Thioredoxin System in Cell Death Progression

Abstract

Significance: The thioredoxin (Trx) system, comprising nicotinamide adenine dinucleotide phosphate, Trx reductase (TrxR), and Trx, is critical for maintaining cellular redox balance and antioxidant function, including control of oxidative stress and cell death. Recent Advances: Here, we focus on the research progress that is involved in the regulation of apoptosis by Trx systems. In mammalian cells, cytosolic Trx1 and mitochondrial Trx2 systems are the major disulfide reductases supplying electrons to enzymes for cell proliferation and viability. The reduced/dithiol form of Trxs binds to apoptosis signal-regulating kinase 1 (ASK1) and inhibits its activity to prevent stress- and cytokine-induced apoptosis. When Trx is oxidized, it dissociates from ASK1 and apoptosis is stimulated. The binding of Trx by its inhibitor Trx interacting protein (TXNIP) also contributes to the apoptosis process by removing Trx from ASK1. TrxRs are large homodimeric selenoproteins with an overall structure which is similar to that of glutathione reductase, and contain an active site GCUG in the C-terminus. Critical Issues and Future Directions: In the regulation of cell death processes, Trx redox state and TrxR activities are key factors that determine the cell fate. The high reactivity of Sec in TrxRs and its accessible location make TrxR enzymes emerge as targets for pharmaceutic drugs. TrxR inactivation by covalent modification does not only change the redox state and activity of Trx, but may also convert TrxR into a reactive oxygen species generator. Numerous electrophilic compounds including some environmental toxins and pharmaceutical drugs inhibit TrxR. We have classified these compounds into four types and propose some useful principles to understand the reaction mechanism of the TrxR inhibition by these compounds. Antioxid. Redox Signal. 17, 1738–1747.

Introduction

P rotein thiols are involved in the catalytic activity of many proteins and may be changed to disulfides under oxidation. This thiol-disulfide change can affect the enzyme activity and, therefore, regulate the cellular functions. Thioredoxin (Trx), a 12 kDa dithiol protein, which is conserved from archaea and bacteria to man, is itself a key factor that maintains the protein dithiol/disulfide homeostasis. Along with Trx reductase (TrxR), Trx can provide electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to a very large number of critical cellular proteins and is, thus, involved in a wide range of cellular functions (Fig. 1). For example, Trx was originally discovered as being the electron donor for ribonucleotide reductase (RNR) from Escherichia coli (24). The ubiquitous RNR catalyzes the de novo synthesis of 2′-deoxyribonucleotides from corresponding ribonucleotides and is essential for DNA replication and repair. RNR from vertebrates and E. coli are a complex consisting of dimeric R1 and R2 subunits. The R2 subunit harbors a stable tyrosyl radical in its oxygen-linked diferric iron center, which is required for the catalytic reaction. The R1 subunit contains a substrate-binding site, an allosteric site, a dithiol active site, and two shuttle sulfhydryl groups in the C-terminus (26). The dithiol in the active site is converted to a disulfide after a cycle of catalytic reactions, but is reduced by the C-terminal sulfhydryl groups via thiol-disulfide exchange. In turn, the disulfide in the C-terminus is reduced by Trx or glutaredoxin (Grx) (6, 26). Other well-known Trx substrates are the peroxiredoxins (Prxs), Trx-dependent peroxidases, which are widely distributed in various subcellular organelles. This enables them to scavenge H₂O₂ locally and control signal transduction more specifically (30). Methionine-S-sulfoxide reductases, which catalyze the reduction of free or protein-bound methionine sulfoxides back to methionine, are also the substrates of Trx system (68). Besides being a disulfide reductase, Trx participates in the regulation of cellular processes by mediating protein S-denitrosylation. This has been recently reviewed (57).

FIG. 1.

Trx systems serve as a general disulfide reductase for many critical proteins in the cells. Along with TrxR, Trx can provide the electrons from NADPH to many critical cellular proteins. Trx can reduce the disulfide in the C-terminus of RNR R1 subunit; subsequently, the C-terminus with dithiols swings to the central active site and reduce the active site disulfide to dithiols, which is essential for the RNR catalytic reaction to synthesize 2′-deoxyribonucleotide from corresponding ribonucleotides. The Trx system can also reduce the disulfide bond in Prxs, one of the most abundant proteins in the cell. Most of the Prxs are Trx-dependent peroxidases and play critical roles in H₂O₂ scavenging and redox signaling. MsrA is another well-known Trx substrate that catalyzes the free or protein-bound methionine sulfoxides back to methionine. The disulfide bonds in PTEN and HDAC4 may also be reduced by the Trx system. MsrA, methionine-S-sulfoxide reductase; NADPH, nicotinamide adenine dinucleotide phosphate; RNR, ribonucleotide reductase; Prx, peroxiredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase.

Role of Trxs in Mammalian Cell Death Progression

Mammalian Trx systems

Both cytosolic Trx1 and mitochondrial Trx2 in mammalian cells contain an active site Trp-Cys-Gly-Pro-Cys- in a typic Trx-fold structure (Fig. 2) (32). Human Trx1 with 105 amino-acid residues has three structural Cys residues at positions 62, 69, and 73, apart from Cys32 and Cys35 in the active site. Cys62 and Cys69 in Trx-S₂ can form a second disulfide bond under oxidative stress conditions (Fig. 2). This two-disulfide bond form of Trx-S₂ is poorly characterized but cannot be reduced by TrxR directly, and its formation leads to the inactivation of Trx1 (20, 23, 40, 67). In addition, these extra structural Cys residues have been suggested to be involved in many post-translational modifications of human Trx1, such as S-nitrosylation, glutathionylation, and dimerization (70).

FIG. 2.

Comparison of the mammalian Trx system with the Escherichia coli Trx system. All the three Trxs, cytosolic Trx1, mitochondrial Trx2, and E. coli. Trx1 are small proteins and contain the active site WCGPC. Human Trx1 has three structural cysteines at the positions 62, 69, and 73, apart from the active sites Cys32 and Cys35. Cys62 and Cys69 can form a disulfide bond under oxidative stress conditions. All these Cys may be involved in mediating the S-nitrosylation and S-denitrosylation of some key protein in apoptosis-like caspase 3. Both human TrxR1 and E. coli. TrxR1 are homodimmeric flavoproteins with FAD and NADPH binding domains. However, mammalian TrxRs are large proteins with 55 kDa or larger subunits; instead, bacterial TrxRs are smaller proteins with 33 kDa subunits. Mammalian TrxR contains both an N-terminal active site CVNVGC and a conserved C-terminal sequence Gly-Cys-Sec-Gly with a wide range of substrates. In contrast, bacterial TrxR has only one active site sequence CXXC with a high substrate specificity. FAD, flavin adenine dinucleotide.

Regulation of cell death by Trx1

Trx1 is a central redox regulator that mediates the activation of many transcription factors which are involved in cell growth, apoptosis, and inflammation, such as NF-κB, activator protein 1 (AP-1), p53, hypoxia-inducible factor 1, and redox factor 1 (Ref-1) (25, 32). The reduction status of some Cys residues in the DNA binding site of the transcription factors, for example, Cys62 of NF-κB p50, is critical for the DNA binding. Under oxidative stress conditions, Trx1 is found to be translocated from the cytosol to the nucleus (22). The presence of Trx can maintain the cysteine in its reduced state and promote the DNA binding activity of NF-κB and AP-1 (22). Moreover, Ref-1 can also translocate from the cytosol to the nucleus, and it interacts with Trx1 physically. The association of Ref-1 with Trx1 can increase the DNA binding activity of transcription factors such as AP-1 (21).

Trx1 can prevent the cell apoptosis process by a direct association with apoptosis signal regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase (MAP3K) (53). This MAP3K activates the c-Jun N-terminal kinase and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-α-induced apoptosis. Trx1 can bind with the N-terminal noncatalytic region of ASK1. The interaction between Trx1 and ASK1 is highly dependent on the redox status of Trx1 and only reduced Trx binds. Reactive oxygen species (ROS) such as H₂O₂ caused by stress or cytokine treatment will lead to the oxidation of Trx either directly or via Prxs, the dissociation of Trx1 from ASK1, and the subsequent activation of ASK1 and subsequent apoptosis (Fig. 3) (53). In addition, Trx1 can induce ASK1 ubiquitination and degradation to inhibit ASK1 activity in endothelial cells. In the process, the Trx1 active site Cys32 or Cys35 is necessary and sufficient for the association of Trx1 with Cys250 in ASK1 (33, 76).

FIG. 3.

Trx is involved in the cell death via an ASK1-Trx-TXNIP signaling pathway. Trx1 can prevent the cell apoptosis process by direct association with the N-terminal noncatalytic region of ASK1, a mitogen-activated protein kinase kinase kinase. The interaction between Trx1 and ASK1 is highly dependent on the redox status of Trx1. Under oxidative stress, H₂O₂ oxidizes Trx1, enables Trx1 to be released from ASK1, and then induces the activation of ASK1 and subsequent apoptosis. Trx1 active sites Cys32 or Cys35 are involved in the association of Trx1 with ASK1. TXNIP, an endogenous inhibitor of Trx, may participate in this process by helping the release of Trx1 from ASK1. TXNIP can attack reduced Trx1, via a disulfide exchange interaction between the disulfide bond of Cys63 and Cys247 in TXNIP and Trx active site dithiols, and results in an oxidized Trx1. Recently, it was proposed that the Trx2-ASK1 signaling pathway involves an intracellular shuttling of TXNIP (76). Under normal conditions, TXNIP is located in the cytosol and primarily in the nucleus. Mitochondrial Trx2 is bound with ASK1 and inhibits its activity. On oxidative stress, TXNIP translocates from the nucleus to the mitochondria, competes for the binding with Trx2, and results in the apoptotic process. ASK1, apoptosis signal regulating kinase 1; TXNIP, thioredoxin interacting protein.

Since Trx1 exerts an anti-apoptotic function, it is not surprising that the inhibition of Trx1 induces cell death. Trx interacting protein (TXNIP, TBP2, or vitamin D3 up regulated protein 1) is an endogenous inhibitor of Trx1 that was identified in a yeast two-hybrid system (44). TXNIP forms a complex with reduced Trx1, but not oxidized Trx1. This process may involve a disulfide exchange interaction between the disulfide bond of Cys63 and Cys247 in TXNIP and reduced Trx active site dithiols (Fig. 3) (48). TXNIP, thus, binds and inhibits Trx1, and this binding results in cellular oxidative stress. [For a review on the role of Trx-TXNIP in apoptosis, see the reference (72)]. Indeed, the overexpression of TXNIP made fibroblasts, cardiomyocytes, and pancreatic β-cells more susceptible to apoptosis. Glucose-caused β-cell death has been shown to be due to the stimulation of the overexpression of TXNIP by glucose (10). However, it should be pointed out that TXNIP is a member of the α-arrestin protein superfamily, and it does not only act as the inhibitor of Trx, but also emerges as a metabolic regulatory protein (74). TXNIP plays a dual role in apoptosis in redox-dependent (29, 51) and -independent regulatory (10, 12, 73) manners, which is reviewed by Spindel et al. (58). More recently, it is reported that the binding of Trx with TXNIP can prevent the TXNIP degradation and adipocyte differentiation (14, 75).

In agreement with an unknown role of Trx1 in embryogenesis, probably involving oxidative stress defense and transcription regulation, homozygotes with a targeted disruption of the mouse TXN gene died shortly after implantation, indicating that Trx1 is essential for early differentiation and morphogenesis of the mouse embryo (41).

Regulation of cell death by Trx2

Mitochondria are considered a major site for ROS production. ROS level is a determining factor for cell death. The low level of ROS promotes the cells into apoptosis, while the high level of ROS causes cell necrosis. Along with Prx3, the Trx2 system is a major player that controls ROS levels in mitochondria and, therefore, plays a key role in regulating cell apoptosis. The deficiency of Trx2 resulted in the elevation of cellular ROS level, cytochrome c release from mitochondria, and the activation of caspase 3 and 9 in chicken DT40 (61). Very interestingly, the transfection of hTrx2 or redox-inactive hTrx2CS can rescue the Trx2-deficeint DT40 from cell death. This may be due to the fact that Trx2 maintains the Bcl-xL protein level and controls the mitochondrial outer membrane permeabilization in a redox-active, site-independent mechanism (64). Trx2 can also bind and inhibit mitochondrial-located ASK1 apoptotic activity. Cys30 in the N-terminal domain of TXNIP is critical for the binding with Trx2 (76). Recently, it has been proposed that the Trx2-ASK1 signaling pathway also involves an intracellular shuttling of TXNIP. Under normal conditions, TXNIP is located in the cytosol and also primarily in the nucleus. Mitochondrial Trx2 is bound with ASK1 and inhibits its protein kinase activity. On oxidative stress, TXNIP translocates from the nucleus to the mitochondria, competes for the binding to Trx2, and results in the apoptotic process (Fig. 3) (55). Another possible mechanism of Trx2 protection against cell death is via the regulation of p66^Shc, a lifespan regulator (42). p66^Shc has been revealed to be a disulfide substrate of Trxs (18). Thus, the p66^Shc tetramer that is able to cause cell death can be reduced by the mitochondrial Trx system into the inactive form p66^Shc dimer (18).

Mitochondrial Trx2 is essential for the normal development of the mouse embryo. Homozymous mutants embryos with the silencing TXN2 gene showed massive increased apoptosis at 10.5 days postcoitus and died in the Theiler stage 15/16 (45). The timing of the embryonic lethality coincides with the maturation of the mitochondria. The embryonic fibroblasts from Trx2-null embryos were not viable. The heterozygous mice are fertile, but theTrx2 decrease mice showed increased oxidative damage to nuclear DNA, lipid, and proteins in the liver (49).

The role of Trx2 in the regulation of apoptosis has been demonstrated by the protection of Trx2 against oxidant-induced cell death. The liver from the Trx2^+/− mice has an increased apoptosis compared with wild type (WT) mice on diquat treatment (49). WT Trx2 overexpression, but not a C93S Trx2 mutant protein, can significantly inhibit TNF-α-induced apoptosis in HeLa cells (19). The redox statue of Trx2 is a key factor for many oxidant-induced cell death processes, and Trx2 get oxidized after the treatment of peroxides and diamide (11). Most recently, we found that some cationic triphenylmethanes such as brilliant green and gentian violet, the long time-used antifungal and antibacterial agents, accumulate in the mitochondria and cause the oxidation and degradation of Trx2. The disruption of the mitochondrial Trx system resulted in a subsequent release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol. Very interestingly, HeLa cells were more sensitive to brilliant green than to fibroblasts. In HeLa cells, Trx2 down-regulation by small interfering RNA (siRNA) resulted in an increased sensitivity, whereas for normal fibroblasts, the Trx2 or Trx1 down-regulation had no effects (77).

Besides Trx1 and Trx2, the Trx family contains many other thiol-disulfide oxidoreductases with a CXXC active site such as Grxs, an endoplasmic reticulum-localized transmembrane Trx-related protein, and a macrophage migration inhibitory factor. These proteins have also been indicated as playing critical roles in the regulation of apoptosis, which have been summarized in reviews (32, 62, 72).

Role of TrxRs in Mammalian Cell Death Progression

Structure and reaction mechanism of mammalian TrxR

Three TrxRs, cytosolic TrxR1, mitochondrial TrxR2, and testis-specific Trx glutathione reductase (GR) have been found in mammalian cells, corresponding to the localization of three types of Trxs (31, 40, 59). All the three mammalian TrxRs are selenoenzymes, with a selenocysteine (Sec, U) residue in their C-terminus (4, 37). Sec is known to be the 21st genetically translated amino acid that is encoded by a UGA codon, which, in most circumstances, acts as stop codon. The Sec incorporation into the selenoprotein polypeptide chain requires a complex machinery containing the Sec insertion sequence (SECIS) element, tRNA^[Ser]Sec, SECIS binding protein 2 (SBP2), and other components in mammalian cells and the availability of selenium (46). The Sec incorporation efficiency affects the activity of selenoprotein TrxR (56). The deficiency of Sec synthesis machinery can also change the existing form of TrxR (39). For example, selenium deficiency causes the Sec residue to be replaced by Cys in the rat liver TrxR (39). On the other hand, the Trx system can reduce disulfide bonds and/or glutathione (GSH) mixed disulfides in oxidized SBP2, and participate in the regulation of SBP2 compartmental location and selenoprotein synthesis efficiency (47).

TrxRs from mammalian cells possess different properties as compared with the TrxR from E. coli, yeast, or plants (Fig. 2) (69). The TrxR from mammalian cells and higher eukaryotes are large homodimmeric flavoproteins with 55 kDa or larger subunits, instead of the bacterial TrxRs, which are smaller dimeric flavoproteins with 33 kDa subunits (69). Mammalian TrxRs have a very broad range of substrates, including selenite, which could be reduced to selenide for selenoprotein synthesis (34, 36). In contrast, TrxRs in prokaryotes, yeast, and plants have a narrow substrate specificity (36).

In contrast to the single active site with a CXXC motif in the dimeric bacterial TrxR, mammalian TrxRs are a head to tail dimer and have an N-terminal active site disulfide CVNVGC as well as a conserved C-terminal sequence Gly-Cys-Sec-Gly (78). The structures of the mammalian TrxRs have been solved by X-ray crystallography (7, 13, 17, 54). The overall structure of TrxR is similar to that of GR with an identical N-terminal active site, NADPH and flavin adenine dinucleotide (FAD) binding domains (Fig. 2). However, mammalian TrxR1 uniquely contains a 16-residue C-terminal tail. Mechanisms of the action of mammalian TrxR have been proposed that electrons from NADPH are transferred to the N-terminal redox-active dithiols via FAD, then to the selenenylsulfide of the other subunit, and, subsequently, to the substrates (Fig. 4) (13, 78, 79). Crystal structures of oxidized and NADPH-reduced mouse TrxR2 have shown a similar overall structure as that of rat TrxR1 (7).

FIG. 4.

Inhibition of TrxR can convert TrxR to be a source for ROS production. As a unique Sec-containing flavoprotein, TrxR is a very suitable target for drug development. The C-terminal active site Sec makes the enzyme highly reactive with electronphilic agents due to its low pKa value and easily accessible location. The inhibition of TrxR does not only decrease the activity of TrxR but also makes TrxR become an ROS production site and results in the oxidation of Trx. ROS, reactive oxygen species.

Role of TrxRs in mammalian cell death progression

To elucidate the roles of TrxR in vivo, several TrxR knock-out models have been studied (8, 15, 27). Mice with a ubiquitous inactivation of TrxR2 were dead at embryonic day 13 (15). TrxR2-deficient embryologic fibroblasts grew slower than their WT counterparts and were highly sensitive to buthionine sulfoximine treatment to block GSH synthesis (15). The inactivation of TrxR1 also led to early embryonic lethality around E9.5 (8) or E10.5 (27). Surprisingly, mice with a heart-specific inactivation of TrxR1 developed normally and appeared healthy (27), and the mice with TrxR-null hepatocyte also survived for more than 1 year (52, 60). These results indicate that TrxR1 and TrxR2 are critical in embryo development, but they are not essential for cell proliferations. The nonessential role of TrxR1 in cell proliferation is consistent with the observation that the down-regulation of TrxR1 by siRNA did not change Trx1 redox status and caused cell toxicity (66, 77). The mechanistic details are unknown as yet.

One reason that TrxR is not essential for cell proliferation may be due to the compensatory effects of another disulfide reductase system, GSH and Grx system, and this cellular response process may be regulated by the Nrf2 pathway (60). The decrease in TrxR1 activity resulting in Nrf2 activation was also observed under other conditions such as selenium deficiency (Fig. 5) (9, 39). Grxs are Trx-fold proteins with a CPYC or CGFS active site motif. In the GSH-Grx system, electrons transfer from NADPH to GR, then to GSH, and, subsequently, to Grx. The functions of Trx and GSH-Grx systems are partially overlapping (35). In particular, when TrxR is absent, Trx may get the electrons from the GSH-Grx system to maintain its reduced status (Y. Du, J. Lu, A. Holmgren, unpublished results).

FIG. 5.

Compensation of GSH-Grx system under the conditions of the deficiency of TrxR1. The activity of TrxR1 can be decreased under various conditions such as the inhibition by inhibitors, selenium deficiency, Sec synthesis machinery such as tRNA^[Ser]Sec, SECIS binding protein 2 mutations, and siRNA treatments. The decrease in TrxR1 activity always results in the Nrf2 pathway activation and compensatory effects from the GSH-Grx system. Grx, glutaredoxin; GSH, glutathione; SECIS, Sec insertion sequence; siRNA, small interfering RNA.

Regulation of Activity in Trx System and Its Medical Applications

Mammalian TrxR as an anticancer target

Though TrxR1 is not essential for cell proliferation, TrxR emerges as a new target for anticancer drug development, because many malignant cells appear to be more dependent on an efficient Trx system. TrxR and Trx are overexpressed in aggressive cancers (5). The correlation between the Trx system and cancer hallmarks has been reviewed (5). Moreover, mouse lung carcinoma cells with TrxR stable-knockdown exhibited a similar cell morphology and anchorage-independent growth property as normal cells (71). The cells transfected with the siTrxR1 construct grew in a monolayer, whereas the control cancer cells grew in a multilayer and loosely attached to the culture dish. Growth of the TrxR1-knockdown cells in soft agar was inhibited compared with control cells (71). Growing unanchored in soft agar is a characteristic of many malignant cells (71). When these TrxR-knockdown cells were injected into mice, the tumor progression and metastasis were dramatically reduced (71). The requirement of protein disulfide reductase for tumor growth was originally reported by Apffel et al. (2), even before the identification of the enzymes to be Trx and TrxR by Holmgren (23). These results strongly suggest that TrxR is critical for cancer cell growth in vivo. In addition, a main potential substrate of the Trx system, RNR, is also a well-known anticancer target. The blockage of the Trx system activity will decrease RNR activity (43). More importantly, the inhibition of TrxR does not only decrease the activity of TrxR but also results in the elevation of ROS production and the oxidation of Trx (Fig. 5).

As a unique Sec-containing flavoprotein, TrxR is very suitable as a target for drug development. The C-terminal active site Sec makes the enzyme highly reactive with electrophilic agents due to its low pKa value and easily accessible location. Indeed, many TrxR inhibitors selectively exhibit the inhibition of TrxR against GR (3). This is also verified in vivo (65). Moreover, the inhibition of TrxR may result in a modified TrxR, which induces rapid cell death (1). Indeed, many clinically used anticancer compounds (3, 63), including alkylating and platinum-containing drugs, arsenic trioxide (35), and chemoprevention agents such as flavonoids (38) and curcumin (16), have been found to possess TrxR inhibitory effects. However, since TrxR is involved in a wide range of cell activities, the inhibition of TrxR may also cause some side effects in normal cells. Thus, further investigation that elucidates the different roles of TrxR along with Trx in normal and cancer cells and the reaction mechanism of TrxR inhibitors may facilitate and enlarge the therapeutic windows and selectivity in treatment.

Principles for some TrxR inhibition reactions

We have classified TrxR inhibitors as four types (36) (Fig. 6). The first type is metal- or metalloid-containing compounds, including gold, platinum-, mercury-, arsenic-containing compounds, some other organometallic complexes, and so on. “Hard and soft acids and bases” theory provides a useful principle for understanding the reaction mechanism between these compounds with TrxR (28). Since Se²⁻ is a softer base than S²⁻, it reacts preferentially with soft bases such as gold(I), platinum(II), mercury(II), and arsenic(III). This principle may also explain why Sec-containing TrxR shows a priority to react with arsenic compounds than Cys-containing proteins. Though the metal- or metalloid-containing compounds have a high priority to attack the C-terminal Sec, the N-terminal CVNVGC active site in TrxR is also involved in the reaction (35). Zinc or lead compounds only show their inhibitory effects in the absence of EDTA in vitro. In cells or in vivo, they do not display the selective inhibition of TrxR as arsenic and mercury compounds do. The reason may be that arsenic and mercury compounds are softer acids and, thus, have a higher affinity with TrxR. For this type of inhibitors, one interesting observation is that some inhibitor has the activity to cross-link TrxR with Trx or other small proteins (50).

FIG. 6.

Classification of TrxR inhibitors and general principles for the inhibition reaction. Numerous compounds exhibit inhibitory effects on mammalian TrxR. We have classified TrxR inhibitors as four types and list some general principles to understand the inhibition mechanism in the right panel. “Hard and soft acids and bases” theory is a good principle that explains the reaction between type I metal- or metalloid- containing compounds and TrxR, because R-Se⁻ is a soft base and reacts preferentially with soft bases, including gold, platinum-, mercury-, and arsenic-containing compounds. The reaction of type II Michael acceptors with TrxR is closely linked to ROS production. The inhibition of TrxR with many flavonoids myricetin, quercetin, quinols, and quinones may involve a direct reaction between the TrxR and semiquinone. Some quinones may act as “suicide inhibitors.” They can be reduced by TrxR, and, at the same time, the reduced product semiquinones inhibit TrxR. Type III inhibitors are sulfur, selenium, or telluride-containing compounds. BBSKE, a compound structurally similar to the bridge combination of two ebselen compounds, is a reversible inhibitor on TrxR. The type IV compound DNCB is an aromatic alkylation agent. This compound can form a covalent bond with C-terminal Sec and convert the antioxidant enzyme into a pro-oxidant. BBSKE, 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane; DNCB, dinitrochlorobenzene.

The second type of TrxR inhibitors are Michael acceptors (α, β-unsaturated carbonyl compounds), including flavonoids, quinols, quinines, and so on. The Michael acceptor curcumin has an α, β-unsaturated ketone structure that is equilibrated with its enol form. The latter is a highly active electrophilic agent that can have a Michael conjugation with the selenide of Sec in TrxR (16). The inhibition of TrxR by the flavonoids myricetin, quercetin, quinols, and quinones may involve a direct reaction between the TrxR and semiquinone (38). This may also indicate that many quinones act as “suicide inhibitors.” They can be reduced by TrxR, and, at the same time, the reduced product semiquinones inhibit TrxR.

The third type is sulfur, selenium, or telluride-containing compounds. The inhibition of TrxR by 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane (BBSKE) is reversible, which is different from most of the other inhibitors. The fourth type is alkylation agents. Dinitrochlorobenzene is an aromatic alkylation agent. This compound can have a covalent bonding with C-terminal Sec and also converts the antioxidant enzyme into a pro-oxidant (Fig. 4).

Concluding Remarks

In summary, the mammalian Trx system is obviously a key player in cell death for a large number of cellular activities they are involved in. The redox status of Trx is a determining factor for the cell death, which is controlled by TrxR activity and the cellular ROS level. Mammalian TrxR is a suitable anticancer target due to its unique structural properties. Various types of compounds can exert inhibitory effects on TrxR. The challenges are how to design and synthesize the compounds to possess higher selectivity related to the inhibitory efficiency and ROS production capacity via TrxR.

Footnotes

Acknowledgments

The authors acknowledge the support from the Swedish Research Council Medicine (3529), the Swedish Cancer Society (961), the K.A. Wallenberg Foundation, Åke Wiberg Stiftelse, and the Karolinska Institutet.

Abbreviations Used

References

Anestal

, Arnér

. Rapid induction of cell death by selenium-compromised thioredoxin reductase 1 but not by the fully active enzyme containing selenocysteine. J Biol Chem, 278:15966–15972. 2003.

Apffel

, Walker

, Nathanson

. Tumor growth and disulfide reduction: possible dependence on protein-disulfide reductase. J Natl Cancer Inst, 51:575–583. 1973.

Arnér

. Focus on mammalian thioredoxin reductases—important selenoproteins with versatile functions. Biochim Biophys Acta, 1790:495–526. 2009.

Arnér

, Holmgren

. Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem, 267:6102–6109. 2000.

Arnér

, Holmgren

. The thioredoxin system in cancer. Semin Cancer Biol, 16:420–426. 2006.

Avval

, Holmgren

. Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for Mammalian s phase ribonucleotide reductase. J Biol Chem, 284:8233–8240. 2009.

Biterova

, Turanov

, Gladyshev

, Barycki

. Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism. Proc Natl Acad Sci U S A, 102:15018–15023. 2005.

Bondareva

, Capecchi

, Iverson

, Li

, Lopez

, Lucas

, Merrill

, Prigge

, Siders

, Wakamiya

, Wallin

, Schmidt

. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome. Free Radic Biol Med, 43:911–923. 2007.

Burk

, Hill

, Nakayama

, Mostert

, Levander

, Motley

, Johnson

, Freeman

, Austin

. Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not. Free Radic Biol Med, 44:1617–1623. 2008.

10.

Chen

, Hui

, Couto

, Mungrue

, Davis

, Attie

, Lusis

, Davis

, Shalev

. Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes. FASEB J, 22:3581–3594. 2008.

11.

Chen

, Cai

, Jones

. Mitochondrial thioredoxin in regulation of oxidant-induced cell death. FEBS Lett, 580:6596–6602. 2006.

12.

Chen

, Lopez-Ramos

, Yoshihara

, Maeda

, Masutani

, Sugie

, Maeda

, Yodoi

. Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation. Leukemia, 25:440–448. 2011.

13.

Cheng

, Sandalova

, Lindqvist

, Arnér

. Crystal structure and catalysis of the selenoprotein thioredoxin reductase 1. J Biol Chem, 284:3998–4008. 2009.

14.

Chutkow

, Lee

. Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability. J Biol Chem, 286:29139–29145. 2011.

15.

Conrad

, Jakupoglu

, Moreno

, Lippl

, Banjac

, Schneider

, Beck

, Hatzopoulos

, Just

, Sinowatz

, Schmahl

, Chien

, Wurst

, Bornkamm

, Brielmeier

. Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function. Mol Cell Biol, 24:9414–9423. 2004.

16.

Fang

, Lu

, Holmgren

. Thioredoxin reductase is irreversibly modified by curcumin: A novel molecular mechanism for its anticancer activity. J Biol Chem, 280:25284–25290. 2005.

17.

Fritz-Wolf

, Kehr

, Stumpf

, Rahlfs

, Becker

. Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat Commun, 2:383. 2011.

18.

Gertz

, Fischer

, Wolters

, Steegborn

. Activation of the lifespan regulator p66Shc through reversible disulfide bond formation. Proc Natl Acad Sci U S A, 105:5705–5709. 2008.

19.

Hansen

, Zhang

, Jones

. Mitochondrial thioredoxin-2 has a key role in determining tumor necrosis factor-alpha-induced reactive oxygen species generation, NF-kappaB activation, and apoptosis. Toxicol Sci, 91:643–650. 2006.

20.

Hashemy

, Holmgren

. Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues. J Biol Chem, 283:21890–21898. 2008.

21.

Hirota

, Matsui

, Iwata

, Nishiyama

, Mori

, Yodoi

. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci U S A, 94:3633–3638. 1997.

22.

Hirota

, Murata

, Sachi

, Nakamura

, Takeuchi

, Mori

, Yodoi

. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kappaB. J Biol Chem, 274:27891–27897. 1999.

23.

Holmgren

. Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. J Biol Chem, 252:4600–4606. 1977.

24.

Holmgren

. Thioredoxin. Annu Rev Biochem, 54:237–271. 1985.

25.

Holmgren

, Lu

. Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun, 396:120–124. 2010.

26.

Holmgren

, Sengupta

. The use of thiols by ribonucleotide reductase. Free Radic Biol Med, 49:1617–1628. 2010.

27.

Jakupoglu

, Przemeck

, Schneider

, Moreno

, Mayr

, Hatzopoulos

, de Angelis

, Wurst

, Bornkamm

, Brielmeier

, Conrad

. Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development. Mol Cell Biol, 25:1980–1988. 2005.

28.

Jungwirth

, Kowol

, Keppler

, Hartinger

, Berger

, Heffeter

. Anticancer activity of metal complexes: involvement of redox processes. Antioxid Redox Signal, 15:1085–1127. 2011.

29.

Junn

, Han

, Im

, Yang

, Cho

, Um

, Kim

, Lee

, Han

, Rhee

, Choi

. Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function. J Immunol, 164:6287–6295. 2000.

30.

Kang

, Rhee

, Chang

, Jeong

, Choi

. 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol Med, 11:571–578. 2005.

31.

Lee

, Kim

, Kwon

, Yoon

, Levine

, Ginsburg

, Rhee

. Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver. J Biol Chem, 274:4722–4734. 1999.

32.

Lillig

, Holmgren

. Thioredoxin and related molecules-from biology to health and disease. Antioxid Redox Signal, 9:25–47. 2007.

33.

Liu

, Min

. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner. Circ Res, 90:1259–1266. 2002.

34.

, Berndt

, Holmgren

. Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase. Biochim Biophys Acta, 1790:1513–1519. 2009.

35.

, Chew

, Holmgren

. Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide. Proc Natl Acad Sci U S A, 104:12288–12293. 2007.

36.

, Holmgren

. Selenoprotein and the thioredoxin system. Selenium: Its Molecular Biology and Role in Human Health. Hatfield

, Berry

, Gladyshev

. New York: Springer Verlag, 2011; 153–165.

37.

, Holmgren

. Selenoproteins. J Biol Chem, 284:723–727. 2009.

38.

, Papp

, Fang

, Rodriguez-Nieto

, Zhivotovsky

, Holmgren

. Inhibition of Mammalian thioredoxin reductase by some flavonoids: implications for myricetin and quercetin anticancer activity. Cancer Res, 66:4410–4418. 2006.

39.

, Zhong

, Lonn

, Burk

, Hill

, Holmgren

. Penultimate selenocysteine residue replaced by cysteine in thioredoxin reductase from selenium-deficient rat liver. FASEB J, 23:2394–2402. 2009.

40.

Luthman

, Holmgren

. Rat liver thioredoxin and thioredoxin reductase: purification and characterization. Biochemistry, 21:6628–6633. 1982.

41.

Matsui

, Oshima

, Takaku

, Maruyama

, Yodoi

, Taketo

. Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev Biol, 178:179–185. 1996.

42.

Migliaccio

, Giorgio

, Mele

, Pelicci

, Reboldi

, Pandolfi

, Lanfrancone

, Pelicci

. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature, 402:309–313. 1999.

43.

Muniyappa

, Song

, Mathews

, Das

. Reactive oxygen species-independent oxidation of thioredoxin in hypoxia: inactivation of ribonucleotide reductase and redox-mediated checkpoint control. J Biol Chem, 284:17069–17081. 2009.

44.

Nishiyama

, Matsui

, Iwata

, Hirota

, Masutani

, Nakamura

, Takagi

, Sono

, Gon

, Yodoi

. Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem, 274:21645–21650. 1999.

45.

Nonn

, Berggren

, Powis

. Increased expression of mitochondrial peroxiredoxin-3 (thioredoxin peroxidase-2) protects cancer cells against hypoxia and drug-induced hydrogen peroxide-dependent apoptosis. Mol Cancer Res, 1:682–689. 2003.

46.

Papp

, Lu

, Holmgren

, Khanna

. From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid Redox Signal, 9:775–806. 2007.

47.

Papp

, Lu

, Striebel

, Kennedy

, Holmgren

, Khanna

. The redox state of SECIS binding protein 2 controls its localization and selenocysteine incorporation function. Mol Cell Biol, 26:4895–4910. 2006.

48.

Patwari

, Higgins

, Chutkow

, Yoshioka

, Lee

. The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange. J Biol Chem, 281:21884–21891. 2006.

49.

Perez

, Lew

, Cortez

, Webb

, Rodriguez

, Liu

, Qi

, Li

, Chaudhuri

, Van Remmen

, Richardson

, Ikeno

. Thioredoxin 2 haploinsufficiency in mice results in impaired mitochondrial function and increased oxidative stress. Free Radic Biol Med, 44:882–892. 2008.

50.

Prast-Nielsen

, Cebula

, Pader

, Arner

. Noble metal targeting of thioredoxin reductase—covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin. Free Radic Biol Med, 49:1765–1778. 2010.

51.

Rani

, Mehta

, Barron

, Doolan

, Jeppesen

, Clynes

, O'Driscoll

. Decreasing Txnip mRNA and protein levels in pancreatic MIN6 cells reduces reactive oxygen species and restores glucose regulated insulin secretion. Cell Physiol Biochem, 25:667–674. 2010.

52.

Rollins

, van der Heide

, Weisend

, Kundert

, Comstock

, Suvorova

, Capecchi

, Merrill

, Schmidt

. Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration. J Cell Sci, 123:2402–2412. 2010.

53.

Saitoh

, Nishitoh

, Fujii

, Takeda

, Tobiume

, Sawada

, Kawabata

, Miyazono

, Ichijo

. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J, 17:2596–2606. 1998.

54.

Sandalova

, Zhong

, Lindqvist

, Holmgren

, Schneider

. Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. Proc Natl Acad Sci U S A, 98:9533–9538. 2001.

55.

Saxena

, Chen

, Shalev

. Intracellular shuttling and mitochondrial function of thioredoxin-interacting protein. J Biol Chem, 285:3997–4005. 2010.

56.

Schoenmakers

, Agostini

, Mitchell

, Schoenmakers

, Papp

, Rajanayagam

, Padidela

, Ceron-Gutierrez

, Doffinger

, Prevosto

, Luan

, Montano

, Lu

, Castanet

, Clemons

, Groeneveld

, Castets

, Karbaschi

, Aitken

, Dixon

, Williams

, Campi

, Blount

, Burton

, Muntoni

, O'Donovan

, Dean

, Warren

, Brierley

, Baguley

, Guicheney

, Fitzgerald

, Coles

, Gaston

, Todd

, Holmgren

, Khanna

, Cooke

, Semple

, Halsall

, Wareham

, Schwabe

, Grasso

, Beck-Peccoz

, Ogunko

, Dattani

, Gurnell

, Chatterjee

. Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans. J Clin Invest, 120:4220–4235. 2010.

57.

Sengupta

, Holmgren

. The role of thioredoxin in the regulation of cellular processes by S-nitrosylation. Biochim Biophys Acta, 1820:689–700. 2012.

58.

Spindel

, World

, Berk

. Thioredoxin interacting protein: redox dependent and independent regulatory mechanisms. Antioxid Redox Signal, 16:587–596. 2012.

59.

Sun

, Wu

, Zappacosta

, Jeang

, Lee

, Hatfield

, Gladyshev

. Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J Biol Chem, 274:24522–24530. 1999.

60.

Suvorova

, Lucas

, Weisend

, Rollins

, Merrill

, Capecchi

, Schmidt

. Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes. PLoS One, 4:e6158. 2009.

61.

Tanaka

, Hosoi

, Yamaguchi-Iwai

, Nakamura

, Masutani

, Ueda

, Nishiyama

, Takeda

, Wada

, Spyrou

, Yodoi

. Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis. EMBO J, 21:1695–1703. 2002.

62.

Thiele

, Bernhagen

. Link between macrophage migration inhibitory factor and cellular redox regulation. Antioxid Redox Signal, 7:1234–1248. 2005.

63.

Urig

, Becker

. On the potential of thioredoxin reductase inhibitors for cancer therapy. Semin Cancer Biol, 16:452–465. 2006.

64.

Wang

, Masutani

, Oka

, Tanaka

, Yamaguchi-Iwai

, Nakamura

, Yodoi

. Control of mitochondrial outer membrane permeabilization and Bcl-xL levels by thioredoxin 2 in DT40 cells. J Biol Chem, 281:7384–7391. 2006.

65.

Wang

, Zhang

, Xu

. Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo. Toxicol Appl Pharmacol, 218:88–95. 2007.

66.

Watson

, Heilman

, Hughes

, Spielberger

. Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation. Biochem Biophys Res Commun, 368:832–836. 2008.

67.

Watson

, Pohl

, Montfort

, Stuchlik

, Reed

, Powis

, Jones

. Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. J Biol Chem, 278:33408–33415. 2003.

68.

Weissbach

, Etienne

, Hoshi

, Heinemann

, Lowther

, Matthews

, St John

, Nathan

, Brot

. Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function. Arch Biochem Biophys, 397:172–178. 2002.

69.

Williams

, Arscott

, Muller

, Lennon

, Ludwig

, Wang

, Veine

, Becker

, Schirmer

. Thioredoxin reductase two modes of catalysis have evolved. Eur J Biochem, 267:6110–6117. 2000.

70.

, Parrott

, Fu

, Liu

, Marino

, Gladyshev

, Jain

, Baykal

, Li

, Oka

, Sadoshima

, Beuve

, Simmons

, Li

. Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies. Antioxid Redox Signal, 15:2565–2604. 2011.

71.

Yoo

, Xu

, Carlson

, Gladyshev

, Hatfield

. Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J Biol Chem, 281:13005–13008. 2006.

72.

Yoshihara

, Chen

, Matsuo

, Masutani

, Yodoi

. Thiol redox transitions by thioredoxin and thioredoxin-binding protein-2 in cell signaling. Methods Enzymol, 474:67–82. 2010.

73.

Yoshihara

, Fujimoto

, Inagaki

, Okawa

, Masaki

, Yodoi

, Masutani

. Disruption of TBP-2 ameliorates insulin sensitivity and secretion without affecting obesity. Nat Commun, 1:127. 2010.

74.

Yoshioka

, Chutkow

, Lee

, Kim

, Yan

, Tian

, Lindsey

, Feener

, Seidman

, Lee

. Deletion of thioredoxin-interacting protein in mice impairs mitochondrial function but protects the myocardium from ischemia-reperfusion injury. J Clin Invest, 122:267–279. 2012.

75.

Zhang

, Wang

, Gao

, Wang

, Mao

, An

, Xu

, Wu

, Yu

, Liu

, Yu

. The ubiquitin ligase itch regulates apoptosis by targeting thioredoxin-interacting protein for ubiquitin-dependent degradation. J Biol Chem, 285:8869–8879. 2010.

76.

Zhang

, Al-Lamki

, Bai

, Streb

, Miano

, Bradley

, Min

. Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner. Circ Res, 94:1483–1491. 2004.

77.

Zhang

, Zheng

, Fried

, Du

, Montano

, Sohn

, Lefkove

, Holmgren

, Arbiser

, Holmgren

, Lu

. Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes. Free Radic Biol Med, 50:811–820. 2011.

78.

Zhong

, Arnér

, Holmgren

. Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc Natl Acad Sci U S A, 97:5854–5859. 2000.

79.

Zhong

, Holmgren

. Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. J Biol Chem, 275:18121–18128. 2000.