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Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.

Glutaredoxin (Grx) belongs to the thioredoxin fold superfamily and catalyzes glutathione-dependent oxidoreductions. The recently discovered mitochondrial and nuclear Grx (Grx2) differs from the more abundant cytosolic Grx (Grx1) by its higher affinity toward S -glutathionylated proteins and by being a substrate for thioredoxin reductase. Here, we have successfully established a method to silence the expression of Grx2 in HeLa cells by using short interfering RNA to study its role in the cell. Cells with levels of Grx2 50 for doxorubicin dropped from 40 to 0.7 μM and for phenylarsine oxide from 200 to 5 nM. However, no differences were detected after treatment with cadmium, a known inhibitor of Grx1. These results indicate a crucial role of Grx2 in the regulation of the mitochondrial redox status and regulation of cell death at the mitochondrial checkpoint.

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2 H )-one], a seleno-organic compound with glutathione peroxidase-like activity is used in clinical trials against stroke. Human and bovine TrxR catalyzed the reduction of ebselen to ebselen selenol by NADPH with an apparent K M -value of 2.5 μM and a k cat of 588 min −1 . The addition of thioredoxin (Trx) stimulated the TrxR-catalyzed reduction of ebselen several-fold. This result was caused by a very fast oxidation of reduced Trx by ebselen with a rate constant in excess of 2 × 10 7 M −1 s −1 . This rate is orders of magnitude faster than the reaction of dithiol Trx with insulin disulfides. Ebselen competed with disulfide substrates for reduction by Trx and, therefore, acted as an inhibitor of protein disulfide reduction by the Trx system. The inherent H 2 O 2 reductase activity of mammalian TrxR dependent on its active-site selenocysteine residue was stimulated 10-fold by 2 μM ebselen and 25-fold in the additional presence of 5 μM Trx. Furthermore, the apparent K M -value of TrxR for H 2 O 2 was lowered 25-fold to about 100 μM. Our results demonstrate that ebselen is a TrxR peroxidase which, in the presence of Trx, acted as a mimic of a peroxiredoxin. The activity with TrxR and oxidation of reduced Trx offer mechanistic explanations for the in vivo effects of ebselen as an antioxidant and anti-inflammatory agent. Our results demonstrate that the mechanism of action of ebselen may be predominantly via the Trx system rather than via glutathione.

Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP + has been determined to 3.0-Å resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: ( i ) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and ( ii ) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.

A fetal form of somatomedin (insulin-like growth factor) that crossreacts in the fetal brain radioreceptor assay has been proposed to exist in humans. Using this assay to monitor activity during purification, we have isolated a variant form of insulin-like growth factor I (IGF-I) from human fetal brain tissue. The variant IGF-I showed potent crossreaction in the fetal brain radioreceptor assay and stimulated DNA synthesis in fetal brain cells in vitro. Structural analysis revealed the variant IGF-I to have a truncated NH2-terminal region compared to IGF-I isolated from serum. An additional peptide, which displayed less potent crossreaction in the fetal brain radioreceptor assay, was also isolated from the human fetal brain. Partial amino acid sequence analysis revealed identity to insulin-like growth factor II.

Purified calf thymus ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), showed an absolute requirement for a dithiol as hydrogen donor, whereas the natural monothiol glutathione (GSH) was inactive per se. However, a protein partially purified from thymus coupled the oxidation of GSH to the formation of deoxyribonucleotides by ribonucleotide reductase. In analogy with the ribonucleotide reductase system of Escherichia coli this protein was called glutaredoxin [Holmgren, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2275-2279]. Thymus glutaredoxin had the following properties: (i) its molecular weight determined by gel chromatography was about 12,000; (ii) it was active iwth ribonucleotide reductase in the presence of GSH, NADPH, and glutathione reductase but had no activity with NADPH and thioredoxin reductase; and (iii) it was immunologically different from thioredoxin because it did not bind to antithioredoxin immunoadsorbents. Experiments on the crossreactivity of thymus and E. coli ribonucleotide reductases and the corresponding thioredoxin and glutaredoxin systems showed essentially no specificity for the homologous thioredoxin but a high species specificity for the homologous glutaredoxin.