The thioredoxin system in aging muscle: key role of mitochondrial thioredoxin reductase in the protective effects of caloric restriction?

Cellular redox balance is maintained by various antioxidative systems. Among those is the thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH. In the present study, we examined the effects of caloric restriction (2 mo) on the expression of the cytosolic and mitochondrial thioredoxin system in skeletal muscle and heart of senescent and young rats. Mitochondrial thioredoxin reductase (TrxR2) is significantly reduced in aging skeletal and cardiac muscle and renormalized after caloric restriction, while the cytosolic isoform remains unchanged. Thioredoxins (mitochondrial Trx2, cytosolic Trx1) are not influenced by caloric restriction. In skeletal and cardiac muscle of young rats, caloric restriction has no effect on the expression of thioredoxins or thioredoxin reductases. Enforced reduction of TrxR2 (small interfering RNA) in myoblasts under exposure to ceramide or TNF-alpha causes a dramatic enhancement of nucleosomal DNA cleavage, caspase 9 activation, and mitochondrial reactive oxygen species release, together with reduced cell viability, while this TrxR2 reduction is without effect in unstimulated myoblasts under basal conditions. Oxidative stress in vitro (H2O2 in C2C12 myoblasts and myotubes) results in different changes: TrxR2, Trx2, and Trx1 are induced without alterations in the cytosolic thioredoxin reductase isoforms. Thus aging is associated with a TrxR2 reduction in skeletal muscle and heart, which enhances susceptibility to apoptotic stimuli but is renormalized after short-term caloric restriction. Exogenous oxidative stress does not result in these age-related changes of TrxR2.     

Deletion of Thioredoxin Reductase and Effects of Selenite and Selenate Toxicity in Caenorhabditis elegans

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0–2 mM Se as selenite results in inverse, sigmoidal response curves with an LC₅₀ of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and ⁷⁵Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC₅₀ 0.23 mM Se) were identical to selenite, but selenate was 1/4^th as toxic (LC₅₀ 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.     

Differential effect of calcium ions on the cytosolic and mitochondrial thioredoxin reductase

The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.     

Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization.

We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal. Northern-blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2.     

Thioredoxin and thioredoxin reductase: Current research with special reference to human disease

Thioredoxin (Trx) and thioredoxin reductase (TrxR) plus NADPH, comprising the thioredoxin system, has a large number of functions in DNA synthesis, defense against oxidative stress and apoptosis or redox signaling with reference to many diseases. All three isoenzymes of mammalian TrxR contain an essential selenocysteine residue, which is the target of several drugs in cancer treatment or mercury intoxication. The cytosolic Trx1 acting as the cells’ protein disulfide reductase is itself reversibly redox regulated via three structural Cys residues. The evolution of mammalian Trx system compared to its prokaryotic counterparts may be an adaptation to the use of hydrogen peroxide and nitric oxide in redox regulation and signal transduction.     

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.     

Adjustments,extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.     

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q(9) and instead accumulate demethoxy-Q(9) (DMQ(9)). DMQ(9) has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ(6) in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q(6) and producing solely DMQ were respiratory deficient and unable to support (6)either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ(6) failed to protect cells against oxidative stress generated by H(2)O(2) or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.     

A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress

Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.     

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.     

Tetrahydropteridine deficiency impairs mitochondrial function in Dictyostelium discoideum Ax2

A putative cellular function of tetrahydropteridines (l-erythro-tetrahydrobiopterin and d-threo-tetrahydrobiopterin) was investigated in Dictyostelium discoideum Ax2 using a mutant disrupted in the gene encoding sepiapterin reductase (SR). The SR mutant, which produces about 3% of tetrahydropteridines if compared to wild-type, was elucidated to have several functional defects related to mitochondria and oxidative stress: retarded growth, poor spore viability, impaired mitochondrial function, and increased susceptibility to oxidative stress induced by hydroxylamine or cumene-hydroperoxide. However, the physiological defects were almost completely rescued by extrachromosomal expression of Dictyostelium SR. The results strongly suggested that tetrahydropteridines in Dictyostelium are associated with mitochondrial function, probably via direct protection against oxidative stress.     

Characterization of mitochondrial thioredoxin reductase from C. elegans

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.     

Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition

The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.