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

Western Australian Institute for Medical Research, Centre for Medical Research, University of Western Australia, Perth, WA 6000, Australia.
Free Radical Biology and Medicine (Impact Factor: 5.74). 03/2011; 50(6):689-99. DOI: 10.1016/j.freeradbiomed.2010.12.015
Source: PubMed


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.

Download full-text


Available from: Oliver Rackham
    • "In fact, SeCys-decient variants of TrxR1 reduce Fe(III)(Tp) 2 faster than wild-type TrxR1[45]. The Cys59 motif of TrxR1 is important for Fe(III)thiosemicarbazone reduction[45], and higher concentrations of ANF might block this activity by binding this motif[63]. Regardless of which TrxR motifs are targeted by ANF, however, ANF markedly enhances the cytotoxicity of Tp in A549 cells (Fig. 7), and blockade of either the SeCys or Cys59 will compromise the ability of TrxR to support Prx. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Peroxiredoxin-3 (Prx3) accounts for about 90% of mitochondrial peroxidase activity, and its marked upregulation in many cancers is important for cell survival. Prx3 oxidation can critically alter peroxide signaling and defense and can be a seminal event in promoting cell death. Here it is shown that this mechanism can be exploited pharmacologically by combinations of clinically available drugs that compromise Prx3 function in different ways. Clinically relevant levels of the thiosemicarbazone iron chelators triapine (Tp) and 2,2'-Dipyridyl-N,N-dimethylsemicarbazone (Dp44mT) promote selective oxidation of mitochondrial Prx3, but not cytosolic Prx1, in multiple human lung and ovarian cancer lines. Decreased cell survival closely correlates with Prx3 oxidation. However, Prx3 oxidation is not merely an indicator of cell death as cytotoxic concentrations of cisplatin do not cause Prx3 oxidation. The siRNA-mediated suppression of either Prx3 or thioredoxin-2, which supports Prx3, enhances Tp's cytotoxicity. Tp-mediated Prx3 oxidation is driven by enhanced peroxide generation, but not by nitric oxide. Many tumors overexpress thioredoxin reductase (TrxR) which supports Prx activity. Direct inhibitors of TrxR (e.g. auranofin, cisplatin) markedly enhanced Tp's cytotoxicity, and auranofin enhanced Prx3 oxidation by low dose Tp. Together, these results support an important role for Prx3 oxidation in the cytotoxicity of Tp, and demonstrate that TrxR inhibitors can significantly enhance Tp's cytotoxicity. Thiosemicarbazone-based regimens could prove effective for targeting Prx3 in a variety of cancers.
    No preview · Article · Dec 2015 · Free Radical Biology and Medicine
  • Source
    • "(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 "
    [Show abstract] [Hide abstract]
    ABSTRACT: During cellular uptake, diphtheria toxin delivers its catalytic domain DTA from acidified endosomes into the cytosol, which requires reduction of the disulfide linking DTA to the transport domain. In vitro, thioredoxin reduces this disulfide and thioredoxin reductase (TrxR) is part of a cytosolic complex facilitating DTA-translocation. We found that the TrxR-specific inhibitor auranofin prevented DTA delivery into the cytosol and intoxication of HeLa cells with diphtheria toxin, offering perspectives for novel pharmacological strategies against diphtheria. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Apr 2015 · Toxicon
  • Source
    • "(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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.
    Full-text · Article · Sep 2014 · Cell Reports
Show more