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Glutamate is excitotoxic to neurons. The entry of glutamine or glutamate from the blood into the brain is limited. To overcome this, branched-chain amino acids (BCAAs) catabolism replenishes the glutamate in brain cells. Branched-chain amino acid transaminase 1 (BCAT1) activity is silenced by epigenetic methylation in IDH mutant gliomas. However, glioblastomas (GBMs) express wild type IDH. Here, we investigated how oxidative stress promotes BCAAs’ metabolism to maintain intracellular redox balance and, consequently, the rapid progression of GBMs. We found that reactive oxygen species (ROS) accumulation promoted the nuclear translocation of lactate dehydrogenase A (LDHA), which triggered DOT1L (disruptor of telomeric silencing 1-like)-mediated histone H3K79 hypermethylation and enhanced BCAA catabolism in GBM cells. Glutamate derived from BCAAs catabolism participates in antioxidant thioredoxin (TxN) production. The inhibition of BCAT1 decreased the tumorigenicity of GBM cells in orthotopically transplanted nude mice, and prolonged their survival time. In GBM samples, BCAT1 expression was negatively correlated with the overall survival time (OS) of patients. These findings highlight the role of the non-canonical enzyme activity of LDHA on BCAT1 expression, which links the two major metabolic pathways in GBMs. Glutamate produced by the catabolism of BCAAs was involved in complementary antioxidant TxN synthesis to balance the redox state in tumor cells and promote the progression of GBMs.

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m is also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, with growth retardation noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin–Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system., This work was funded by grant PID2020-115156GB-100, funded by Agencia Estatal de Investigación (AEI), Ministerio de Ciencia e Innovación (MICIN), Spain (MICIN/AEI/10.13039/501100011033).

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1’s ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers.

SIGNIFICANCE The presence of a large number of thioredoxin superfamily members suggests a complex mechanism of redox-based regulation in mammalian cells. However, whether these members are functionally redundant or play separate and distinct roles in each cellular compartment remains to be elucidated. Recent advances: In the mammalian endoplasmic reticulum (ER), approximately 20 thioredoxin-like proteins have been identified. Most ER oxidoreductases are soluble proteins located in the luminal compartment, while a small family of five thioredoxin-related transmembrane proteins (TMX) also reside in the ER membrane and play crucial roles with specialized functions. CRITICAL ISSUES In addition to the predicted function of ER protein quality control, several independent studies have suggested the diverse roles of TMX family proteins in the regulation of cellular processes, including calcium homeostasis, bioenergetics, and thiol-disulfide exchange in the extracellular space. Moreover, recent studies have provided evidence of their involvement in the pathogenesis of various diseases. FUTURE DIRECTIONS Extensive research is required to unravel the physiological roles of TMX family proteins. Given that membrane-associated proteins are prime targets for drug discovery in a variety of human diseases, expanding our knowledge on the mechanistic details of TMX action on the cell membrane will provide molecular basis for developing novel diagnostic and therapeutic approaches as a potent molecular target in a clinical setting.

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA–substrate thiol–disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

C⁴-substituted fluoroprolines (4R)-fluoroproline ((4R)-Flp) and (4S)-fluoroproline ((4S)-Flp) have been used in protein engineering to enhance the thermodynamic stability of peptides and proteins. The electron-withdrawing effect of fluorine can bias the pucker of the pyrrolidine ring, influence the conformational preference of the preceding peptide bond, and can accelerate the cis/trans prolyl peptide bond isomerisation by diminishing its double bond character. The role of 4,4-difluoroproline (Dfp) in the acceleration of the refolding rate of globular proteins bearing a proline (Pro) residue in the cis conformation in the native state remains elusive. Moreover, the impact of Dfp on the thermodynamic stability and bioactivity of globular proteins has been seldom described. In this study, we show that the incorporation of Dfp caused a redox state dependent and position dependent destabilisation of the thioredoxin (Trx) fold, while the catalytic activities of the modified proteins remained unchanged. The Pro to Dfp substitution at the conserved cisPro76 in the thioredoxin variant Trx1P did not elicited acceleration of the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond. Our results show that pucker preferences in the context of a tertiary structure could play a major role in protein folding, thus overtaking the rules determined for cis/trans isomerisation barriers determined in model peptides., ChemBioChem, 22 (23), ISSN:1439-4227, ISSN:1439-7633

Glutathione (Grx) is a glutathione-dependent oxidoreductase belonging to the thioredoxin (Trx) superfamily. It maintains redox homeostasis in organisms and plays a significant role in resisting oxidative stress. Nevertheless, Grx-related sequence information and functional validation are greatly inadequate in crustaceans. In this study, the Grx3 of Penaeus monodon (PmGrx3) was cloned. The full-length cDNA of PmGrx3 was 1546 bp, which consists of 113-bp 5′-UTR, 458-bp 3′-UTR, and 975-bp ORF, encoding a 324 amino acid putative protein. The putative PmGrx3 protein was 36.04 kDa with a theoretical isoelectric point of 4.98. Sequence alignment indicated that PmGrx3 had the highest amino acid sequence homology with the Grx3 of Penaeus vannamei at 96.57% and was clustered with the crustaceans. Quantitative real-time PCR (qRT-PCR) analysis revealed that PmGrx3 was detected in all tissues examined, with the highest expression in the muscle, stomach, and testis, as well as the lowest expression in abdominal nerve. In addition, PmGrx3 was expressed during the developmental period and was the most highly expressed in the zygote. Both ammonia-N stress and bacterial infection caused different degrees of upregulation or inhibition of the PmGrx3 expression in the hepatopancreas and gills. The present study indicates that PmGrx3 may be a key oxidoreductase in the defense system of shrimp against environmental stress and pathogen infection.

Selenium (Se) is a micronutrient with essential physiological functions achieved through the production of selenoproteins. Adequate Se intake has health benefits and reduces mercury (Hg) toxicity, which is important due to its neurotoxicity. This study determined the Se status and redox enzyme, including selenoproteins’, activity in pregnant women highly exposed to Hg (between 1 to 54 µg Hg/L blood) via fish consumption. A cross-sectional study enrolling 513 women between the first and third trimester of pregnancy from Madeira, Portugal was conducted, encompassing collection of blood and plasma samples. Samples were analyzed for total Se and Hg levels in whole blood and plasma, and plasma activity of redox-active proteins, such as glutathione peroxidase (GPx), thioredoxin reductase (TrxR) and thioredoxin (Trx). Enzyme activities were related to Se and Hg levels in blood. Se levels in whole blood (65.0 ± 13.1 µg/L) indicated this population had a sub-optimal Se status, which translated to low plasma GPx activity (69.7 ± 28.4 U/L). The activity of TrxR (12.3 ± 5.60 ng/mL) was not affected by the low Se levels. On the other hand, the decrease in Trx activity with an increase in Hg might be a good indicator to prevent fetal susceptibility.

The stabilization of natural proteins is a long-standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged-to-hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by partial least squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stabilities reported for a protein with a natural occurring fold (137°C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo., European Regional Development Fund funds and grants, Grant/Award Numbers: BIO2012-34937, CSD2009-00088; Junta de Andalucia, Grant/Award Number: P09-CVI-5073; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: BIO2015-66426-R

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host‐induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference‐induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1‐based host‐induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS‐A) and Nicotiana benthamiana (HIGS‐N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum., Normal expression of SsTrx1 is crucial for pathogenicity, sclerotial development, and oxidative stress tolerance of Sclerotinia sclerotiorum.

Significance: Glutathione (GSH) represents the most abundant and the main antioxidant in the body with important functions in the brain related to Alzheimer's disease (AD). Recent Advances: Oxidati...

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Rationale Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo. Objective A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip's pro-oxidative or cytotoxic effects in the heart. Methods and results Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip's inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms. Conclusion Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.

Acetaminophen (APAP) is one of the most widely used drugs with antipyretic and analgesic effects, and thus hepatotoxicity from the overdose of APAP becomes one of the most common forms of drug-induced liver injury. The reaction towards thiol molecules, such as GSH by APAP metabolite, N-acetyl-p-benzo-quinonimine (NAPQI), is the main cause of APAP-induced hepatotoxicity. However, the role of many other thiol-related regulators in toxicity caused by APAP is still unclear. Here we have found that knockout of the Glrx2 gene, which encodes mitochondrial glutaredoxin2 (Grx2), sensitized mice to APAP-caused hepatotoxicity. Glrx2 deletion hindered Nrf2-mediated compensatory recovery of thiol-dependent redox systems after acetaminophen challenge, resulting in a more oxidized cellular state with a further decrease in GSH level, thioredoxin reductase activity, and GSH/GSSG ratio. The weakened feedback regulation capacity of the liver led to higher levels of protein glutathionylation and thioredoxin (both Trx1 and Trx2) oxidation in Glrx2−/− mice. Following the cellular environment oxidation, nuclear translocation of apoptosis-inducing factor (AIF) was elevated in the liver of Glrx2−/− mice. Taken together, these results demonstrated that mitochondrial Grx2 deficiency deteriorated APAP-induced hepatotoxicity by interrupting thiol-redox compensatory response, enhancing the AIF pathway-mediated oxidative damage.

Soybean (Glycine max (L.) Merr.) establishes symbiosis with rhizobacteria, developing the symbiotic nodule, where the biological nitrogen fixation (BNF) occurs. The redox control is key for guaranteeing the establishment and correct function of the BNF process. Plants have many antioxidative systems involved in ROS homeostasis and signaling, among them a network of thio- and glutaredoxins. Our group is particularly interested in studying the differential response of nodulated soybean plants to water-deficit stress. To shed light on this phenomenon, we set up an RNA-seq experiment (for total and polysome-associated mRNAs) with soybean roots comprising combined treatments including the hydric and the nodulation condition. Moreover, we performed the initial identification and description of the complete repertoire of thioredoxins (Trx) and glutaredoxins (Grx) in soybean. We found that water deficit altered the expression of a greater number of differentially expressed genes (DEGs) than the condition of plant nodulation. Among them, we identified 12 thioredoxin (Trx) and 12 glutaredoxin (Grx) DEGs, which represented a significant fraction of the detected GmTrx and GmGrx in our RNA-seq data. Moreover, we identified an enriched network in which a GmTrx and a GmGrx interacted with each other and associated through several types of interactions with nitrogen metabolism enzymes.

Thioredoxins (TRXs) f and m are redox proteins that regulate key chloroplast processes. The existence of several isoforms of TRXs f and m indicates that these redox players have followed a specialization process throughout evolution. Current research efforts are focused on discerning the signalling role of the different TRX types and their isoforms in chloroplasts. Nonetheless, little is known about their function in non-photosynthetic plastids. For this purpose, we have carried out comprehensive expression analyses by using Arabidopsis thaliana TRXf (f1 and f2) and TRXm (m1, m2, m3 and m4) genes translationally fused to the green fluorescence protein (GFP). These analyses showed that TRX m has different localisation patterns inside chloroplasts, together with a putative dual subcellular localisation of TRX f1. Apart from mesophyll cells, these TRXs were also observed in reproductive organs, stomatal guard cells and roots. We also investigated whether photosynthesis, stomatal density and aperture or root structure were affected in the TRXs f and m loss-of-function Arabidopsis mutants. Remarkably, we immunodetected TRX m2 and the Calvin–Benson cycle fructose-1,6-bisphosphatase (cFBP1) in roots. After carrying out in vitro redox activation assays of cFBP1 by plastid TRXs, we propose that cFBP1 might be activated by TRX m2 in root plastids., This work has been funded by research projects BIO2009-07297 and BIO2015-65272 from the Spanish Ministry of Science and Innovation and the European Fund for Regional Development; project P07-CVI-2795 and BIO 154 from the Andalusian Regional Government, Spain; project BIO2012-33292 from the Spanish Ministry of Economy and Competitiveness; and by Grant PGC2018-096851-B-C21, from FEDER/Spanish Ministry of Science, Innovation and University: AEI, Spain.

Nitric oxide (NO) is a free radical with a signaling capacity. Its cellular functions are achieved mainly through S-nitrosation where thioredoxin (hTrx) is pivotal in the S-transnitrosation to specific cellular targets. In this study, we use NMR spectroscopy and mass spectrometry to follow the mechanism of S-(trans)nitrosation of hTrx. We describe a site-specific path for S-nitrosation by measuring the reactivity of each of the 5 cysteines of hTrx using cysteine mutants. We showed the interdependence of the three cysteines in the nitrosative site. C73 is the most reactive and is responsible for all S-transnitrosation to other cellular targets. We observed NO internal transfers leading to C62 S-nitrosation, which serves as a storage site for NO. C69-SNO only forms under nitrosative stress, leading to hTrx nuclear translocation.

Cisplatin is one of the most active chemotherapy drugs to treat solid tumors. However, it also causes various side effects, especially nephrotoxicity, in which oxidative stress plays critical roles. Our previous studies found that cisplatin selectively inhibited selenoenzyme thioredoxin reductase1 (TrxR1) in the kidney at an early stage and, subsequently, induced the activation of Nrf2. However, the effects of selenium on cisplatin-induced nephrotoxicity are still unclear. In this study, we established mice models with different selenium intake levels to explore the effects of selenoenzyme activity changes on cisplatin-induced nephrotoxicity. Results showed that feeding with a selenium-deficient diet sensitize the mice to cisplatin-induced damage, whereas selenium supplementation increased the activities of selenoenzymes TrxR and glutathione peroxidase (GPx), changed the renal cellular redox environment to a reduced state, and exhibited protective effects. These results demonstrated the correlation of selenoenzymes with cisplatin-induced side effects and provided a basis for the potential approach to alleviate cisplatin-induced renal injury.

Significance: Thioredoxin (Trx) and thioredoxin reductase are two core members of the Trx system. The system bridges the gap between the universal reducing equivalent NADPH and various biological molecules and plays an essential role in maintaining cellular redox homeostasis and regulating multiple cellular redox signaling pathways. Recent Advance: In recent years, the Trx system has been well documented as an important regulator of many diseases, especially tumorigenesis. Thus, the development of potential therapeutic molecules targeting the system is of great significance for disease treatment. Critical Issues: We herein first discuss the physiological functions of the Trx system and the role that the Trx system plays in various diseases. Then, we focus on the introduction of natural small molecules with potential therapeutic applications, especially the anticancer activity, and review their mechanisms of pharmacological actions via interfering with the Trx system. Finally, we further discuss several natural molecules that harbor therapeutic potential and have entered different clinical trials. Future Directions: Further studies on the functions of the Trx system in multiple diseases will not only improve our understanding of the pathogenesis of many human disorders but also help develop novel therapeutic strategies against these diseases. Antioxid. Redox Signal. 34, 1083-1107.

In this study, a higher metal ions-resistant bacterium, Stenotrophomonas rhizophila JC1 was isolated from contaminated soil in Jinchang city, Gansu Province, China. The Pb2+ (120 mg/L) and Cu2+ (80 mg/L) removal rate of the strain reached at 76.9% and 83.4%, respectively. The genome comprises 4268161 bp in a circular chromosome with 67.52% G + C content and encodes 3719 proteins. The genome function analysis showed czc operon, mer operon, cop operon, arsenic detoxification system in strain JC1 were contributed to the removal of heavy metals. Three efflux systems (i.e., RND, CDF, and P-ATPase) on strain JC1 genome could trigger the removal of divalent cations from cells. cAMP pathway and ABC transporter pathway might be involved in the transport and metabolism of heavy metals. The homology analysis exhibited multi-gene families such as ABC transporters, heavy metal-associated domain, copper resistance protein, carbohydrate-binding domain were distributed across 410 orthologous groups. In addition, heavy metal-responsive transcription regulator, thioredoxin, heavy metal transport/detoxification protein, divalent-cation resistance protein CutA, arsenate reductase also played important roles in the heavy metals adsorption and detoxification process. The complete genome data provides insight into the exploration of the interaction mechanism between microorganisms and heavy metals.

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.

Thioredoxin (Trx) plays a critical role in maintaining redox balance in various cells and exhibits anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, whether exogenous Trx can inhibit intracellular oxidative damage has not been investigated. In previous study, we have identified a novel Trx from the jellyfish Cyanea capillata, named CcTrx1, and confirmed its antioxidant activities in vitro. Here, we obtained a recombinant protein, PTD-CcTrx1, which is a fusion of CcTrx1 and protein transduction domain (PTD) of HIV TAT protein. The transmembrane ability and antioxidant activities of PTD-CcTrx1, and its protective effects against H2O2-induced oxidative damage in HaCaT cells were also detected. Our results revealed that PTD-CcTrx1 exhibited specific transmembrane ability and antioxidant activities, and it could significantly attenuate the intracellular oxidative stress, inhibit H2O2-induced apoptosis, and protect HaCaT cells from oxidative damage. The present study provides critical evidence for application of PTD-CcTrx1 as a novel antioxidant to treat skin oxidative damage in the future.

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii’s response to oxidative stress. The role of the large TrxR, however, remains elusive.

Atherosclerosis is the leading cause of death worldwide and has in part an inflammatory basis. Since epicardial adipose tissue (EAT) is in close contact with coronary arteries we hypothesized that an imbalance in thioredoxin-1 (TRX-1) and thioredoxin interacting protein (TXNIP) in EAT, activates NLRP3 inflammasome and promotes production of IL-1β, leading to the development of atherosclerosis. Thirty-eight patients with coronary artery disease (CAD) and thirty patients with no clinical signs of atherosclerosis who underwent open-heart surgery for valve replacement were classified as CAD and control groups, respectively. Biopsy samples from EAT were collected and expression of TXNIP, TRX-1, NLRP3 and IL-1β genes were assessed using qRT-PCR. Tissue protein levels of TXNIP and TRX-1 were determined by Western blotting while ELISA was applied to measure IL-1β. Haematoxylin and eosin staining was used for histological examination. mRNA and protein levels of TXNIP in EAT were significantly higher in patients with CAD compared with control group, whereas CAD patients showed lower TRX-1 gene and protein expression. In addition, in CAD patients the NLRP3 gene expression was almost doubled and IL-1β significantly increased at the both mRNA and protein levels. Enhancment in NLRP3 gene expression and TXNIP protein levels were accompanied with the increase in IL-1β protein level whereas TRX-1 protein content showed a negative correlation with IL-1β level. Concurrent increase in TXNIP, NLRP3, and IL-1β suggests possible involvement of thioredoxin system in the activation of NLRP3 inflammasome, production of IL-1β, and the presence of inflammation in CAD patients.

Short synthetic peptide molecules which bind to a specific target protein with a high affinity to exert its function are known as peptide aptamers. The high specificity of aptamers with small-molecule targets (metal ions, dyes and theophylline; ATP) is within 1 pM and 1 μM range, whereas with the proteins (thrombin, CD4 and antibodies) it is in the nanomolar range (which is equivalent to monoclonal antibodies). The recently identified coronavirus (SARS-CoV-2) genome encodes for various proteins, such as envelope, membrane, nucleocapsid, and spike protein. Among these, the protein necessary for the virus to enter inside the host cell is spike protein. The work focuses on designing peptide aptamer targeting the spike receptor-binding domain of SARS-CoV-2. The peptide aptamer has been designed by using bacterial Thioredoxin A as the scaffold protein and an 18-residue-long peptide. The tertiary structure of the peptide aptamer is modeled and docked to spike receptor-binding domain of SARS CoV2. Molecular dynamic simulation has been done to check the stability of the aptamer and receptor-binding domain complex. It was observed that the aptamer binds to spike receptor-binding domain of SARS-CoV-2 in a similar pattern as that of ACE2. The aptamer–receptor-binding domain complex was found to be stable in a 100 ns molecular dynamic simulation. The aptamer is also predicted to be non-antigenic, non-allergenic, non-hemolytic, non-inflammatory, water-soluble with high affinity toward ACE2 than serum albumin. Thus, peptide aptamer can be a novel approach for the therapeutic treatment for SARS-CoV-2. Graphical abstract

Suppressors of cytokine signaling (SOCS) exhibit diverse antiinflammatory effects. Since ROS acts as a critical mediator of inflammation, we have investigated the anti-inflammatory mechanisms of SOCS via ROS regulation in monocytic/macrophagic cells. Using PMA-differentiated monocytic cell lines and primary BMDMs transduced with SOCS1 or shSOCS1, the LPS/TLR4-induced inflammatory signaling was investigated by analyzing the levels of intracellular ROS, antioxidant factors, inflammasome activation, and pro-inflammatory cytokines. The levels of LPS-induced ROS and the production of pro-inflammatory cytokines were notably down-regulated by SOCS1 and up-regulated by shSOCS1 in an NAC-sensitive manner. SOCS1 up-regulated an ROS-scavenging protein, thioredoxin, via enhanced expression and binding of NRF-2 to the thioredoxin promoter. SOCS3 exhibited similar effects on NRF-2/thioredoxin induction, and ROS downregulation, resulting in the suppression of inflammatory cytokines. Notably thioredoxin ablation promoted NLRP3 inflammasome activation and restored the SOCS1-mediated inhibition of ROS and cytokine synthesis induced by LPS. The results demonstrate that the anti-inflammatory mechanisms of SOCS1 and SOCS3 in macrophages are mediated via NRF-2-mediated thioredoxin upregulation resulting in the downregulation of ROS signal. Thus, our study supports the anti-oxidant role of SOCS1 and SOCS3 in the exquisite regulation of macrophage activation under oxidative stress. [BMB Reports 2020; 53(12): 640-645].

Ferredoxin-thioredoxin reductase (FTR) is an iron-sulfur protein that supplies electrons from photochemically reduced ferredoxin (Fd) to thioredoxin (Trx) in the ferredoxin/thioredoxin system in chloroplasts. The FTR is a heterodimer with a variable subunit (FTRv) and a catalytic subunit (FTRc). The function of FTRv is not well known. In petunia (Petunia hybrida), FTRv is a single-copy gene, which is named PhFTRv. In this study, the spatio-temporal expression of PhFTRv in petunia was analyzed, and PhFTRv transcription was found to be high in leaves and stems. A tobacco rattle virus gene silencing was used in this study. Virus induced gene silencing-mediated PhFTRv silencing resulted in large yellow-green leaves, delayed flowering, and reduced cold tolerance in petunia plants.

The thiol-based glutathione reductase (GR) and thioredoxin reductase (TrxR) are the major antioxidant enzymes present in various organisms that maintain the internal redox homeostasis. The thioredoxin system has attracted the attention of researchers from diverse investigation fields of biological sciences. Apart from redox regulation, this system is thought to be the major regulator of various biological processes including transcription, apoptosis, etc. Identification and physicobiochemical characterization of the reductase enzyme i.e. Thioredoxin reductase (TrxR) revealed the potency of it to become a promising target. Novel therapeutic interventions by selective targeting of TrxR in parasitic organisms as well as in the cancer cells have now become a usual treatment approach. However, different isoforms and their variation in the penultimate amino acid (Selenocysteine or cysteine) present in the catalytic site of the enzyme have made this enzyme to respond differently towards various drugs and synthetic and/or natural compounds. Therefore, the present article seeks to highlight the importance and the detailed molecular mechanism, functional perspective underlying the TrxR inhibition in various parasitic protozoans, helminthes as well as in cancer cells for devising suitable anti-TrxR candidates.

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.

Peritoneal dialysis (PD) is an effective method of renal replacement therapy, providing a high level of patient autonomy. Nevertheless, the long-term use of PD is limited due to deleterious effects of PD fluids to the structure and function of the peritoneal membrane leading to loss of dialysis efficacy. PD patients show excessive oxidative stress compared to controls or chronic kidney disease (CKD) patients not on dialysis. Therefore, defense systems against detrimental events play a pivotal role in the integrity of the peritoneal membrane. The thioredoxin-interacting-protein (TXNIP)/thioredoxin (TRX) system also plays a major role in maintaining the redox homeostasis. We hypothesized that the upregulation of TXNIP negatively influences TRX activity, resulting in enhanced oxidative DNA-damage in PD patients. Therefore, we collected plasma samples and human peritoneal biopsies of healthy controls and PD patients as well. Using ELISA-analysis and immunohistochemistry, we showed that PD patients had elevated TXNIP levels compared to healthy controls. Furthermore, we demonstrated that PD patients had a reduced TRX activity, thereby leading to increased oxidative DNA-damage. Hence, targeting the TXNIP/TRX system as well as the use of oxidative stress scavengers could become promising therapeutic approaches potentially applicable in clinical practice in order to sustain and improve peritoneal membrane function.

Glioblastoma multiforme (GBM) is the most aggressive and common form of glioma. GBM, like many other tumors, expresses high levels of redox proteins, such as thioredoxin (Trx) and thioredoxin reductase (TrxR), allowing tumor cells to cope with high levels of reactive oxygen species (ROS) and resist chemotherapy and radiotherapy. Thus, tackling the activity of these enzymes is a strategy to reduce cell viability and proliferation and most importantly achieve tumor cell death. Mercury (Hg) compounds are among the most effective inhibitors of TrxR and Trx due to their high affinity for binding thiols and selenols. Moreover, organomercurials such as thimerosal, have a history of clinical use in humans. Thimerosal effectively crosses the blood–brain barrier (BBB), thus reaching effective concentrations for the treatment of GBM. Therefore, this study evaluated the effects of thimerosal (TmHg) and its metabolite ethylmercury (EtHg) over the mouse glioma cell line (GL261), namely, the inhibition of the thioredoxin system and the occurrence of oxidative cellular stress. The results showed that both TmHg and EtHg increased oxidative events and triggered cell death primarily by apoptosis, leading to a significant reduction in GL261 cell viability. Moreover, the cytotoxicity of TmHg and ETHg in GL261 was significantly higher when compared to temozolomide (TMZ). These results indicate that EtHg and TmHg have the potential to be used in GBM therapy since they strongly reduce the redox capability of tumor cells at exceedingly low exposure levels., This study was funded by FCT—Portugal through projects Target_Cancer (PTDC/MED-FAR/31136/2017) and by iMed.ULisboa UIB04138/2020 both from FCT. Vasco Branco was financed by national funds via FCT through Norma Transitória—DL57/2016/CP1376/CT002. MA was supported in part by a grant from the National Institute of Environmental Health Sciences (NIEHS) R01 ES07331.

International audience; Deinococcus species possess remarkable tolerance to extreme environmental conditions that generate oxidative damage to macromolecules. Among enzymes fulfilling key functions in metabolism regulation and stress responses, thiol reductases (TRs) harbour catalytic cysteines modulating the redox status of Cys and Met in partner proteins. We present here a detailed description of Deinococcus TRs regarding gene occurrence, sequence features, and physiological functions that remain poorly characterised in this genus. Two NADPH-dependent thiol-based systems are present in Deinococcus. One involves thioredoxins, disulfide reductases providing electrons to protein partners involved notably in peroxide scavenging or in preserving protein redox status. The other is based on bacillithiol, a low-molecular-weight redox molecule, and bacilliredoxin, which together protect Cys residues against overoxidation. Deinococcus species possess various types of thiol peroxidases whose electron supply depends either on NADPH via thioredoxins or on NADH via lipoylated proteins. Recent data gained on deletion mutants confirmed the importance of TRs in Deinococcus tolerance to oxidative treatments, but additional investigations are needed to delineate the redox network in which they operate, and their precise physiological roles. The large palette of Deinococcus TR representatives very likely constitutes an asset for the maintenance of redox homeostasis in harsh stress conditions.

Cys-based redox regulation was long regarded a major adjustment mechanism of photosynthesis and metabolism in plants, but in the recent years, its scope has broadened to most fundamental processes of plant life. Drivers of the recent surge in new insights into plant redox regulation have been the availability of the genome-scale information combined with technological advances such as quantitative redox proteomics and in vivo biosensing. Several unexpected findings have started to shift paradigms of redox regulation. Here, we elaborate on a selection of recent advancements, and pinpoint emerging areas and questions of redox biology in plants. We highlight the significance of (1) proactive H2O2 generation, (2) the chloroplast as a unique redox site, (3) specificity in thioredoxin complexity, (4) how to oxidize redox switches, (5) governance principles of the redox network, (6) glutathione peroxidase-like proteins, (7) ferroptosis, (8) oxidative protein folding in the ER for phytohormonal regulation, (9) the apoplast as an unchartered redox frontier, (10) redox regulation of respiration, (11) redox transitions in seed germination and (12) the mitochondria as potential new players in reductive stress safeguarding. Our emerging understanding in plants may serve as a blueprint to scrutinize principles of reactive oxygen and Cys-based redox regulation across organisms.

Plastids are specialized organelles found in plants, which are endowed with their own genomes, and differ in many respects from the intracellular compartments of organisms belonging to other kingdoms of life. They differentiate into diverse, plant organ-specific variants, and are perhaps the most versatile organelles known. Chloroplasts are the green plastids in the leaves and stems of plants, whose primary function is photosynthesis. In response to environmental changes, chloroplasts use several mechanisms to coordinate their photosynthetic activities with nuclear gene expression and other metabolic pathways. Here, we focus on a redox-based regulatory network composed of thioredoxins (TRX) and TRX-like proteins. Among multiple redox-controlled metabolic activities in chloroplasts, tetrapyrrole biosynthesis is particularly rich in TRX-dependent enzymes. This review summarizes the effects of plastid-localized reductants on several enzymes of this pathway, which have been shown to undergo dithiol-disulfide transitions. We describe the impact of TRX-dependent control on the activity, stability and interactions of these enzymes, and assess its contribution to the provision of adequate supplies of metabolic intermediates in the face of diurnal and more rapid and transient changes in light levels and other environmental factors.

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g., cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded β-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the β7-β8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

AhpC is a bacterial representative of 2-Cys peroxiredoxins (Prxs) with broad substrate specificity and functional plasticity. However, details underpinning these two important attributes of AhpC remain unclear. Here, we studied the functions and mechanisms of regulation of AhpC in the facultative Gram-negative anaerobic bacterium Shewanella oneidensis, in which AhpC's physiological roles can be conveniently assessed through its suppression of a plating defect due to the genetic loss of a major catalase. We show that successful suppression can be achieved only when AhpC is produced in a dose- and time-dependent manner through a complex mechanism involving activation of the transcriptional regulator OxyR, transcription attenuation, and translation reduction. By analyzing AhpC truncation variants, we demonstrate that reactivity with organic peroxides (OPs) rather than H2O2 is resilient to mutagenesis, implying that OP reduction is the core catalytic function of AhpC. Intact AhpC could be recycled only by its cognate reductase AhpF, and AhpC variants lacking the Prx domain or the extreme C-terminal five residues became promiscuous electron acceptors from the thioredoxin reductase TrxR and the GSH reductase Gor in addition to AhpF, implicating an additional dimension to functional plasticity of AhpC. Finally, we show that the activity of S. oneidensis AhpC is less affected by mutations than that of its Escherichia coli counterpart. These findings suggest that the physiological roles of bacterial AhpCs are adapted to different oxidative challenges, depending on the organism, and that its functional plasticity is even more extensive than previously reported.

The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.

Nitro-oxidative stress and lowered antioxidant defences play a key role in neuropsychiatric disorders such as major depression, bipolar disorder and schizophrenia. The first part of this paper details mitochondrial antioxidant mechanisms and their importance in reactive oxygen species (ROS) detoxification, including details of NO networks, the roles of H2O2 and the thioredoxin/peroxiredoxin system, and the relationship between mitochondrial respiration and NADPH production. The second part highlights and identifies the causes of the multiple pathological sequelae arising from self-amplifying increases in mitochondrial ROS production and bioenergetic failure. Particular attention is paid to NAD+ depletion as a core cause of pathology; detrimental effects of raised ROS and reactive nitrogen species on ATP and NADPH generation; detrimental effects of oxidative and nitrosative stress on the glutathione and thioredoxin systems; and the NAD+-induced signalling cascade, including the roles of SIRT1, SIRT3, PGC-1α, the FOXO family of transcription factors, Nrf1 and Nrf2. The third part discusses proposed therapeutic interventions aimed at mitigating such pathology, including the use of the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside, both of which rapidly elevate levels of NAD+ in the brain and periphery following oral administration; coenzyme Q10 which, when given with the aim of improving mitochondrial function and reducing nitro-oxidative stress in the brain, may be administered via the use of mitoquinone, which is in essence ubiquinone with an attached triphenylphosphonium cation; and N-acetylcysteine, which is associated with improved mitochondrial function in the brain and produces significant decreases in oxidative and nitrosative stress in a dose-dependent manner.

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H(2)S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser–His–Asp triad, proposed to serve a general acid–base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 μmol·kg(−1) in brain to 1.4 μmol·kg(−1) in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the K(M) for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 μm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.

Significance: The pathogenesis and progression of allergic inflammation in the respiratory system are closely linked to oxidative stress. Thioredoxin (TRX) is an essential redox balance regulator in organisms and is induced by various oxidative stress factors, including ultraviolet rays, radiation, oxidation, viral infections, ischemia reperfusion, and anticancer agents. Recent Advances: We demonstrated that systemic administration and transgenic overexpression of TRX is useful in a wide variety of in vivo inflammatory respiratory diseases models, such as viral pneumonia, interstitial lung disease, chronic obstructive pulmonary disease, asthma, acute respiratory distress syndrome, and obstructive sleep apnea syndrome, by removing reactive oxygen species, blocking production of inflammatory cytokines, inhibiting migration and activation of neutrophils and eosinophils, and regulating the cellular redox status. In addition, TRX's anti-inflammatory mechanism is different from the mechanisms associated with anti-inflammatory agents, such as glucocorticoids, which regulate the inflammatory reaction in association with suppressing immune responses. Critical Issues: Understanding the molecular mechanism of TRX is very helpful for understanding the role of TRX in respiratory diseases. In this review, we show the protective effect of TRX in various respiratory diseases. In addition, we discuss its anti-allergic and anti-inflammatory molecular mechanism in detail. Future Directions: The application of TRX may be useful for treating respiratory allergic inflammatory disorders. Antioxid. Redox Signal. 32, 785-801.