Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

Abstract

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.

Selenocysteine in Thiol/Disulfide-Like Exchange Reactions

“…The present data in the field indicates that the cytosolic TR is much more dependent on selenium for catalyzing the reduction of a diverse array of substrates (91). Furthermore, a recent study (65) showed that mitochondrial and cytosolic TRs present significantly different responses to metal compounds, for example, various gold(I) compounds were found to inhibit either one or the other (and not both) isozymes. The reason for this is still unclear: it may be that cytosolic and mitochondrial TRs have different local features (e.g., structure around their active sites, local flexibility, etc.)…”

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

“…(JUG), auranofin (AUR), myricetin (MYR), and curcumin (CUR) (Cai et al, 2012;Fang et al, 2005;Lu and Holmgren, 2012;Lu et al, 2006;Omata et al, 2006;Rackham et al, 2011). Figure 2B shows that the inhibition of the BoNT/A-mediated cleavage of SNAP25 by these inhibitors is dose dependent.…”

Glutaredoxin 2 Reduces Both Thioredoxin 2 and Thioredoxin 1 and Protects Cells from Apoptosis Induced by Auranofin and 4-Hydroxynonenal

“…However, both the cytosolic and mitochondrial forms of TrxR possess a super active selenocysteine that is very sensitive to inactivation by electrophilic compounds (35,44). Upon extensive inactivation of TrxR, Trx will not be reduced effectively, and increased levels of oxidized Trx will result in cell death.…”