and phosphorylation and Trx-1 expression were measured by quantitative blot immunolabeling using specific antibodies. Key findings: The expression of HSP27, pHSP27, Trx-1, total TH and pTH in the right ventricle was increased after binge ethanol or MDMA alone. In addition, the combination of binge ethanol + MDMA enhanced TH expression and phosphorylation versus their individual administration. Significance: These results indicate that this combination could produce higher activation of sympathetic pathways, which could trigger an increased cell stress. On the other hand, increased HSP27, pHSP27 and Trx-1 expression in the right ventricle by ethanol + MDMA could be a protective mechanism to reduce the adverse effects of oxidative stress caused by both drugs of abuse. The funders of this research have been the Spanish Ministry of Economy and Innovation and FEDER (SAF/FEDER 2013-49076-P, SAF2017-85679-R and SAF2013-41761-R), the Spanish Ministry of Health, Social Affairs and Equality (PNSD 2014-020) and Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Salud -ISCIII-FEDER- RETIC-Trastornos Adictivos RTA, RD12/0028/0003 and RD12/0028/0024).

Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni2+ in protein samples purified by IMAC. Both qualitative and quantitative colorimetric methods detect the amount of Ni2+ via the color change produced when a [Ni(PAR)n]2+ (PAR = 4-(2-pyridylazo)resorcinol, n = 1, 2) complex is formed. The qualitative assay provides a rapid visual test for the presence of Ni2+ in the low micromolar range in a sample of interest. The usefulness of the spectroscopic quantitative assay is illustrated by: (i) detecting a 12 μM Ni2+ contamination in an NMR sample containing 950 μM of the 7.5 kDa α3W protein purified by a standard His-tag Ni2+/IMAC approach and (ii) showing that the 15N-HSQC spectrum of the α3W NMR sample, containing 1 paramagnetic Ni2+ ion per 80 protein molecules, displays clear line broadening of both water and protein spectral lines. We also (iii) measured Ni2+ release during the equilibration, wash, and elution steps of three commonly used Ni2+/IMAC resins when following manufacturer's protocols. The concentration of Ni2+ detected in elutes of the three resins ranged from 2 μM to nearly 1 mM.

20 páginas. -- The final version is available at http://www.elsevier.com, Thioredoxins (Trxs) are low-molecular-weight proteins that participate in the reduction of target enzymes. Trxs contain a redox-active disulfide bond, in the form of a WCGPC amino acid sequence motif, that enables them to perform dithiol-disulfide exchange reactions with oxidized protein substrates. Widely distributed across the three domains of life, Trxs form an evolutionarily conserved family of ancient origin. Thioredoxin reductases (TRs) are enzymes that reduce Trxs. According to their evolutionary history, TRs have diverged, thereby leading to the emergence of variants of the enzyme that in combination with different types of Trxs meet the needs of the cell. In addition to participating in the regulation of metabolism and defense against oxidative stress, Trxs respond to environmental signals—an ability that developed early in evolution. Redox regulation of proteins targeted by Trx is accomplished with a pair of redox-active cysteines located in strategic positions on the polypeptide chain to enable reversible oxidative changes that result in structural and functional modifications target proteins. In this review, we present a general overview of the thioredoxin system and describe recent structural studies on the diversity of its components., MB acknowledges financial support from the Spanish Ministerio de Ciencia, Innovación y Universidades [Project Reference BFU2016_80343-P]

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., Highlights • C. elegans expressing endogenous “redox-dead” TRX-1 are viable. • The extended lifespan extension of worm daf-2 and eat-2 mutants and the food-leaving behaviour of C. elegans males requires a redox-active TRX-1. • The SKN-1 nuclear translocation and increased lips-6 expression upon TRX-1 deficiency is redox-independent. • TRX-1 regulates dauer formation by both redox-dependent and redox-independent mechanisms. • C. elegans is an ideal model to interrogate on the molecular mechanisms underlying the redox-independent functions of metazoan thioredoxins.

Mitochondrial uncoupling protein 2 (UCP2) has been well characterized to control the production of reactive oxygen species (ROS) and astrocytes are the major cells responsible for the ROS production and the inflammatory responses in the brain. However, the function of UCP2 in astrocytes and the contribution of astrocytic UCP2 to depression remain undefined. Herein, we demonstrated that UCP2 knockout (KO) mice displayed aggravated depressive-like behaviors, impaired neurogenesis, and enhanced loss of astrocytes in the chronic mild stress (CMS)-induced anhedonia model of depression. We further found that UCP2 ablation significantly enhanced the activation of the nod-like receptor protein 3 (NLRP3) inflammasome in the hippocampus and in astrocytes. Furthermore, UCP2 deficiency promoted the injury of mitochondria, the generation of ROS and the physical association between thioredoxin-interacting protein (TXNIP) and NLRP3 in astrocytes. Moreover, transiently expressing exogenous UCP2 partially rescued the deleterious effects of UCP2 ablation on the astrocytes. These data indicate that UCP2 negatively regulates the activation of NLRP3 inflammasome and inhibited the ROS-TXNIP-NLRP3 pathway in astrocytes. Collectively, our findings reveal that UCP2 regulates inflammation responses in astrocytes and plays an important role in the pathogenesis of depression and that UCP2 may be a promising therapeutic target for depression., Graphical abstract fx1, Highlights • UCP2 knockout exacerbates depressive behaviors in mice. • UCP2 knockout aggravates CMS-induced the inhibition of neurogenesis. • UCP2 deletion enhances NLRP3 inflammasome activation in hippocampus and in astrocyte. • UCP2 inhibits the ROS-TXNIP-NLRP3 signaling.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes., Graphical abstract fx1, Highlights • Trx1 and Grx1 Cys redox targets are abundant among Glycolytic enzymes. • PRDX6-Cys91 and PGAM-Cys153 are specific targets of Grx1. • Down regulation of thioredoxin and glutaredoxin have different metabolic outcomes. • Glutathione synthesis and membrane lipid composition are sensitive to Trx1 and Grx1 down regulation. • Redoxins down regulation also induce target Cys reductive changes under NOS3 overexpression.

The overall redox potential of a cell is primarily determined by oxidizable/reducible chemical pairs, including glutathione–glutathione disulfide, reduced thioredoxin–oxidized thioredoxin, and NAD+–NADH (and NADP–NADPH). Current methods for evaluating oxidative stress rely on detecting levels of individual byproducts of oxidative damage or by determining the total levels or activity of individual antioxidant enzymes. Oxidation–reduction potential (ORP), on the other hand, is an integrated, comprehensive measure of the balance between total (known and unknown) pro-oxidant and antioxidant components in a biological system. Much emphasis has been placed on the role of oxidative stress in chronic diseases, such as Alzheimer's disease and atherosclerosis. The role of oxidative stress in acute diseases often seen in the emergency room and intensive care unit is considerable. New tools for the rapid, inexpensive measurement of both redox potential and total redox capacity should aid in introducing a new body of literature on the role of oxidative stress in acute illness and how to screen and monitor for potentially beneficial pharmacologic agents., Graphical abstact

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20 mM, 24–48 h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300 µM, 24–48 h) increased clonogenic cell killing in both human prostate (PC-3 and DU145) and human breast (MDA-MB231) cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH) synthesis (l-buthionine sulfoximine; BSO, 1 mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48 h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1 µM), was combined with 2DG+DHEA or DHEA-alone for 24 h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20 mM). Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1) oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC) were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231). Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing., Graphical abstract, Highlights • Inhibition of both glycolysis and pentose cycle causes oxidative stress in human breast and prostate cancer cells. • Combining inhibition of glycolysis and pentose cycle with inhibition of thioredoxin reductase enhances cell killing of these human cancer cells. • The toxicity and oxidative stress is selective for cancer vs. normal cells.

Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance., This work was supported by grants BIO2012-34937, CSD2009-00088, and BIO2015-66426-R (J.M.S.-R.) from MINECO/FEFER and grant P09-CVI-5073 (B.I.-M.) from the “Junta de Andalucía” and Feder Funds.

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues. Fondation pour la Recherche Medicale DBI2014231801 Agence Nationale de la Recherche 12-BSV5-0019 REDPRO2

Mutations in the DJ-1 gene have been shown to cause a rare autosomal-recessive genetic form of Parkinson’s disease (PD). The function of DJ-1 and its role in PD development has been linked to multiple pathways, however its exact role in the development of PD has remained elusive. It is thought that DJ-1 may play a role in regulating reactive oxygen species (ROS) formation and overall oxidative stress in cells through directly scavenging ROS itself, or through the regulation of ROS scavenging systems such as glutathione (GSH) or thioredoxin (Trx) or ROS producing complexes such as complex I of the electron transport chain. Previous work in this laboratory has demonstrated that isolated brain mitochondria consume H2O2 predominantly by the Trx/Thioredoxin Reductase (TrxR)/Peroxiredoxin (Prx) system in a respiration dependent manner (Drechsel et al., Journal of Biological Chemistry, 2010). Therefore we wanted to determine if mitochondrial H2O2 consumption was altered in brains from DJ-1 deficient mice (DJ-1-/-). Surprisingly, DJ-1-/- mice showed an increase in mitochondrial respiration-dependent H2O2 consumption compared to controls. To determine the basis of the increased H2O2 consumption in DJ1-/- mice, the activities of Trx, Thioredoxin Reductase (TrxR), GSH, glutathione disulfide (GSSG) and glutathione reductase (GR) were measured. Compared to control mice, brains from DJ-1-/- mice showed an increase in (1) mitochondrial Trx activity, (2) GSH and GSSG levels and (3) mitochondrial glutaredoxin (GRX) activity. Brains from DJ-1-/- mice showed a decrease in mitochondrial GR activity compared to controls. The increase in the enzymatic activities of mitochondrial Trx and total GSH levels may account for the increased H2O2 consumption observed in the brain mitochondria in DJ-1-/- mice perhaps as an adaptive response to chronic DJ-1 deficiency., Highlights • Brain mitochondria from DJ-1-/- mice show increased mitochondrial H2O2 consumption rates. • DJ-1-/- mice show increased brain mitochondrial thioredoxin (Trx) activity. • DJ-1-/- mice show increased total brain glutathione levels. • Increased Trx activity may be the link to the increased H2O2 consumption rates., Graphical Abstract

In fission yeast, the transcription factor Pap1 undergoes H2O2-dependent oxidation that promotes its nuclear accumulation and the activation of an antioxidant gene program. However, the mechanisms that regulate the sensitivity and selectivity of Pap1 activation by peroxides are not fully understood. Here, we demonstrate that the peroxiredoxin Tpx1, the sensor of this signaling cascade, activates the otherwise unresponsive Pap1 protein once the main cytosolic reduced thioredoxin, Trx1, becomes transiently depleted. In other words, Pap1 works as an alternative electron donor for oxidized Tpx1. We have trapped the very transient Tpx1-Pap1 intermediate in cells depleted in Trx1, as we show here using mass spectrometry. Recycling of Tpx1 by Trx1 is required for the efficient signaling to Pap1, suggesting that the complete cycle of H2O2 scavenging by Tpx1 and further recycling of oxidized Tpx1 by Trx1 is required for full downstream activation of the redox cascade. This work was supported by the Spanish Ministry of Science and Innovation (BFU2009-06933 and BFU2012-32045), PLAN E and FEDER, by the Spanish program Consolider-Ingenio 2010 grant CSD 2007-0020, and by SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of ICREA Academia Awards (Generalitat de Catalunya)

Thioredoxin (TRX) is an oxidative stress-inducible biological antioxidant that is highly expressed in the synoviocytes of rheumatoid arthritis (RA) patients. There is much evidence that oxidative stress plays a key role in the inflammation and destruction of RA joints; the functional relationship between TRX and RA remains unknown, however. We therefore investigated the role played by TRX in the inflammatory and joint-damaging processes of RA using a murine model in which arthritis was induced by administering a mixture of anti-type II collagen monoclonal antibodies (mAb) and lipopolysaccharide (LPS). In Wt mice mAb/LPS injection induced neutrophil infiltration, cartilage destruction, and chondrocyte apoptosis within the joints, all of which were dramatically suppressed in TRX transgenic (TRX-Tg) mice. Moreover, the 8-hydoxy-2'-deoxyguanosine (8-OHdG) expression seen in Wt mice after mAb/LPS injection was almost completely inhibited in TRX-Tg mice. The administration of recombinant TRX also suppressed mAb/LPS-induced joint swelling in Wt mice. Taken together, these results suggest that TRX protects against arthritis and is a plausible candidate with which to develop novel therapies for the treatment of RA.

Thiol-based redox reactions are involved in the regulation of a variety of biological functions, such as protection against oxidative stress, signal transduction and protein folding. Some proteins involved in redox regulation have been shown to modulate life span in organisms from yeast to mammals. To assess the role of thiol oxidoreductases in aging on a genome-wide scale, we analyzed the replicative life span of yeast cells lacking known and candidate thiol oxidoreductases. The data suggest the role of several pathways in controlling yeast replicative life span, including thioredoxin reduction, protein folding and degradation, peroxide reduction, PIP3 signaling, and ATP synthesis. © 2010 Elsevier Ireland Ltd., DPT-2003K120690; TÜBA-GEBİP and NIH AG021518

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His 6 -tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

27 páginas, 6 figuras. Datos suplementarios., The thioredoxin system, composed of thioredoxin (Trx) and thioredoxin reductase (TrxR), emerges as one of the most important thiol-based systems involved in the maintenance of the cellular redox balance. Thioredoxin-like-1 (TXL-1) is a highly conserved protein comprising an N-terminal Trx domain and a C-terminal domain of unknown function. Here we show that TXL-1 is a substrate for the cytosolic selenoprotein TrxR-1. In situ hybridization experiments demonstrates high expression of Txl-1 mRNA in various areas of central nervous system and also in some reproductive organs. Glucose deprivation, but not hydrogen peroxide treatment, reduced the levels of endogenous TXL-1 protein in HEK-293 cell line. Conversely, overexpression of TXL-1 protects against glucose deprivation-induced cytotoxicity. Taken together, the finding that Txl-1 mRNA is highly expressed in tissues which use glucose as a primary energy source and the modulation of TXL-1 levels upon glucose deprivation indicate that TXL-1 might be involved in the cellular response to sugar starvation stress., This work was supported by the Swedish Medical Research Council (Projects 03P-14096, 03X-14041, and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet and Medical Research Fund of Tampere University Hospital. A. Jiménez was supported by a postdoctoral fellowship (EX2003-0390) from the Spanish Ministerio de Educación, Cultura y Deporte.

28 páginas, 3 figuras más 1 suplementaria, 4 tablas más 1 suplementaria., Thioredoxins function as general protein disulphide reductases. Mammalian male germ cells are equipped with a set of three testis-specific thioredoxins (named Sptrx-1, -2, and -3, respectively) that are expressed either in different structures within the sperm cell or at different stages of sperm development. Previous studies based on qualitative northern-blot and in situ hybridization analyses restricted the presence of Sptrx mRNAs to adult testis, but nothing is known about their transcriptional regulation or relative expression levels in this tissue. In this report, we investigate the transcriptional profiles of the mouse Sptrx genes in terms of the germ cell-specific regulation by promoter analysis in GC-2spd(ts) cells. Besides, we perform a comprehensive quantification of the Sptrx mRNA molecules by real-time PCR in whole-animal experiments. By these means, we show that transcription is differentially regulated for each Sptrx gene and identify the 5′-flanking regions anticipated to contain the cis-regulatory elements responsible, at least in part, for the transcriptional silencing and/or activation of the Sptrx genes. In addition, we show remarkable age-associated variations between the Sptrx mRNA expression patterns., This work was supported by the Swedish Medical Research Council (Projects 03P-14096, 03X-14041, and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet, and the Spanish Ministerio de Ciencia y Tecnología (Grant BMC2002-00179). A. Jiménez was supported by a postdoctoral fellowship (EX2003-0390) from the Spanish Ministerio de Educación, Cultura y Deporte. M.-J. Prieto-Álamo and J. Jurado were recipients of postdoctoral contracts (Programa Ramón y Cajal) from the Spanish Ministerio de Ciencia y Tecnología.

6 páginas, 5 figuras., Thioredoxins comprise a growing family of proteins that function as general protein-disulfide reductases and are maintained in their reduced active form by the flavoenzyme thioredoxin reductase. Human Trx-1 is mainly a cytosolic protein, although it has been shown to translocate into the nucleus upon certain stimuli and can also be secreted. We report here the expression and characterization of delta3Trx-1, a splicing variant of human Trx-1, lacking exon 3, which spans from residues 44 to 63 in the wild-type protein. Structure-based prediction of this splicing form indicates that delta3Trx-1 lacks helix alpha2 and strand beta3, which are implicated in substrate positioning and three-dimensional stabilization of the active site residues. Recombinant human delta3Trx-1 is recognized by polyclonal antibodies raised against full-length human Trx-1. However, delta3Trx-1 retains no enzymatic activity either with DTT or thioredoxin reductase and NADPH as reducing systems. Delta3Trx-1 competes with full-length Trx-1 for the interaction with thioredoxin reductase. The absence of helix alpha2 and strand beta3 in delta3Trx-1 is consistent with the lack of enzymatic activity and its potential dominant negative properties., This work was supported by grants from the Swedish Medical Research Council (Projects 03P-14096-01A and 03X-14041-01A), the Åke Wibergs Stiftelse, and the Karolinska Institutet, and the Fundación Margit y Folke Pehrzon.

The reduction/oxidation (redox) state of the cell is a consequence of the balance between the levels of oxidising and reducing equivalents. A reducing intracellular environment is often associated with cell survival; however, redox unbalance is necessary since it represents a regulatory sensor for several nuclear transcription factors. Activator protein 1 (AP-1), nuclear factor-kappaB (NF-kappaB) and protein tyrosine phosphatases 1-B (PTP-1B) are some of the well-known molecular factors for which a redox modulation of their activity has been demonstrated. The glutathione buffer system modulates cell response to redox changes induced by either external or intracellular stimuli. This paper summarises recent knowledge on the role played by several redox modulators in inducing signalling events that finally regulate cell cycle progression.

Background IgE-mediated cross-reactivity between fungal antigens and human proteins has been described in patients with atopic dermatitis (AD), but it remains to be elucidated whether there is also cross-reactivity at the T-cell level. Objective We sought to explore cross-reactivity at the T-cell level between the fungal thioredoxin (Mala s 13) of the skin-colonizing yeast Malassezia sympodialis and its homologous human thioredoxin (hTrx). Methods T-cell lines (TCLs) were generated in the presence of rMala s 13 from the peripheral blood and from skin biopsy specimens of positive patch test reactions of patients with AD sensitized to Mala s 13 and hTrx. Patients with AD not sensitized to Malassezia species, healthy subjects, and patients with psoriasis served as control subjects. Mala s 13–specific T-cell clones (TCCs) were generated from TCLs. TCCs were characterized by antigen specificity, phenotype, and cytokine secretion pattern. Human keratinocytes were stimulated with IFN-γ, TNF-α, and IL-4, and the release of hTrx was determined by means of ELISA. Results Mala s 13–specific TCLs and TCCs from the blood and skin of patients with AD sensitized to Mala s 13 and hTrx were fully cross-reactive with hTrx. Mala s 13– and hTrx–specific TCCs could not be generated from control subjects. The majority of cross-reactive TCCs were CD4 + and coexpressed cutaneous lymphocyte antigen. In addition to T H 1 and T H 2 TCCs, we could also identify TCCs secreting IL-17 and IL-22. After stimulation with IFN-γ and TNF-α, keratinocytes released substantial amounts of thioredoxin. Conclusion In patients with AD sensitized to Malassezia species, cross-reactivity at the T-cell level to Mala s 13 and the homologous hTrx is detectable. hTrx autoreactive skin-homing T cells might be relevant for cutaneous inflammation in patients with AD.