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Cells constantly face the challenge of managing oxidants. In aerobic organisms, oxygen (O2) is used for energy production, generating reactive oxygen species (ROS) as byproducts of enzymatic reactions. To protect against oxidative damage, cells possess an intricate system of redox scavengers and antioxidant enzymes, collectively forming the antioxidant defense system. This system maintains the redox equilibrium and enables the generation of localized oxidative signals that regulate essential cellular functions. One key component of this defense is the thioredoxin (Trx) system, which includes Trx, thioredoxin reductase (TrxR), and NADPH. The Trx system reverses oxidation of macromolecules and indirectly neutralizes ROS via peroxiredoxin (Prx). This dual function protects cells from damage accumulation and supports physiological cell signaling. However, the Trx system also shields tumors from oxidative damage, aiding their survival. Due to elevated ROS levels from their metabolism, tumors often rely on the Trx system. In addition, the Trx system regulates critical pathways such as proliferation and neoangiogenesis, which tumors exploit to enhance growth and optimize nutrient and oxygen supply. Consequently, the Trx system is a potential target for cancer therapy. The challenge lies in selectively targeting malignant cells without disrupting the redox equilibrium in healthy cells. The aim of this review article is threefold: first, to elucidate the function of the Trx system; second, to discuss the Trx system as a potential target for cancer therapies; and third, to present the possibilities for inhibiting key components of the Trx system, along with an overview of the latest clinical studies on these inhibitors. [ABSTRACT FROM AUTHOR]

The phytotoxic mechanisms of patulin (PAT) and penicillic acid (PA) have not been identified unambiguously. This study aims to clarify their effects on thioredoxins (Trxs). Aflatoxin B1 (AFB1), PAT and PA were isolated by solvent extraction and chromatographic techniques from the cultures of Aspergillus flavus Z2 (LC171449), Penicillium vulpinum CM1 and Aspergillus ochraceus EMCC516, respectively. The three mycotoxins showed phytotoxicity to the germination of maize seeds, which was manifested by inhibiting radicle and coleoptile emergence, in addition to their toxic effects on fresh weights and root and shoot lengths. The phytotoxicity of AFB1 and PAT was greater than that of PA. Due to the central roles exhibited by plant Trxs in cellular metabolic activities, they were tested as target proteins for PAT and PA using AFB1 as positive control. In vivo studies showed that the mycotoxins significantly reduced Trx activity measured in the roots and shoots of maize seedlings. PAT showed greater Trx-inhibiting activity than PA and AFB1. In vitro studies of the mycotoxins on Trx y1 (from Arabidopsis thaliana) and thioredoxin reductase (Tr, from rat liver) activities confirm the results of in vivo studies. The inactivation of Trx with PAT and PA was reduced in the presence of glutathione (GSH). Data obtained suggest that lactone mycotoxins are more highly reactive with simple low-molecular-weight thiols (like GSH) than with complex ones (like Trx). [ABSTRACT FROM AUTHOR]

AIM: The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides in Escherichia coli. Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP). METHODS: The vectors were transformed into E. coli TOP10F' for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl ß-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays. RESULTS: The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct. CONCLUSION: The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELPintein construct. [ABSTRACT FROM AUTHOR]

Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana . Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o -type Trx (Trx- o ) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx- o -deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K i . Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. [ABSTRACT FROM AUTHOR]

Oxidant stimulation has been suggested to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our study aimed to investigate the role and mechanisms of thioredoxin (Trx) nitration during the development of IPF. A rat model of IPF was established by intratracheal instillation of bleomycin (BLM). Male Wistar rats were randomly distributed among the control group and BLM-treated group, in which rats were intratracheally instilled with a single dose of BLM (5.0 mg/kg body mass in 1.0 mL phosphate-buffered saline). At 7 or 28 days after instillation the rats were euthanized. Histopathological and biochemical examinations were performed. The activity and protein level of thioredoxin were assessed. The thioredoxin nitration level was determined using immunoprecipitation and immunoblotting techniques. Our results demonstrated that protein tyrosine nitration increased in the BLM-treated group compared with the control group. Trx activity decreased in the BLM group compared with control group, whereas Trx expression and nitration level increased dramatically in the BLM group compared with the control group. Our results indicated that Trx nitration might be involved in the pathogenesis of IPF. [ABSTRACT FROM AUTHOR]

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF1-γ subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF1-γ and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts. [ABSTRACT FROM PUBLISHER]

Abstract: Thioredoxins are small (12–13kDa) ubiquitous proteins containing a redox active disulfide bridge. The primary structure of one of the rice Trx isoforms, OsTrx20, in which Thr is substituted for the largely conserved Gly in position 55 in the active site and Lys is substituted for the conserved Asp/Asn in position 48 is considerably different with other h-type Trx isoforms. In order to probe the functional roles of Thr-55 and Lys-48 in OsTrx20, Thr was replaced with Gly and Lys with Asp using site-directed mutagenesis. The wild type OsTrx20 as well as single mutants T55GOsTrx20, K48DOsTrx20 and the double mutant T55G-K48DOstrx20 were heterologously expressed in Escherichia coli and purified. The changes in the ability to reduce insulin for OsTrx20 and mutants as well as OsTrx23 which has a Trx typical active site were monitored in the pH range 6.5-8. The results showed that whereas the activity of wild type OsTrx20 is dependent on pH and decreases remarkably at high pH values, the activities of mutants T55GOsTrx20, K48DOsTrx20, T55G-K48DOsTrx20 and wild type OsTrx23 slightly change under different pH conditions. These results support the significant involvement of residues Thr-55 and Lys-48 in instability of OsTrx20 activity under pH variations. [Copyright &y& Elsevier]

Abstract: T cells are required for an effective adaptive immune response. The principal function of T cells is to promote efficient removal of foreign material by identifying and mounting a specific response to nonself. A decline in T cell function in aging is thought to contribute to reduced response to infection and vaccination and an increase in autoimmunity. This may in part be due to the age-related decrease in naïve CD4+ T cells and increase in antigen-experienced CD4+ T cells, loss of redox homeostasis, and impaired metabolic switching. Switching between subsets is triggered by the integration of extracellular signals sensed through surface receptors and the activation of discrete intracellular metabolic pathways. This article explores how metabolic programming and loss of redox homeostasis during aging may contribute to age-associated changes in T cell phenotype and function. [Copyright &y& Elsevier]

Abstract: Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II through its functional α,β-unsaturated ketone group serving as a Michael acceptor. A similar group is found in SK053, a compound recently developed by our group to target the thioredoxin–thioredoxin reductase (Trx–TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets additional proteins including thioredoxin–thioredoxin reductase system as well as protein disulfide isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold. Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes containing readily accessible, nucleophilic cysteines. This observation might be of importance in considering potential therapeutic applications of adenanthin to include a range of diseases, where aberrant activity of Prdx, Trx–TrxR and PDI is involved in their pathogenesis. [Copyright &y& Elsevier]

Abstract: Glutaredoxins (Grxs) and thioredoxins (Trxs) play important roles in maintaining intracellular thiol-redox homeostasis by scavenging reactive oxygen species. However, few Grxs and Trxs have been functionally characterized in Apis cerana cerana. In this study, we identified three genes, AccGrx1, AccGrx2, and AccTrx1, and investigated their connection to antioxidant defense. AccGrx1 and AccGrx2 were mainly detected in dark-eyed pupae, whereas AccTrx1 was highly concentrated in 15-day postemergence adults. The expression levels of AccGrx1 and AccTrx1 were the highest in fat body and epidermis, respectively. However, the expression level of AccGrx2 was the highest in muscle, followed by the epidermis. AccGrx1, AccGrx2, and AccTrx1 were induced by 4, 16, and 42°C; H2O2; and pesticide (acaricide, paraquat, cyhalothrin, and phoxime) treatments and repressed by UV light. AccGrx1 and AccGrx2 were upregulated by HgCl2 treatment, whereas AccTrx1 was downregulated. We investigated the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 in A. cerana cerana and surprisingly found that knockdown of the these four genes enhanced the enzymatic activities of CAT and POD; the metabolite contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP+/NADPH. In addition, we also analyzed the transcripts of other antioxidant genes and found that some were upregulated and others were downregulated, revealing that the upregulated genes may be involved in compensating for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1. Taken together, these results suggest that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 may play critical roles in antioxidant defense. [Copyright &y& Elsevier]

Abstract: Long-term treatment with ambroxol (ABX), a bronchial expectorant, was found to prevent acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The underlying mechanism remains unclear. To address this, we have investigated the effect of ABX on critical antioxidant proteins thioredoxin (Trx) and thioredoxin reductase (TrxR) that are decreased in patients with AECOPD. Trx, TrxR and NADP(H) form Trx system, which is involved in regulating numerous oxidative stress-related events. In human bronchial epithelial cells, treatment with ABX from 0 to 200 μM gradually increased mRNA and protein levels of TrxR/Trx. At these ABX concentrations, TrxR activity was elevated progressively, whereas Trx activity exhibited a dose-dependent biphasic response, increasing at 50 and 75 μM, but decreasing at ABX over 150 μM. Pre-treatment with 75 μM ABX enhanced the capacity of the cells to eliminate reactive oxygen species, which was largely prevented by knockdown of cytosolic Trx (hTrx1). In a purified system, ABX shortened the initial lag phase during the reduction of insulin disulfide by Trx system. Pre-treatment of NADPH-reduced TrxR with ABX caused a dose- and time-dependent increase in thiolate/selenolate species, i.e. the catalytically active form of TrxR. Kinetic analysis demonstrated that the reduction of H2O2 by TrxR or Trx system were enhanced by 100 or 200 μM ABX. When hTrx1 was mixed with ABX in a molar ratio of 1:1 to 1:100 (which could occur in human plasma), changes in intrinsic Trp fluorescence occurred, and the response of reduced hTrx1 was especially remarkable. These data reveal an ABX-sensing mechanism of TrxR/Trx. We therefore conclude that the antioxidant actions of ABX at physiological concentrations are, at least partially, mediated by TrxR and/or Trx system. [Copyright &y& Elsevier]

Abstract: Recent studies have revealed that reactive oxygen species (ROS) are actively generated in cells and function as second messengers to mediate physiological intracellular signaling. ROS exert their effects on intracellular signaling via ROS effector proteins, which are sensitively and reversibly oxidized by ROS. Among various ROS effector proteins, the protein tyrosine phosphatase (PTP) family is of special interest. In the catalytic pocket, PTP proteins commonly possess a highly reactive cysteine (Cys) residue, which is susceptible to oxidation by ROS. Phosphatase of regenerating liver (PRL) belongs to the PTP family and is oxidized by ROS to form an intramolecular disulfide bond. In general, disulfide bonds in proteins can be reduced in cells with the help of various reducing enzymes, which enables the reversible redox regulation of PRL proteins. In the case of PRL proteins, thioredoxin-related protein 32 specifically catalyzes the reducing reaction, indicating the importance of redox regulation for ROS effector proteins. [Copyright &y& Elsevier]

Abstract: The thioredoxin (Trx) system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin, is a key antioxidant system in defense against oxidative stress through its disulfide reductase activity regulating protein dithiol/disulfide balance. The Trx system provides the electrons to thiol-dependent peroxidases (peroxiredoxins) to remove reactive oxygen and nitrogen species with a fast reaction rate. Trx antioxidant functions are also shown by involvement in DNA and protein repair by reducing ribonucleotide reductase, methionine sulfoxide reductases, and regulating the activity of many redox-sensitive transcription factors. Moreover, Trx systems play critical roles in the immune response, virus infection, and cell death via interaction with thioredoxin-interacting protein. In mammalian cells, the cytosolic and mitochondrial Trx systems, in which TrxRs are high molecular weight selenoenzymes, together with the glutathione-glutaredoxin (Grx) system (NADPH, glutathione reductase, GSH, and Grx) control the cellular redox environment. Recently mammalian thioredoxin and glutathione systems have been found to be able to provide the electrons crossly and to serve as a backup system for each other. In contrast, bacteria TrxRs are low molecular weight enzymes with a structure and reaction mechanism distinct from mammalian TrxR. Many bacterial species possess specific thiol-dependent antioxidant systems, and the significance of the Trx system in the defense against oxidative stress is different. Particularly, the absence of a GSH-Grx system in some pathogenic bacteria such as Helicobacter pylori, Mycobacterium tuberculosis, and Staphylococcus aureus makes the bacterial Trx system essential for survival under oxidative stress. This provides an opportunity to kill these bacteria by targeting the TrxR-Trx system. [Copyright &y& Elsevier]

Highlights: [•] Gatekeeper site is in a hydrophobic groove of catalytic cleft of protein kinases. [•] Gatekeeper site is therefore a least probable target for phosphorylation. [•] AhSymRK has tyrosine (Y670) as gatekeeper. [•] Y670 in AhSymRK is accessible for autophosphorylation and dephosphorylation. [•] Gatekeeper tyrosine phosphorylation was not a prerequisite for AhSymRK activity. [ABSTRACT FROM AUTHOR]

Abstract: Protein S-nitrosylation is a dynamic post-translational modification (PTM) of specific cysteines within a target protein. Both proteins and small molecules are known to regulate the attachment and removal of this PTM, and proteins exhibiting such a function are transnitrosylase or denitrosylase candidates. With the advent of the biotin switch technique coupled to high-throughput proteomics workflows, the identification and quantification of large numbers of S-nitrosylated proteins and peptides is now possible. Proper analysis and interpretation of high throughout and quantitative proteomics data will help identify specific transnitrosylase and denitrosylase target peptide sequences and contribute to an understanding of the function and regulation of specific S-nitrosylation events. Here we describe the application of a quantitative proteomics approach using isotope-coded affinity tags (ICAT) in the biotin switch approach for the identification of transnitrosylation and denitrosylation targets of thioredoxin 1, an enigmatic protein with both reported transnitrosylase and denitrosylase activities. [Copyright &y& Elsevier]

Abstract: Cystathionine β-synthase (CBS) domains are small intracellular modules that can act as binding domains for adenosine derivatives, and they may regulate the activity of associated enzymes or other functional domains. Among these, the single CBS domain-containing proteins, CBSXs, from Arabidopsis thaliana, have recently been identified as redox regulators of the thioredoxin system. Here, the crystal structure of CBSX2 in complex with adenosine monophosphate (AMP) is reported at 2.2Å resolution. The structure of dimeric CBSX2 with bound-AMP is shown to be approximately flat, which is in stark contrast to the bent form of apo-CBSXs. This conformational change in quaternary structure is triggered by a local structural change of the unique α5 helix, and by moving each loop P into an open conformation to accommodate incoming ligands. Furthermore, subtle rearrangement of the dimer interface triggers movement of all subunits, and consequently, the bent structure of the CBSX2 dimer becomes a flat structure. This reshaping of the structure upon complex formation with adenosine-containing ligand provides evidence that ligand-induced conformational reorganization of antiparallel CBS domains is an important regulatory mechanism. [Copyright &y& Elsevier]

The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system. [ABSTRACT FROM AUTHOR]

Numerous studies indicate that reactive oxygen species (ROS) are not merely cellular by-products of respiration, but are able to modulate various signalling pathways and play certain physiological roles. Recent studies have revealed the importance of translating ROS-generation to activation/suppression of specific signalling pathways. The Wnt signalling pathway, which is essential for early development and stem cell maintenance, is also regulated by ROS. A thioredoxin-related protein, nucleoredoxin (NRX), governs ROS-stimulated Wnt signalling in a temporal manner. NRX usually interacts with Dishevelled (Dvl), an essential adaptor protein for Wnt signalling, and blocks the activation of the Wnt pathway. Oxidative stress causes dissociation of NRX from Dvl, which enables Dvl to activate the downstream Wnt signalling pathway. This study also presents the latest research findings on NRX and its related molecules [ABSTRACT FROM AUTHOR]

Abstract: A thioredoxin (Trx) gene was cloned from the silkworm, Bombyx mori. The B. mori Trx (BmTrx) cDNA contains an open reading frame of 318 bp encoding 106 amino acid residues with a conserved active site (CGPC). Northern blot analysis revealed the presence of BmTrx transcripts in all tissues examined. The cDNA encoding BmTrx was expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells. The recombinant BmTrx proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from B. mori. When H2O2 or paraquat was injected into the body cavity of B. mori larvae, BmTrx mRNA expression was upregulated in the fat body tissue. In addition, the expression levels of BmTrx mRNA in the fat body were greatly increased when B. mori larvae were exposed to low or high temperatures, or injected with microorganisms. These results suggest that BmTrx possibly protects against oxidative stress caused by extreme temperatures and microbial infection as well as by intracellularly generated reactive oxygen species during metabolism. [Copyright &y& Elsevier]

Neuroprotective effects of dehydroepiandrosterone (DHEA) have been shown to be associated with its antioxidant properties, but the mechanisms remain unknown. Considering that the thioredoxin (Trx) system, an important cellular redox modulation system, changes under oxidative stress and could exert protective effects, the relationship between the antioxidant effects of DHEA and the Trx system regulation was explored. Using MTT assay and morphological observation, the effects of DHEA in the model of H2O2-induced oxidative stress in SH-SY5Y cells were analyzed, then RT-PCR and Western blot assay were used to detect the alteration in mRNA and protein level of Trx. The results showed that a pre-treatment of DHEA (10–100 nM) protected cells against the toxicity induced by H2O2 in a dose-dependent manner, which could be confirmed in morphological observation by phase-contract microscope. In addition, Trx mRNA transcription was inhibited by H2O2 (300μM), which could be reversed by the pre-administration of DHEA in various concentrations (0.1–100 nM). Western blot assay confirmed that protein level of Trx could be elevated by the pre-treatment of DHEA (10–100 nM) with the exposure of H2O2. Taken together, these data suggest that DHEA may be useful in treating age-related neurodegenerative diseases based on its up-regulating effects on an antioxidant and neuroprotective protein thioredoxin, a substrate in the Trx redox system. [ABSTRACT FROM AUTHOR]

Abstract: NADPH-dependent thioredoxin reductases (NTR) are homodimeric enzymes that reduce thioredoxins. Two genes encoding NADPH-dependent thioredoxin reductases (AtNTRA and AtNTRB) were found in the genome of Arabidopsis thaliana. These originated from a recent duplication event and the encoded proteins are highly homologous. Previously, AtNTRA was shown to encode a dual targeted cytosol and mitochondrial protein. Here, we show that the AtNTRB gene encodes two mRNAs, presumably by initiating transcription at two different sites. The longer mRNA encodes a precursor polypeptide that is actively imported into mitochondria by a cleavage-associated mechanism, while the shorter mRNA encodes a cytosolic isoform. Isolation of Arabidopsis mutants with knocked-out AtNTRA or AtNTRB genes allowed us to prove that both genes encode cytosolic and mitochondrial isoforms. Interestingly, AtNTRB appeared to express the major mitochondrial NTR, while AtNTRA expresses as the major cytosolic isoform, suggesting that these two recently duplicated genes are evolving towards a specific function. [Copyright &y& Elsevier]

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS–PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell. [Copyright &y& Elsevier]

Changes in the components of the Jun N-terminal kinase (JNK) signalling pathway were investigated in human A549 lung carcinoma cells treated with sodium dichromate. Sodium dichromate (100 μM, 0–6 h) failed to activate nuclear factor kappa B (NF-κB) as determined by a lack of nuclear translocation of p65 but resulted in Jun N-terminal kinase activation as assessed by phospho-Jun N-terminal kinase Western blotting in a dose-dependent (>25 μM) and time-dependent (>1 h) manner. In addition, c-Jun, a downstream target of Jun N-terminal kinase signalling was also activated with a similar dose- and time-dependency at the level of both protein expression and degree of phosphorylation. In contrast, sodium dichromate treatment had no effect on levels of phospho-p38. Immunoprecipitation demonstrated that apoptosis signal regulating kinase-1 (ASK-1), an upstream activator of Jun N-terminal kinase was dissociated from its inhibitory partner thioredoxin (Trx) in response to sodium dichromate (100 μM, 4 h) treatment. This treatment was also associated with a transient (2 h) increase in cytosolic levels of thioredoxin but no nuclear translocation of thioredoxin was observed. In conclusion, sodium dichromate had a stimulatory effect on the Jun N-terminal kinase signalling pathway in A549 cells, resulting in activation of downstream effector molecules. We hypothesise that dissociation of apoptosis signal regulating kinase-1 from thioredoxin may be at least partially responsible for Jun N-terminal kinase activation. [Copyright &y& Elsevier]

In plants, thioredoxins h are encoded by a multigenic family of genes (eight in Arabidopsis thaliana, at least five in Populus sp.). The multiplicity of these isoforms raises the question of their specificity. This review focuses on thioredoxins h in two plant models: Arabidopsis and poplar. Thioredoxins h can be divided into three different subgroups according to the analysis of their primary structure. This paper describes the biochemical properties of each subgroup. Recent data in the field indicate that subgroup members differ by their subcellular localization as well as their reduction pathways suggesting specific functions for each subgroup. The development of proteomic tools has also increased considerably the number of potential thioredoxin targets, showing the importance of thioredoxins h in plants. [Copyright &y& Elsevier]

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4+-T cells derived from MIF −/− mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression. [Copyright &y& Elsevier]

Three full-length cDNA clones, designated TRX1, TRX2, and TRX3 encoding different but similar thioredoxin h polypeptides, were isolated from sweet potato (Ipomoea batatas [L.] Lam ‘Tainong 57’) storage roots. These three thioredoxin h clones were similar to each other and contained the canonical WCGPC active site and the important structural and functional amino acids that were conserved in thioredoxin sequences.Recombinant thioredoxin h (TRX2) overproduced in Escherichia coli (M15) was purified by Ni2+-chelated affinity chromatography. The molecular mass of TRX2 is ca. 1.4 kDa determined by SDS–PAGE. TRX2 carries an N-terminal 17-amino acid extension enriched in hydrophobic residues. In Northern blot analysis, mRNAs corresponding to all three thioredoxin genes were found to have the highest level in the storage roots; those corresponding to TRX2 and TRX3 were detected at the next higher level in flowers. All three transcripts were detected at very low levels in sprouts of storage roots, roots, veins, and leaves. In Western blot analysis, the thioredoxins were found to have the highest level in the storage roots and veins; higher level in leaves; and very low levels in sprouts of storage root and roots. Active recombinant TRX2 protein is able to reduce the TI proteins in NTR system. Thioredoxin TRX2 was not reduced efficiently when NTR was removed from the NTR system. Three thioredoxin h genes of sweet potato storage roots display differential gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues. [Copyright &y& Elsevier]

Selenium treatment of the doxorubicin-resistant cell line, U-1285dox, derived from human small cell carcinoma of the lung, resulted in massive apoptosis. This effect appeared maximal at 2 days after addition of selenite. The apoptosis was caspase-3 independent as revealed by Western blot analysis, activity measurement and by using caspase inhibitors. Induction of apoptosis was significantly more pronounced and occurred after addition of lower concentrations of selenite in the doxorubicin-resistant cells compared to the parental doxorubicin-sensitive cells. High levels of selenite caused necrosis in the doxorubicin-sensitive cells. Analysis of enzymatic activity (insulin reduction) of thioredoxin reductase (TrxR) and TrxR protein concentration, measured by ELISA, revealed increasing activity and protein levels after treatment with increasing concentrations of selenium. Maximum relative increase was induced up to 1 μM in both sublines and at this selenium level the concentrations of TrxR measured as insulin reducing activity or ELISA immunoreactivity were nearly identical. Increasing concentrations of selenite up to 10 μM resulted in increased activity and concentration of TrxR in the sensitive subline but decreasing levels in the resistant subline. The level of truncated Trx (tTrx) was higher in the resistant U-1285dox cells but the level did not change with increasing selenite concentrations. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy-resistant cells and suggest that redox regulation through the thioredoxin system is an important target for cancer therapy. [Copyright &y& Elsevier]

The multifunctional enzyme thioredoxin-glutathione reductase (TGR) was purified to homogeneity from the soluble fraction of Taenia crassiceps metacestode (cysticerci). Specific activities of 17.5 and 4.7 U mg−1 were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3 μM were respectively calculated for thioredoxin, GSSG and NADPH. The kcat/Km ratio of T. crassiceps TGR for both thioredoxin and GSSG falls in the range observed for typical thioredoxin reductases and glutathione reductases. Purified enzyme also showed glutaredoxin activity, with a specific activity of 19.2 U mg−1 with hydroxyethyl disulfide as substrate. Both thioredoxin and GSSG disulfide reductase activities were fully inhibited by nanomolar concentrations of the gold compound auranofin, supporting the existence of an essential selenocysteine residue. Relative molecular mass of native enzyme was 136,000±3000, while the corresponding value per subunit, obtained under denaturing conditions, was 66,000±1000. These results suggest TGR exists as a dimeric protein. Isoelectric point of the enzyme was at pH 5.2. Moderate or high concentrations of GSSG, but neither thioredoxin nor NADPH, resulted in a markedly hysteretic kinetic, characterized by a lag time before the steady state velocity was reached. The magnitude of the lag time was dependent on GSSG and enzyme concentration. Preincubation of the enzyme with micromolar concentrations of GSH or DTT abolished the hysteresis, suggesting that a thiol–disulfide exchange mechanism is involved. [Copyright &y& Elsevier]

In recent years, redox control has emerged as a fundamental mechanism of gene regulation through transcriptional control. Thioredoxin (Trx) is a dithiol-reducing enzyme known to be involved in the redox regulation of a number of transcription factors, and in this study, we have investigated the redox-dependent regulation of the DNA binding activity of Sp1 by thioredoxin. Electrophoretic mobility shift assays were used to show that both recombinant Sp1 produced in Escherichia coli and endogenous Sp1 expressed by MDA-MB-231 breast cancer cells is subject to redox regulation. We found that thioredoxin alone or in conjunction with the full thioredoxin system (comprising thioredoxin, thioredoxin reductase [TR], and α-nicotinamide adenine dinucleotide phosphate [NADPH]) can increase Sp1 DNA binding activity in vitro to an oligonucleotide containing the Sp1 consensus sequence. Furthermore, we have provided evidence that recombinant Sp1 can bind to Sp1 consensus sequences within a 330-base pair (bp) thioredoxin promoter fragment and that this interaction can also be enhanced by the presence of thioredoxin. Luciferase reporter assays using this same minimal thioredoxin promoter region demonstrated that both Sp1 and Sp3 can bind to the promoter and act to enhance transcription. When the three identified Sp1 consensus sequences within the reporter construct were deleted, there was a loss of basal promoter activity, showing that these closely positioned sites are important for regulation of thioredoxin gene expression. [Copyright &y& Elsevier]

A novel human gene, named as hTLP19, was isolated and characterized as secretory protein by combining bioinformatics tools with experiments. The hTLP19 encodes 172 amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laevis, etc. Moreover, the result from insulin reduction assay indicated that the hTLP19 protein has Trx enzymatic activity. By comparing full-length cDNA with human genomic DNA, the hTLP19 gene might have seven coding exons spanning 35 kb of genomic DNA on the human chromosome 1p32.3. Northern blot analysis showed that human hTLP19 is expressed widely in many tissues with 1.6 kb band and extra 1.2 kb band in placenta. Subcellular localization and immunoblotting assays indicated that hTLP19 might be secreted out of cell through trans-Golgi network (TGN). [Copyright &y& Elsevier]

Peroxiredoxins (Trx-Px) are ubiquitous antioxidant enzymes that catalyse the thioredoxin-dependent reduction of hydroperoxides. The number of characteristic active site (VCP/T) motifs defines these proteins as 1-Cys and 2-Cys Trx-Px. Steady-state kinetic parameters of Plasmodium falciparum 2-Cys Trx-Px (PfTrx-Px1) were determined using stopped flow rapid kinetics. The bi-substrate reaction displays ping-pong kinetics and the Km values for H2O2 and thioredoxin were determined to be 0.78±0.14 μM and 18.94±3.01 μM, respectively. The Vmaxapp and kcatapp for H2O2 were found to be 4±0.6 U mg−1 and 1.67±0.25 s−1, respectively and those for thioredoxin are 23.0±0.2 U mg−1 and 9.65±0.1 s−1, emphasising the specificity of the enzyme for the substrate H2O2. After subjection to exogenous and endogenous oxidative stress, P. falciparum blood stage forms showed a marked elevation of PfTrx-Px1 mRNA and protein levels consistent with the hypothesis that it is an important component of the parasite’s antioxidant machinery. Gel filtration, cross-linking and electron microscopy (EM) revealed that the protein forms decamers consisting of pentamers of homodimers that have a doughnut-like shape consistent with the structures of related proteins. No dimeric forms of the protein were detectable after gel filtration suggesting that PfTrx-Px1 predominantly exists as an oligomer. [Copyright &y& Elsevier]

The completion of the sequencing of the Arabidopsis genome revealed the multiplicity of plant thioredoxins, dithiol proteins involved in redox regulation, due to their ability to undergo thiol-disulfide interchanges with numerous proteins. An examination of the expressed sequence tag (EST) databanks of the unicellular green alga Chlamydomonas reinhardtii reveals also multiple thioredoxin types, but with fewer isoforms in each type than for higher plants. The pioneering work on the Chlamydomonas thioredoxin systems, mainly based on biochemical approaches, is summarised and the EST databanks analysed from a qualitative (different ESTs) and quantitative (EST abundance) viewpoints. It is proposed that Chlamydomonas can be a suitable organism to uncover basic functions of each thioredoxin type. [Copyright &y& Elsevier]

The chloroplastic drought-induced stress protein of 32 kDa (CDSP32) is composed of two thioredoxin modules and participates in plastid defence against oxidative damage. Previous data indicated that the CDSP32 gene is induced at the transcript level by environmental and oxidative stress conditions. We further investigated CDSP32 expression in relation to leaf age and to stress conditions in Solanum tuberosum wild-type (WT) and CDSP32-overexpressing plants. Under control conditions, no noticeable variation in CDSP32 mRNA level was observed depending on leaf age in both plant types. A higher protein amount was noticed in developing leaves of WT and the protein abundance decreased in a leaf age-dependent manner. Similar data with a higher magnitude in the protein level were observed in CDSP32-overexpressing plants. Immunoprecipitation experiments revealed no difference between the CDSP32 synthesis rates in young and in mature leaves in WT as well as in transgenic plants. Under water deficit conditions, a strong increase in CDSP32 mRNA level was observed in leaves from WT whereas no change in transcript abundance was noticed in transgenic plants. Similar CDSP32 levels were observed whatever the leaf developmental stage in water-stressed WT and transgenic plants. These data show that, under normal growth conditions, post-transcriptional events likely related to protein turnover play a fundamental role in regulating the expression of CDSP32 during leaf development. Further, they also indicate that the CDSP32 thioredoxin likely participates in the protection of the photosynthetic structures in developing leaves. [Copyright &y& Elsevier]

Thioredoxin (TRX) is an ubiquitous protein disulfide reductase, which is known to be involved in the implantation development of mouse embryos. In the present study, recombinant human TRX was used to evaluate its effect on the promotion of preimplantation development of bovine embryos derived from in vitro maturation and fertilization. Supplementation of the medium 24 h post insemination with TRX significantly (P<0.05) enhanced the frequency of development to the blastocyst stage in 5% O2 concentration. The optimal concentration was 0.5 μg/ml (P<0.05, compared with 0, 0.1 and 1.0 μg/ml). This effect of TRX was evident only when added around the time of the first cleavage stage (24 h post insemination); no promotion was found with treatment at 6 h (one-cell) or 44 h (six- to eight-cell) after insemination. Moreover, it is of interest that even with the best combination of the dose and timing of TRX treatment (0.5 μg/ml, at 24 h post insemination), no promotion of development was observed when embryos were cultured under 20% O2. However, a preincubation of TRX in the culture medium under 20% oxygen for 24 h did not diminish the promoting effect in the subsequent TRX treatment under optimal conditions, thus suggesting that the possible oxidation of TRX alone may not be the reason for the disappearance of the effect under a high oxygen concentration. These results indicate that TRX does improve the development of bovine embryos in vitro, though unlike the general reducing reagents such as β-mercaptoethanol or cysteamine, TRX may have to exert its effect at specific times and in more physiologic oxygen environments. [Copyright &y& Elsevier]

There is no doubt that oxidative stress occurs in patients with rheumatoid arthritis (RA) and play an important role in both inflammation and destruction of RA joints. Thioredoxin (TRX) is a ubiquitous redox-active protein and is known to be induced in several cells against oxidative stress and to be secreted extracellularly. To clarify whether plasma thioredoxin levels could be a marker for oxidative stress in patients with RA, we measured plasma TRX levels in patients with RA using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and investigated its relationship to TRX concentrations in the inflammatory joints.We have found that the plasma TRX levels of RA patients were significantly higher than those of normal subjects (86.8±54.1 ng/ml versus 38.6±18.5 ng/ml, P<0.0001). The plasma levels were correlated with the disease activity of RA and also with serum C-reactive protein (CRP) values (P<0.01). The concentration of TRX in synovial fluid (SF) from RA was 353.3±220.1 ng/ml (mean±S.D.) which was significantly higher than that in SF from osteoarthritis patients (70.6±31.0 ng/ml, P<0.0001). The SF TRX concentration was significantly correlated with the number of leukocytes infiltrating in SF and with the serum CRP levels. The serum TRX levels were significantly positively correlated with the SF TRX concentrations in RA patients (P<0.05). By the histological examination for synovial tissue of RA patients, TRX was shown to be present on the surface of synovial lining layer as well as in the leukocytes.Moreover, urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage by endogenously generated oxygen radicals, was significantly higher in RA patients than in healthy subjects (11.55±4.71 versus 7.76±2.26 ng/mg creatinine, P<0.0001). Plasma TRX levels were significantly correlated with urinary excretion of 8-OHdG (P<0.005). We concluded that plasma TRX level is a new biomarker for the disease activity of RA and may reflect higher levels of oxidative stress in RA patients. [Copyright &y& Elsevier]

We examined the effects of continuous overexpression of thioredoxin (Trx) 1 on aging in Trx1 transgenic mice [Tg(TXN)+/0]. This study was conducted to test whether increased thioredoxin expression over the lifespan in mice would alter aging and age-related pathology because our previous study demonstrated that Tg(act-TXN)+/0 mice had no significant maximum life extension, possibly due to the use of actin as a promoter, which may have resulted in loss of Trx1 overexpression during aging. To test this hypothesis, we generated new Trx1 transgenic mice using a fragment of the human genome containing the TXN gene with an endogenous promoter to ensure continuous overexpression of Trx1 throughout the lifespan. Universal overexpression of Trx1 was observed, and Trx1 overexpression was maintained during aging (up to 22-24 months old) in the Tg(TXN)+/0 mice. The levels of Trx1 are significantly higher (approximately 4 to 31 fold) in all of the tissues examined in the Tg(TXN)+/0 mice compared to the wild-type (WT) littermates. The overexpression of Trx1 did not cause any changes in the levels of Trx2, glutaredoxin, glutathione, or other major antioxidant enzymes. The survival study demonstrated that male Tg(TXN)+/0 mice slightly extended the earlier part of the lifespan compared to WT littermates, but no significant life extension was observed over the lifespan. The cross-sectional pathological analysis (22-25 months old) showed that Tg(TXN)+/0 mice had a significantly higher severity of lymphoma and more tumor burden than WT mice, which was associated with the suppression of the apoptosis signal-regulating kinase 1 (ASK1) pathway. Our findings suggest that the increased levels of Trx1 over the lifespan in Tg(TXN)+/0 mice showed some beneficial effects (slight extension of lifespan) in the earlier part of life but had no significant effects on median or maximum lifespans, and increased Trx1 levels enhanced tumor development in old mice. [ABSTRACT FROM AUTHOR]