Differences in substrate preferences and metal compound inhibition of cytosolic and mitochondrial human thioredoxin reductases

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In this study we made a side-by-side comparison of substrate and inhibition specificities of pure recombinant human TrxR2 and TrxR1 and found significant differences between the two human enzymes that were previously not observed and are beyond those determined solely by their different subcellular compartments. The divergent sensitivities of TrxR1 and TrxR2 to different inhibitors should underpin inhibitor-specific differences in cytotoxicity profiles of metal-based anticancer drugs that target cellular TrxR as part of their molecular mechanisms for toxicity.

Although we used established protocols for recombinant expression of TrxRs [48], this is the first study comparing the two human isoenzymes and the first to express full-length recombinant human TrxR2. Earlier attempts to express recombinant human TrxR1 gave much lower yields than expressing rat TrxR1, which was explained by higher frequency of rare codon usage in the human gene and a higher instability of the human enzyme [69, 70], which we also noted. We were surprised for the near-complete enrichment of the full-length enzymes following PAO Sepharose purification, considering that earlier studies found rat TrxR1 to be purified as heterodimers of truncated and full-length subunits still displaying a specific activity of 40 U/mg or more [41]. That suggests that homodimeric full length rat TrxR1 would have significantly higher specific activity than 40 U/mg [41], while we found that homogeneously full-length human TrxR1 and TrxR2 had specific activities of about 40 U/mg in the DTNB model assay, which is the commonly denoted inherent activity of mammalian TrxR [50].

Human TrxR2 had a clear preference for its native Trx2 substrate and its catalytic efficiency was higher at pH 8, indicating that the mitochondrial localization of TrxR2, where the pH is generally one unit higher compared to the cytosol, has tailored its differences in affinity and activity for its natural substrate Trx2. Importantly, the differences in catalytic efficiency between the mitochondrial and cytosolic TrxRs as found here should have implications for their intracellular functions. It is clear that TrxR1 can reduce the mitochondrial Trx2 equally well as its native Trx1, as observed before for Trxs from other species [43],  but the question remains whether TrxR1 would ever have the possibility to reduce Trx2 in vivo considering the different subcellular compartments of the two proteins.

Human TrxR1 clearly reduced DTNB and lipoamide more efficiently than TrxR2, while the selenenylsulfide active center motif of TrxR2 was not essential for their reduction as shown before for mouse TrxR2 [21], but in contrast to that study, we found that human TrxR2 could not reduce lipoic acid. Although lipoic acid is a substrate for human TrxR1 we still found that its efficiency was two-fold lower compared to that for lipoamide. Endogenous lipoic acid is a covalently bound dithiol cofactor for the α-keto acid dehydrogenase enzyme complexes, such as pyruvate dehydrogenase and α-ketoglutarate dehydrogenase [71] and is negatively charged compared to the neutral charge of lipoamide. This charge difference should play an important role in substrate discrimination for human TrxR2 and in its reduced efficiency of lipoic acid reduction, as compared to the lipoamide reduction by human TrxR1. It may be thus that in humans lipoic acid is not reduced by TrxR2 and that other enzymes such as lipoamide dehydrogenase and glutathione reductase catalyze this reaction [72].

We found that human TrxR1 had a three-fold higher affinity for the quinone substrate juglone compared to TrxR2 and the TrxR2D suggesting that the N-terminal motif alone can reduce juglone. Juglone can be rapidly reduced by the selenenylsulfide/ selenolthiol active center of rat TrxR1, which can result in the formation of a nucleophilic arylating juglone derivative that can target selenocysteine and irreversibly inactivate this motif. Nevertheless, juglone may continue to efficiently redox cycle directly with the N-terminal CVNVGC/FAD motif of rat TrxR1 and thus show high activity [13, 26, 73].

Our kinetic data indicate that the affinity of TrxR2 for disulfide substrates other than its endogenous Trx2 substrate is significantly lower than the affinity of TrxR1 for these types of substrates. Because of its mitochondrial localization it may obviously still be that TrxR2 is more efficient at using endogenous mitochondria localized substrates such as Trx2, Grx2 and cytochrome c [37, 38] and there may also be other mitochondria specific substrates for which TrxR2 has high affinity yet to be discovered. Our results suggest that the functions of the mitochondrial TrxR2 and cytosolic TrxR1 are less overlapping than generally believed, also when considering their native biochemical and kinetic properties. The previously reported crystal structure of mitochondrial TrxR2 indeed indicates that there are distinct differences in the positioning of residues around the redox active centers of TrxR2 as compared to TrxR1, which may contribute to or be responsible for the divergent reduction of different substrates as well as their affinities [16]. From our data we conclude that, (i) TrxR1 has a broader substrate specificity compared to TrxR2, (ii) TrxR2 has greater affinity for its endogenous substrates that are different to those for TrxR1, which may reflect other functional requirements within the mitochondrial compartment, and (iii) hTrxR2 is catalytically more efficient at the higher endogenous pH of the mitochondrial matrix.

Several emerging anticancer therapies identify TrxR as a target for drug development [74, 75] as altered activities of the Trx system proteins have been observed in several human diseases [18, 76]. Many therapeutically used compounds have been identified as TrxR inhibitors [18, 26, 73, 76-78], including gold(I) compounds that have also shown early promise as anticancer drugs [45, 46, 60, 74, 77, 79, 80]. Auranofin is probably the most effective inhibitor of mammalian TrxR found to date [60, 81] and here we show that at micromolar concentrations it is as an effective inhibitor of both the N-terminal dithiol and C-terminal selenolthiol redox centres of human TrxRs. This indicates that auranofin has high affinity not only for selenocysteines but thiolates as well, suggesting that auranofin has a limited specificity for selenocysteines. The finding that the related compound, aurothioglucose also inhibited all three enzymes was in contrast to a recent finding that it is a better inhibitor of the N-terminal redox center compared to the C-terminal selenenylsulfide/selenolthiol active center of the mouse TrxR2 [21]. These findings may reflect differences between the human and mouse TrxR2 enzymes. Interestingly, at nanomolar concentrations auranofin and aurothioglucose were more specific inhibitors of TrxR1 compared to TrxR2 and less effective at inhibiting TrxR2D, suggesting that the selenolthiol motif of TrxR is inhibited in preference to the dithiol motif. Also, the active site microenvironment around the C-terminal selenolthiol motif of the reduced enzyme must finetune its susceptibility to different inhibitors, as illustrated by the differences in inhibition between TrxR1 and TrxR2. Thus, the tetrapeptide C-terminal active site of TrxR should not only be regarded as an easily accessible Sec-presenting motif targeted indiscriminately by electrophiles, which is a rather simplified view often found in the literature. Cisplatin inhibited the activity of TrxR1 and TrxR2, but not TrxR2D, suggesting that its mechanism of inhibition likely involves coordination of platinum to the Sec-containing redox center. This was suggested to generate selenium compromised thioredoxin reductase-derived apoptotic proteins (SecTRAPs) that may induce cell death by a gain of function [56]. The bis-chelated Au(I) pyridyl phosphine compounds were inspired by auranofin, but designed to lower their thiol reactivities while improving the selectivity for Sec [62, 63]. Consequently we found that the bis-chelated gold(I) compounds [Au(d2pype)₂]Cl and [Au(d2pypp)₂]Cl were effective inhibitors of the selenenylsulfide/ selenolthiol active center of the TrxRs, thus potentially also yielding cytotoxic SecTRAP proteins. However, such proteins have not been shown to form from TrxR2 yet.

The cationic gold(I) compounds could inhibit both TrxR1 and TrxR2 in vitro, however because they are lipophilic and are consequently accumulated inside mitochondria as a result of the high mitochondrial membrane potential (Dy_m), we observed their specific inhibition of TrxR2 in cells and conclude that previously observed cellular TrxR inhibition [46] also likely involved inhibition of TrxR2. Surprisingly, the [Au(d2pype)₂]Cl and [Au(d2pypp)₂]Cl compounds were significantly better inhibitors for TrxR1 in vitro whereas [(iPr₂Im)₂Au]Cl inhibited only TrxR2. This indicates not only that there is a difference in the inhibition mechanism of these compounds, but also that the active centres of the cytosolic and mitochondrial TrxR isoenzymes may have different affinities for these compounds. We are currently attempting to investigate the mechanism of binding of these gold(I) compounds by co-crystallizing them with the purified TrxRs.

At low concentration auranofin led to Bax/Bak dependent cell death likely by preferentially reacting with selenoproteins such as TrxR2 whose inhibition by auranofin has been shown to lead to peroxiredoxin-3 oxidation and Bax/Bax dependent apoptosis [57]. High concentrations of auranofin caused cell death independent of Bax/Bak, most likely by binding both thiols and selenols, causing changes in the mitochondrial thiol redox pool that have been shown to cause apoptosis by inducing mitochondrial permeability transition (MPT) [60, 81]. Therefore the mechanism of action of auranofin via the Bax/Bak pore is concentration dependent and may vary between cell types. Nevertheless, the high thiol reactivity of auranofin is likely to limit its anticancer activity in vivo as its toxicity to cultured cancer cells was reduced 10-fold in the presence of serum proteins, where the loss of activity was attributed to binding to extracellular protein thiols [82]. Although aurothioglucose was an effective inhibitor of recombinant TrxRs, its inhibition of cellular TrxRs was limited and it was not toxic to the two cell lines, most likely because polymeric gold(I) thiolates do not readily enter cells and consequently have very low cytotoxicity and lack anticancer activity [83]. In contrast, cell death induced by cisplatin was found to be entirely dependent of the presence of Bax and Bak, suggesting that TrxR inhibition and/or DNA binding could lead to Bax/Bak dependent cell death. Although the exact mechanism of cisplatin-induced cell death needs to be investigated further, the combined ability of cisplatin to form DNA lesions and inhibit TrxR activity make it an effective chemotherapeutic agent, and may contribute to its success as a clinically used anticancer drug.

The lipophilic cations [Au(d2pypp)₂]Cl and in particular [Au(d2pype)₂]Cl were more TrxR1-specific inhibitors and caused cell death that was independent of the Bax and Bak proteins. We have previously shown that these compounds cause cell death via mitochondria [46] and this may be a result of their high lipophilicity, that can cause generalized permeabilization of the mitochondrial membrane or induce the MPT that dissipates the Dy_m. In addition, a small amount of these compounds may remain in the cytoplasm, as we observed some inhibition of TrxR1 activity that may further contribute to the onset of cell death independent of Bax and Bak. In contrast to these compounds, the [(iPr₂Im)₂Au]Cl compound led to Bax/Bak dependent cell death at lower concentrations. We have shown previously that [(iPr₂Im)₂Au]Cl is a moderately lipophilic cation that selectively accumulates in the mitochondrial matrix driven by the Dy_m [45] and here we showed that it is a TrxR2-specific inhibitor. We suggest that the mechanism of action of this compound involves mitochondrial uptake followed by specific inhibition of the TrxR2 that leads to apoptosis via mitochondria which requires the formation of the Bax/Bak pore. However, at higher concentrations this compound can cause cell death independent of the Bax/Bak pore, much like [Au(d2pype)₂]Cl and [Au(d2pypp)₂]Cl, most likely as a result of its lipophilicity. An alternative explanation may be that the compounds are irreversibly modifying the Sec active site and thereby forming SecTRAPs that induce cell death. Cell death induced by different SecTRAPs may be caused by different mechanisms that may not always require the Bax and Bak proteins. The molecular mechanisms by which the inhibition of TrxRs leads to cell death and whether it requires Bax/Bak pore formation must evidently be specifically studied and resolved for every individual TrxR-targeting drug and for each specific cell type.

In conclusion, we have shown that the human cytosolic TrxR1 and mitochondrial TrxR2 have different affinities for low molecular-weight disulfide substrates and metal-based inhibitors. In combination with cell death assays in Bax/Bak double knockout cells our data enable models to be proposed for the mechanisms of action of these compounds that take into account the physiologically relevant cellular locations of TrxR1 and TrxR2, their compound-specific targeting profiles and the patterns of inhibition determined by different kinetic properties of TrxR1 and TrxR2.