Protein Disulfide Reductase Research Articles

Regulation of the Catalytic Activity and Structure of Human Thioredoxin 1 via Oxidation and S-Nitrosylation of Cysteine Residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.

Matrix-assisted refolding and redox properties of WhiB3/Rv3416 of Mycobacterium tuberculosis H37Rv

Redox stress is one of the major challenges faced by Mycobacterium tuberculosis during early infection and latency. The mechanism of sensing and adaptation to altered redox conditions is poorly understood. whiB family of Mtb is emerging as an important class of stress responsive genes. WhiB3/Rv3416 has been shown to be important for pathogenesis in animal model and was recently shown to co-ordinate a Fe–S cluster. Here, we report a simple, rapid and efficient matrix-assisted refolding method and important redox properties of WhiB3. Similar to other WhiB proteins, WhiB3 also has four conserved cysteine residues, where two of them are present in a CXXC motif. The Fe–S cluster of WhiB3 remained bound in the presence of strong protein denaturant. Upon cluster removal due to oxidation, the four cysteine residues which are ligands of Fe–S cluster, formed two intra-molecular disulfide bridges where one of them is possibly between the cysteines of CXXC motif, an important feature of several thiol-disulfide oxido-reductases. Far-UV CD spectroscopy revealed the presence of both α-helices and β-strands in apo WhiB3. The secondary structural elements of apo WhiB3 were found resistant for thermal denaturation. The results demonstrated that apo WhiB3 functions as a protein disulfide reductase similar to thioredoxins. The importance of WhiB3 in redox sensing and its possible role in mycobacterial physiology has been discussed.

2P-033 小胞体に内在するジスルフィド結合還元酵素ERdj5の構造・機能解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

2P-033 小胞体に内在するジスルフィド結合還元酵素ERdj5の構造・機能解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

The Archaeon Methanosarcina acetivorans Contains a Protein Disulfide Reductase with an Iron-Sulfur Cluster

Methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain Archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain Bacteria. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated. The product of MA3736, overproduced in Escherichia coli, had protein disulfide reductase activity dependent on a C(67)XXC(70) motif not found in carboxymuconolactone decarboxylase. We propose that MA3736 be renamed mdrA (methanosarcina disulfide reductase). Further, unlike carboxymuconolactone decarboxylase, MdrA contained an Fe-S cluster. Binding of the Fe-S cluster was dependent on essential cysteines C(67) and C(70), while cysteines C(39) and C(107) were not required. Loss of the Fe-S cluster resulted in conversion of MdrA from an inactive hexamer to a trimer with protein disulfide reductase activity. The data suggest that MdrA is the prototype of a previously unrecognized protein disulfide reductase family which contains an intermolecular Fe-S cluster that controls oligomerization as a mechanism to regulate protein disulfide reductase activity.

A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin-nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left-right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing.

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C–X–X–C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni 2+–NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as −236 ± 2 mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.

Structural Basis for Target Protein Recognition by the Protein Disulfide Reductase Thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.

An evolutionarily conserved 16-kDa thioredoxin-related protein is an antioxidant which regulates the NF-κB signaling pathway

Thioredoxin (TRX) is generally a 12-kDa protein-disulfide reductase. Here, we report the discovery of a 16-kDa thioredoxin-related protein designated Cr-TRP16, from a “living fossil,” the horseshoe crab ( Carcinoscorpius rotundicauda). Cr-TRP16 contains an atypical WCPPC catalytic motif and possesses the classical thiodisulfide reductase activity, as indicated by the insulin reduction assay. Furthermore, Cr-TRP16 can function as an antioxidant and protect against DNA nicking by reactive oxygen species. Overexpression of Cr-TRP16 regulated the transcription of NF-κB-dependent genes probably by enhancing NF-κB DNA-binding activity, suggesting possible roles for Cr-TRP16 in modulating the NF-κB signaling pathway. In vivo, the antioxidant pyrrolidine dithiocarbamate suppressed the expression of NF-κB-regulated genes such as IκB and inducible nitric oxide synthase. This further supports the notion that oxidative stress is also a regulatory factor of the NF-κB signaling pathway, a phenomenon which has been entrenched for several hundred million years. Furthermore, we demonstrated that the 16-kDa thioredoxins are evolutionarily conserved from Caenorhabditis elegans to human. Interestingly, thioredoxin-like 6, a human homologue of Cr-TRP16, could also enhance NF-κB DNA-binding activity, suggesting that the regulatory role of the 16-kDa thioredoxins on NF-κB is well conserved through evolution.

Changes of endoplasmic reticulum chaperone complexes, redox state, and impaired protein disulfide reductase activity in misfolding αi‐antitrypsin transgenic mice

Alpha1-antitrypsin (AAT) deficiency is characterized by the accumulation of the misfolded mutant, Z form of alpha1-antitrypsin (PiZ) inside the lumen of the hepatic endoplasmic reticulum (ER). Both human patients and PiZ transgenic mice have similar symptoms of hepatic failure culminating in cirrhosis and hepatocellular carcinoma. The involvement of molecular chaperones, as well as the relevance of oxidative stress in this disease is not characterized well yet. Here, we show that, in the PiZ transgenic mice, the 58-kDa protein disulfide isomerase (PDI), the most important oxidoreductase and chaperone of the endoplasmic reticulum, is in a complex with PiZ, which is accompanied by a decrease of protein disulfide reductase activity of the ER. PiZ transgenic mice have a shift toward a more reduced ER environment and an elevation of cytoplasmic chaperones and antioxidant enzymes. Our data suggest that lower availability of PDI and a decreased protein disulfide reductase activity of the ER along with a cytoplasmic stress may contribute to the toxic effects of PiZ aggregation.

Catalysis of Thiol/Disulfide Exchange: GLUTAREDOXIN 1 AND PROTEIN-DISULFIDE ISOMERASE USE DIFFERENT MECHANISMS TO ENHANCE OXIDASE AND REDUCTASE ACTIVITIES

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.

Expression, purification and X-ray crystallographic analysis of thioredoxin from Streptomyces coelicolor.

Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. In turn, they are reduced by electrons obtained from the NADPH-containing thioredoxin reductase. Thioredoxins have been isolated and characterized from a large number of organisms. The Gram-positive bacterium Streptomyces coelicolor contains three thioredoxins that are involved in unknown biological processes. trxA from S. coelicolor was cloned and expressed in Escherichia coli and the protein purified and crystallized using the hanging-drop method of vapour diffusion. The crystal structure of thioredoxin A has been determined at 1.5 A resolution using a synchrotron-radiation source. The protein reveals a thioredoxin-like fold with a typical CXXC active site. The crystal exhibits the symmetry of space group P2(1)2(1)2, with unit-cell parameters a = 43.6, b = 71.8, c = 33.2 A.

Truncated Thioredoxin: Physiological Functions and Mechanism

Human cytosolic thioredoxin (Trx), which is the 12-kDa protein disulfide reductase with the Cys-Gly-Pro-Cys active site and a key component of cellular redox biochemistry and regulation, acts as cocytokine upon leaderless secretion. A 10-kDa C-terminally truncated thioredoxin (Trx80) comprising the 80 or 84 N-terminal amino acids is also secreted and present in plasma, where it originally was purified and identified as eosinophilic cytotoxicity enhancing factor. Recombinant Trx80 was discovered to be a potent mitogenic cytokine that stimulates growth of resting human peripheral blood mononuclear cells (PBMC) in a synthetic medium, an effect that Trx lacks. Trx80 is very different from Trx because it is a dimer lacking reductase activity and the cytokine activity is not dependent on the Cys residues of the Trx active-site motif. The primary targets of Trx80 in PBMC are monocytes that are activated to proliferate and increase expression of CD14, CD40, CD54, and CD86. Trx80 induces secretion of interleukin (IL)-12 in CD40+ monocytes from PBMC. Trx80 and IL-2 together were strongly synergistic to induce secretion of interferon-gamma in PBMC. Trx80 is a potent cytokine for monocytes directing the immune system to a Th1 response via IL-12 production.

Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum.

The persistent and viscous nature of airway secretions in cystic fibrosis (CF) disease leads to airway obstruction, opportunistic infection, and deterioration of lung function. Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reductive processes. To determine whether Trx can alter the rheological properties of mucus, sputum obtained from CF patients was treated with TRX and its reducing system (0.1 microM thioredoxin reductase + 2 mM NADPH), and liquid phase-gel phase ratio (percent liquid phase) was assessed by compaction assay. Exposure to low Trx concentrations (1 microM) caused significant increases in the percentage of liquid phase of sputum. Maximal increases in percent liquid phase occurred with 30 microM Trx. Additional measurements revealed that sputum liquefaction by the Trx reducing system is dependent on NADPH concentration. The relative potency of the Trx reducing system also was compared with other disulfide-reducing agents. In contrast with Trx, glutathione and N-acetylcysteine were ineffective in liquefying sputum when used at concentrations <1 mM. Sputum viscoelasticity, measured by magnetic microrheometry, also was diminished significantly following 20-min treatment with 3, 10, or 30 microM Trx. Similarly, this reduction in viscoelasticty also was dependent on NADPH concentration. Further investigation has indicated that Trx treatment increases the solubility of high-molecular-weight glycoproteins and causes redistribution of extracellular DNA into the liquid phase of sputum. Recognizing that mucins are the major gel-forming glycoproteins in mucus, we suggest that Trx alters sputum rheology by enzymatic reduction of glycoprotein polymers present in sputum.

Redox Control of Hsp70-Co-chaperone Interaction Revealed by Expression of a Thioredoxin-like Arabidopsis Protein

By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that AtTDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of AtTDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the AtTDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity.

Purification and characterization of Δ3Trx-1, a splicing variant of human thioredoxin-1 lacking exon 3

Thioredoxins comprise a growing family of proteins that function as general protein-disulfide reductases and are maintained in their reduced active form by the flavoenzyme thioredoxin reductase. Human Trx-1 is mainly a cytosolic protein, although it has been shown to translocate into the nucleus upon certain stimuli and can also be secreted. We report here the expression and characterization of Δ3Trx-1, a splicing variant of human Trx-1, lacking exon 3, which spans from residues 44 to 63 in the wild-type protein. Structure-based prediction of this splicing form indicates that Δ3Trx-1 lacks helix α2 and strand β3, which are implicated in substrate positioning and three-dimensional stabilization of the active site residues. Recombinant human Δ3Trx-1 is recognized by polyclonal antibodies raised against full-length human Trx-1. However, Δ3Trx-1 retains no enzymatic activity either with DTT or thioredoxin reductase and NADPH as reducing systems. Δ3Trx-1 competes with full-length Trx-1 for the interaction with thioredoxin reductase. The absence of helix α2 and strand β3 in Δ3Trx-1 is consistent with the lack of enzymatic activity and its potential dominant negative properties.

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.

Effects of thioredoxin on the preimplantation development of bovine embryos

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% O 2 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% O 2. 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.

Vitamin D3-up-regulated Protein-1 Is a Stress-responsive Gene That Regulates Cardiomyocyte Viability through Interaction with Thioredoxin

The protein-disulfide reductase thioredoxin is critical for redox signaling during apoptosis and growth. In this study, we demonstrate that vitamin D(3)-up-regulated protein-1 regulates thioredoxin in conditions of biomechanical or oxidative stress and critically regulates cardiomyocyte viability. Expression of vitamin D(3)-up-regulated protein-1 but not of thioredoxin in rat cardiomyocytes was rapidly suppressed by biomechanical strain or hydrogen peroxide at both mRNA and protein levels. Mechanical suppression of vitamin D(3)-up-regulated protein-1 gene expression was blocked by N-acetylcysteine. The half-life of vitamin D(3)-up-regulated protein-1 transcripts in cardiomyocytes was only 1.1 h and remained unchanged after mechanical stimulation, suggesting that rapid responses in vitamin D(3)-up-regulated protein-1 gene expression occur through transcriptional control. Vitamin D(3)-up-regulated protein-1 down-regulation by strain or hydrogen peroxide led to increased thioredoxin activity, whereas adenovirus-mediated overexpression of vitamin D(3)-up-regulated protein-1 suppressed thioredoxin activity. Overexpression of vitamin D(3)-up-regulated protein-1 but not of thioredoxin induced cardiomyocyte apoptosis. Furthermore, overexpression of vitamin D(3)-up-regulated protein-1 sensitized cells to hydrogen peroxide-induced apoptosis, whereas overexpression of thioredoxin protected against injury. These data identify vitamin D(3)-up-regulated protein-1 as a key stress-responsive inhibitory switch of thioredoxin activity in cardiomyocytes and demonstrate that the vitamin D(3)-up-regulated protein-1/thioredoxin axis has an important role in the preservation of cellular viability.

Secretion of 10-kDa and 12-kDa thioredoxin species from blood monocytes and transformed leukocytes.

Thioredoxins (TRX) are ubiquitous, small redox-active proteins with multiple functions, including antioxidant, cytoprotective, and chemoattractant activities. In addition to a 12-kDa intracellular form, extracellular 10-kDa and 12-kDa TRX have been defined. The biological activities of the 10-kDa TRX were previously measured as eosinophil cytotoxicity enhancing activity or B-cell stimulatory activity. Cytotrophoblastic cell lines also release a 10-kDa TRX form. To study the biological role of 10-kDa TRX, we established two highly sensitive enzyme-linked immuno-spot assays (ELISPOT), which detect secreted truncated 10-kDa and full-length 12-kDa TRX at the single cell level. TRX secretion was investigated in several cell lines including the T-helper cell hybridoma MP6, the Jurkat T-cell leukemia, the U-937 myelomonocytic leukemia, and the 3B6, EBV-transformed, lymphoblastoid B-cell line. The highest number of secreting cells was found in 3B6 cultures, median = 34 (quartiles, 27-39) per well (10(5) cells). Peripheral blood monocytes isolated from healthy donors secreted significantly more TRX after stimulation with ionomycin, phorbol myristate acetate (PMA), fMLP, and lipopolysaccharide (LPS), compared to unstimulated cells. Oxidative stress induced by thioloxidant diamide also induced the secretion of both truncated and full-length TRX measured in ELISPOT (p = 0.047 and p = 0.031, respectively). The biological activity of the truncated and full-length forms was tested in a cell migration assay. Truncated TRX was devoid of protein disulfide reductase activity, but retained strong chemoattractant activity for human monocytes, in the same range as full-length TRX, as previously reported (Bertini et al., 1999).