Article

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: 5.71). 03/2011; 50(6):689-99. DOI: 10.1016/j.freeradbiomed.2010.12.015
Source: PubMed

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.

Download full-text

Full-text

Available from: Oliver Rackham, Aug 23, 2015
0 Followers
 · 
160 Views
  • 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.
    Toxicon 04/2015; DOI:10.1016/j.toxicon.2015.04.012 · 2.58 Impact Factor
  • 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.
    Cell Reports 09/2014; 8(6). DOI:10.1016/j.celrep.2014.08.017 · 8.36 Impact Factor
  • Source
    • "It is interesting to note that over-expression of Cys-TR2 tended to enhance viability under low menadione concentrations, yet this effect was not statistically significant. Two factors may contribute to the ability of Cys-TR2 to support viability: (1) mitochondrial and cytosolic redox potentials differ [50], and (2) the substrate specificity for TR1 and TR2 differs [51]. Moreover, co-expression of TRX1 or TRX2 with its cognate reductase did not enhance survival, suggesting the expression levels of TRX1 and TRX2 were not rate limiting survival factors under these experimental conditions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1-4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.
    02/2014; 2:475-84. DOI:10.1016/j.redox.2014.01.021
Show more