Introduction

Proteins in the extracellular environment or on the cell surface are rich in stabilizing disulfides, reflecting oxidizing conditions. In contrast, the inside of the cell is kept reduced and proteins contain many free sulfhydryl groups and disulfides are rare [1]. The major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state is thioredoxin, which is reduced by electrons from NADPH via thioredoxin reductase [2]. The other major factor generally responsible for the low redox potential and high free SH level inside cells is glutathione (GSH) present in millimolar concentrations and kept reduced by NADPH and glutathione reductase [1,3]. GSH-dependent disulfide reductions are catalyzed by glutaredoxins overlapping the functions of thioredoxins, but also uniquely reactive with GSH-mixed disulfides [4].

Thiol–disulfide exchange reactions via redox active disulfides are efficient for electron transport and used in the mechanisms of essential enzymes such as ribonucleotide reductase, which is required to provide deoxyribonucleotides for DNA synthesis. Thiol–disulfide exchange reactions, which are rapid and readily reversible, are also ideally suited to control protein function via the redox state of structural or catalytic SH groups. Oxidation of a critical SH group will generally lead to a changed biological function. This mechanism of thiol redox control [2] is emerging as a major regulatory mechanism in signal transduction. Increased production of reactive oxygen species oxidizing protein thiols and its balance by thioredoxin and glutathione–glutaredoxin-dependent reactions have a wide range of functions in cellular physiology and pathological conditions.

The many roles of the thioredoxin system in different organisms

The general enzymatic reactions of the ubiquitous thioredoxin system have been reviewed previously in detail [2,3,5] and are summarized in Fig. 1. As dithiol–disulfide oxidoreductases all thioredoxins catalyze reduction of disulfides with the rates for proteins being orders of magnitude faster than those for dithiothreitol or GSH. Thioredoxin reductases differ fundamentally between lower and higher organisms. The enzyme in complex eukaryotic organisms is more closely related to glutathione reductase than to bacterial thioredoxin reductase, being larger, having a broader substrate specificity, and an additional redox active motif, which at least in mammals contains selenocysteine (as discussed below and further reviewed by C. H. Williams, Jr., and coworkers in this series of Minireviews).

As every protein is discovered and given a name, its function is linked to a particular biochemical system. It is striking that thioredoxin has been discovered in at least a dozen different such systems, a testimony to the usefulness in nature of this protein. The physiological functions of thioredoxins in different types of organisms have evolved from a common fundamental reaction to a large number of different specialized functions. This includes their conserved role as high-capacity hydrogen donor systems for reductive enzymes, also shared by glutaredoxins, to highly specialized functions in phage T7 DNA replication or filamentous phage assembly of Escherichia coli phages. An early recognized role in chloroplast photosynthetic enzyme regulation by light, has now also been observed in redox regulation of enzymes and transcription factors by thiol redox control. Recent results demonstrate roles of thioredoxin in defense against oxidative stress or in control of apoptosis. Most, but not all, of these functions depend on the disulfide reductase activity of thioredoxin, and thereby on the supply of NADPH and the activity of thioredoxin reductase. Moreover, selective cellular expression observed in immunohistochemical analyses suggests that thioredoxin reductases, of higher organisms, which also reduce many substrates other than thioredoxin [6], may carry out functions unconnected with those of thioredoxin.

Several examples of organism-specific functions of thioredoxins are summarized in Table 1. At present, more than 1900 references in Medline mention ‘thioredoxin’ and it has more than 600 entries in GenBank (excluding ESTs and high-throughput sequences). It has thus become impossible to cover all research on the physiological functions of the thioredoxin system in a short review. However, the references in Table 1 should serve as entry points to the literature for the interested reader. As follows, we shall generally comment upon some of the different types of functions that thioredoxins display in diverse organisms. Thioredoxin appears to be the ultimate ‘moonlighting protein’[7] with many new functions being discovered.

The general oxidoreductase activity of thioredoxins plays two well-known roles: (a) as electron carriers necessary for the catalytic cycles of biosynthetic enzymes, such as ribonucleotide reductases, methionine sulfoxide reductases and sulfate reductases; and (b) generally protecting cytosolic proteins from aggregation or inactivation via oxidative formation of intra- or inter-molecular disulfides.

One function of thioredoxin is to act as a structural component of another enzyme, by forming a complex. This is best characterized for T7 DNA polymerase, where thioredoxin-(SH)₂ binds with high affinity (3 nm) to the DNA polymerase (Gene 5 protein) forming a 1 : 1 complex giving the enzyme high processivity [8]. Remarkably, only thioredoxin-(SH)₂ binds whereas thioredoxin-S₂ is unable to bind despite their closely related 3D structures. A similar structural role is assumed for the role of thioredoxin in phage assembly, because active site mutants were as effective as the wild-type thioredoxin, while the latter had to be reduced by thioredoxin reductase for function in phage assembly [9], thereby creating a situation highly reminiscent to that of the interaction with T7 DNA polymerase. Similarly, in mammalian cells thioredoxin-(SH)₂, but not thioredoxin-S₂, makes an inhibitory complex with apoptosis signaling kinase 1 (ASK1) [10]. This provides a means of redox regulation of apoptosis, with apoptosis inhibited under conditions where thioredoxin-(SH)₂ is the predominant intracellular thioredoxin species, i.e. when oxidative stress is low, and apoptosis is activated when thioredoxin becomes oxidized. This fits well with the general notion of increased oxidative stress as part of the mechanism for induction of apoptosis.

Unique to plants, is the link from light to reduction of disulfide bonds in enzyme regulation, via multiple chloroplast thioredoxins and the ferredoxin:thioredoxin reductase system [11].

Many transcription factors have been shown or are thought to be redox regulated by thioredoxin, with either their activation or inactivation dependent on thioredoxin-catalyzed reduction [12–19]. This gives thioredoxin a central role in thiol redox control of cell function by modulation of the transcription of cell-type specific target genes (Fig. 2). Thioredoxin is, for example, critical for redox regulation of NF-κB [20], a transcription factor that controls expression of numerous inflammatory genes. In addition, thioredoxin has general intracellular antioxidant activity and when upregulated or overexpressed, protects against oxidative stress [21].

Another general and important function in cell signaling and the defense against oxidative damage and stress is the function of thioredoxin as an electron donor for the ubiquitous family of thioredoxin peroxidases or peroxiredoxins (at least six members in mammalian cells) that catalyze the reduction of H₂O₂[22]. Although glutathione peroxidases and catalase reduce H₂O₂, it was demonstrated that at least one of the thioredoxin peroxidases (human TPxII) when overexpressed in cells could inhibit induction of apoptosis by decreasing H₂O₂ levels, thereby constituting a thioredoxin-dependent regulatory step of apoptosis upstream that of Bcl-2 [23].

Extracellular thioredoxin exerts immunomodulatory properties (Fig. 3). Thioredoxin is secreted from cells by a hitherto unknown mechanism that is not dependent on a signal peptide [24](Fig. 3). Human thioredoxin was found as a secreted protein upregulating the IL-2 receptor and having co-cytokine activity from HTLV-I transformed T-lymphocytes, where it was initially called adult T-cell leukemia-derived factor [25]. It was subsequently shown that this factor and human thioredoxin are the same protein [26], and it is now clear that the functions of extracellular human thioredoxin are many. Secretion of thioredoxin under conditions of oxidative stress and inflammation has been observed from many normal or neoplastic cells [24,27]. Clinically, plasma thioredoxin levels are raised in patients undergoing cardiopulmonary bypass operations [28] and in those with reumatoid arthritis [29] or HIV infection[30]. Extracellular thioredoxin has proinflammatory effects by potentiating cytokine release from fibroblasts [29] as well as monocytes [31]. Recently, thioredoxin has been shown to act as a chemotactic protein causing migration of neutrophils, monocytes and T-cells with a potency similar to known chemokines including IL-8 [32]. Mutation of the active site residues to serines resulted in loss of chemotactic activity, suggesting that the redox activity is required. The concentration-dependent activity of thioredoxin as a chemotactic factor is bell-shaped like other chemotactic agents and has a maximum in the range of the thioredoxin plasma concentration (25 ng per mL). However, the effect differs in one important respect from other known chemokines in being G-protein-independent [32]. Thioredoxin may also block the action of other chemokines [32]; therefore if thioredoxin levels are highly upregulated in severe inflammation, this could block cell migration and make the infection more severe. Moreover, a truncated form of thioredoxin (Trx80), found at the surface of monocytic cell lines [33], is most likely identical to the protein described as eosinophilic cytotoxicity enhancing factor (ECEF), which enhances the capacity of eosinophils to kill larvae of Schistosoma mansoni[34,35]. Recent results have shown that cloned Trx80 is a novel mitogenic cytokine for human peripheral blood mononuclear cells (K. Pekkari, R. Gurunath, E. S. J. Arnér & A. Holmgren, unpublished results).

Thioredoxin structure and mechanism

In spite of the many functions of thioredoxins outlined in Table 1, thioredoxins from Archaea to humans have 27–69% sequence identity to that of E. coli Trx1, the best characterized protein, demonstrating that all thioredoxins have the same overall 3D structure (the thioredoxin fold) [5,36]. This consists of a central core of five β strands surrounded by four α helices and the archetypical active site sequence -Cys-Gly-Pro-Cys- [36,37] which is located at the end of a β strand (β₂) and in the beginning of a long α helix. In addition to the active site sequence, a number of residues are highly conserved: Asp26, Ala29, Trp31, Asp61, Pro76 (in the cis configuration), and Gly92 (using the E. coli numbering). High-resolution 3D structures of oxidized and reduced E. coli thioredoxin in solution have been determined by NMR spectroscopy [38]. The overall difference between the oxidized and reduced forms is subtle [5,38] and involves a local conformational change in and around the redox-active disulfide with more conformational substates for the reduced form also reflected in different hydrogen bonding pattern. The N-terminal active site Cys residue has a low pK_a value and is the attacking nucleophile in disulfide reduction of proteins [5]. The mechanism involves a transient mixed disulfide intermediate and fast thiol–disulfide exchange in a hydrophobic environment [5]. The reaction is reversible and thioredoxin may either break or form disulfides depending on the redox potential of its substrate. The low redox potential of thioredoxin (E. coli Trx1 = −270 mV) ensures that thioredoxin-(SH)₂ is the major dithiol reductant in the cytosol, or an advanced equivalent to dithiothreitol of cells [2]. Human thioredoxin may form an inactive homodimer via a disulfide between the structural surface located Cys73 implicated to have a regulatory function [37,39].

There is a large superfamily of proteins that contain one or multiple thioredoxin domains [40,41]. Such proteins containing a thioredoxin fold are found in all organisms and can be grouped into at least six classes: thioredoxins, glutaredoxins, DsbA, protein disulfide isomerase, glutathione transferases, and glutathione peroxidases [40,41]. While sequence homology between these six classes is limited and no function or activity is common to all, there is a functional similarity common to four of these members. Thioredoxins, glutaredoxins, DsbA, protein disulfide isomerases are all redox-active proteins containing a -Cys-Xaa1-Xaa2-Cys-active-site motif (where Xaa represents any of the 19 commonly occuring noncysteine amino acids). Moreover, the remaining two classes of proteins, the glutathione transferases, and glutathione peroxidases, while lacking the -Cys-Xaa1-Xaa2-Cys-active-site motif share with the glutaredoxins a specific interaction with GSH. Among the recent additions to the thioredoxin superfamily is a protein interacting with protein kinase C, coined PICOT (protein kinase C-interacting cousin of thioredoxin) [42], a larger cytoplasmic thioredoxin-like protein of unknown function [43,44] and a nuclear thioredoxin-like protein with oxidoreductase activity, also of unknown function, named nucleoredoxin [45].

Human thioredoxin and thioredoxin reductase

In contrast to bacteria, yeast and particularly plants which have multiple thioredoxins, only one cytoplasmic thioredoxin has so far been found in human cells. However, movements within the cellular compartments or secretion through the cell membrane (Fig. 3) adds to the complexity. A detailed mechanism for thioredoxin secretion and its effects as a co-cytokine and growth factor is an area for further research. Much evidence supports the idea [46] that oxidized forms of thioredoxin are secreted (Fig. 3). Recently, a human mitochondrial thioredoxin (Trx2) has been cloned [47]. This together with the classical cytosolic enzyme and its above mentioned truncated form are the presently known human thioredoxin isoenzymes (Fig. 3).

The serendipitous discovery by T. Stadtman and coworkers about five years ago that human thioredoxin reductase is a selenoprotein [48,49] resulted in much interest and intimately linked the thioredoxin system with the selenium status and function in cells. Interestingly, it was previously shown that the mammalian thioredoxin system catalyzed reduction of selenium compounds such as selenite [50] or selenodiglutathione [51] and, moreover, may function as a hydrogen donor system for the selenoprotein plasma glutathione peroxidase [52] or as a lipid hydroperoxide reductase [53]. The mammalian thioredoxin system is thereby very much at the center of selenium biology and thioredoxin reductase being a selenoprotein may provide a connection between the similar phenotypes of mice lacking the tRNA^Sec, necessary for selenoprotein production, or thioredoxin, both showing a similar early embryonic lethality of the –/– phenotype with failing implantation [54,55].

The selenocysteine residue in thioredoxin reductase (Fig. 4) is conserved between mammalian species in a C-terminal -Gly-Cys-Sec-Gly motif (where Sec is selenocysteine), with the selenocysteine residue being essential for catalytic activity [56,57]. Recently we demonstrated that the selenocysteine and its neighboring cysteine residue constitute the redox active site of the enzyme by forming a selenenylsulfide in the oxidized enzyme that is reduced to a selenolthiol by the redox active dithiol of the other subunit in the dimeric enzyme [58]. This mode of catalysis had already been suggested based upon kinetic analysis and redox titrations by Williams and coworkers [59] and is also in agreement with recent findings of Lee et al. [60].

Recently two novel human thioredoxin reductase isoenzymes were cloned and sequenced, one mitochondrial [61,62] and one predominantly expressed in testis [61], both showing the conserved C-terminal selenocysteine-containing motif and general domain organization of the first characterized classical enzyme. The mitochondrial thioredoxin reductase has been purified from rat liver [63] and bovine adrenal cortex [64] and the first characterizations revealed enzymatic characteristics similar to those of the classical enzyme. A general difficulty in studying mammalian thioredoxin reductases, and other selenoproteins, are species-barriers in the translation machinery for selenocysteine incorporation making it impossible to directly express a mammalian selenoprotein in bacteria or yeast. This difficulty was ,however, recently overcome [65]. This should enable production of pure recombinant forms of the different mammalian thioredoxin reductase isoenzymes for further mechanistic characterization as well as use in enzymatic assays [6].

Thioredoxin reductase may possibly be (transiently or permanently) inactivated by oxidants such as H₂O₂, which has been proposed to give the enzyme the status of a cellular ‘redox sensor’[61]. This is an attractive idea that requires further studies by in vivo experiments. Also, the physiological functions of plasma thioredoxin reductase, the presence of which recently was demonstrated [66] and which may derive from the proximal tubuli of the kidney [67], are still unknown.

Due to the many known physiological roles of the thioredoxin system, it is not surprising that it also may play a role in the pathogenesis of a number of diseases and the effects of clinically used drugs, either being reduced by the thioredoxin system or inhibiting thioredoxin reductase (see Becker et al. in this series of Minireviews). From the first characterizations in the mid-1960s of thioredoxin as a dithiol hydrogen donor cofactor for ribonucleotide reductase in E. coli[68,69], the known physiological roles of the thioredoxin system have grown in number in a remarkable way. It seems safe to state that this number will continue to grow and that we may today only know a limited number of mechanisms by which thioredoxins and thioredoxin reductases are involved in diverse systems such as, e.g. redox signaling, photosynthesis, regulation of apoptosis, immunomodulation, embryonic implantation and developmental biology.

Acknowledgements

We are grateful to Dr Charles Williams and other colleagues for many helpful discussions. Supported by grants from the Swedish Cancer Society projects 0961-24XAC (A. H.), 3775-04XBB and 4056-02PBD (E. A.) and the Swedish Medical Research Council 13X-3529 (A. H.).