Original ContributionDetails in the catalytic mechanism of mammalian thioredoxin reductase 1 revealed using point mutations and juglone-coupled enzyme activities
Graphical abstract
Introduction
As a well-studied selenoprotein flavoenzyme oxidoreductase in mammals, thioredoxin reductase 1 (TrxR1) serves numerous important functions in cells, mainly by virture of its physiological substrate thioredoxin 1 (Trx1). Trx1 is maintained in its reduced state by TrxR1 using NADPH, thereby constituting a cornerstone of the Trx system. Cellular roles of the Trx system include redox regulation through control of the redox state of several disulfide-containing proteins, cytoprotection against oxidative stress through reduction of peroxiredoxins, support of cell growth through reduction of ribonucleotide reductase, and prevention of apoptosis by inhibition of apoptosis signal regulating kinase 1 (ASK1), to mention a few examples of the many cellular roles of the Trx system [1]. The catalytic mechanism of mammalian TrxR1 has been much studied, but still several of its details are topics of debate. It is clear that each subunit of the active homodimeric enzyme contains a FAD close to two redox active Cys residues (Cys59, Cys64) within an N-terminal domain, as well as a selenenylsulfide/selenolthiol motif (Cys497, Sec498) at the C-terminal end, with one of these two motifs of one subunit interacting with the other motif of the other subunit during the reductive half of the catalytic cycle [2], [3]. The Sec-containing C-terminal motif is absolutely required for NADPH-dependent reduction of the active site disulfide of Trx1. The Sec residue within the flexible and surface exposed C-terminus of reduced TrxR1 is highly reactive and suggested to be the target of several electrophilic antitumor agents, explaining parts of their cytotoxic mechanisms [4], [5], [6], [7], [8]. However, the Sec residue in the C-terminal motif of TrxR1 is shielded and non-reactive in the oxidized state of the enzyme, as it then forms a selenenylsulfide with its neighboring Cys residue [3].
In the reductive pathway of TrxR1, NADPH is believed to first reduce the FAD of the enzyme, subsequently reducing the disulfide between Cys59 and Cys64 and forming a charge-transfer complex with Cys64 [3], [9], [10], [11]. The reduced Cys59 thiolate is likely reacting with the C-terminal selenenylsulfide, forming an intermediate mixed disulfide with Cys497 or a mixed selenenylsulfide with Sec498, thereby reducing the C-terminal redox active motif and again forming a disulfide with Cys64, resulting in an enzyme with oxidized FAD and a reduced C-terminal tail selenolthiol motif [3], [10], [11], [12]. Two additional electrons are subsequently required from NADPH to finally produce the four-electron reduced active EH4 state of the enzyme, presumed to be the major active species of the enzyme [3], [9], [10], [11]. It is clear that TrxR1 is highly dependent upon its C-terminal Sec residue, because replacement of selenium to sulfur by a Sec-to-Cys mutation yields loss of both Se-nucleophilicity and Se-electrophilicity [13] and markedly reduces its catalytic activity with most of its substrates [9], [14], [15]. Still, several questions remain regarding the catalytic mechanism of TrxR1, among them why the enzyme is so highly dependent upon Sec, while other TrxR isoforms of the same general structure are rather efficient reductants of at least some substrates also in the absence of Sec. For example, mitochondrial TrxR2 can reduce DTNB with rather high efficiency also in the form of Sec-deficient variants [16], [17]. The TrxR variant in Drosphila melanogaster is furthermore an efficient Trx1 reductase, although its C-terminal active site motif is -SCCS instead of -GCUG as found in mammalian TrxR1 [18]. However, the same -SCCS motif is not active when grafted to the scaffold of mammalian TrxR1, illustrating that not only the C-terminal motif, but also the whole active site environment, affects overall activity as well as Sec dependence [9]. It was underscored by Hondal and coworkers that Sec in TrxR1 must act as both a donor and an acceptor of electrons, during separate phases of the catalytic cycle, whereby it is yet unclear which of these roles of Sec in the catalysis of TrxR1 that must be maintained and cannot be replaced by Cys [12]. Recently the same group reported findings that supported a view that one difference between Sec-containing TrxR1 and other orthologous of the enzyme is the fact that TrxR1 has a so-called “guiding bar”, initially proposed by Becker and coworkers to be formed by amino acids 407-422 to facilitate interactions between the C-terminal redox active site and the N-terminal motif of the enzyme [19], which may, at the same time, restrict many low molecular weight substrates from direct interactions with the non-Sec containing N-terminal FAD/dithiol motif of TrxR1 [13].
Several quinone compounds are good alternative low molecular weight substrates for TrxR1. This includes alloxan [20], ubiquinone Q10 [21], 9,10-phanenthrenequinone (9,10-PQ) [22], and the dietary nutritional component pyrroloquinoline quinone (PQQ) [23]. Those particular quinone substrates of TrxR1 still require the presence of an intact Sec residue in TrxR1 for efficient reduction. In contrast, efficient reduction and also redox cycling with oxygen by TrxR1 is seen with 5-hydroxy-1,4-naphthoquinone (juglone; walnut toxin), a naturally occurring naphthoquinone secreted from the walnut tree (juglans). Its activities with TrxR1 are promoted in a Sec-independent manner, thereby having been proposed to be supported mainly by the N-terminal redox active motif of TrxR1 [22], [24], [25]. The activity of TrxR1 with juglone is also maintained in Sec compromised species of the enzyme and correlate to pro-oxidant derivatives of TrxR1 in the form of SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins), which may contribute to the cytotoxicity of electrophilic compounds targeting the Sec residue of TrxR1 [26], [27], [28], [29], [30], [31], [32]. It was recently proposed that targeting of the Sec residue in TrxR1 may possibly lead to a conformational change of the enzyme, allowing substrates such as juglone to reach the N-terminal active site and thus by-pass the restriction imposed by the guiding bar discussed above [13]. Here we determined to further probe the activities of TrxR1 derivatives with juglone as a means of better understanding the details of the catalytic mechanisms of the enzyme and its unique redox cycling properties when coupled to juglone.
Utilizing a side-by-side comparison of steady-state kinetic analyzes with juglone as well as other substrates using different pure recombinant mutant variants of rat TrxR1, we here found that the nature of the C-terminal motif of TrxR1 is crucial for support of high-efficiency juglone-coupled activity, which occurs in part via one-electron transfer reactions producing superoxide. This hitherto unrecognized involvement of the C-terminal tail in reduction of juglone by TrxR1 yields further insights into the catalytic mechanism of the enzyme, and should have importance for the mechanisms of cytotoxicity of juglone and of Sec-deficient species of TrxR1 that may be formed in a cellular context.
Section snippets
Materials
A BL21 (DE3) gor− host strain and pure recombinant human wild-type Trx1 were generously provided by Dr. Arne Holmgren, Karolinska Institutet, Stockholm, Sweden. The 2′5′-ADP SepharoseTM, SuperdexTM 200, NAPTM-25 and NAPTM-5 columns were purchased from GE Healthcare Life Sciences (Uppsala, Sweden). Ultra-concentration units (30 kDa cut-off and 3 kDa cut-off) were purchased from Merck EMD Millipore (USA). All other chemicals and reagents were purchased from Sigma-Aldrich Chemicals, unless stated
Construction and activities of mutant TrxR1 variants
To further analyze the involvement of the different redox active moieties of TrxR1 in its highly efficient juglone-coupled redox cycling activity [22], we first constructed a number of mutant TrxR1 variants that would be expected to have an affected FAD function. For this we made various combinatorial substitutions of the two redox active cysteines in its N-terminal domain that directly interact with the FAD (Cys59 and Cys64), and the Sec-containing selenothiol motif (Cys497 and Sec498) within
Discussion
It is well known that juglone is a highly cytotoxic compound, with the mechanisms of cytotoxicity involving increased oxidative stress and redox cycling processes with both one- and two-electron transfer reactions [55], [56], [57]. It was also shown that targeting of TrxR1 and Sec-compromised forms of the enzyme may underpin several of these reactions with juglone [24], [25], [26], [53]. In the present study we found that the presence of at least one Cys residue in the C-terminus of TrxR1 is
Author contributions
Jianqiang Xu and Elias Arnér conceived the project and designed experiments. Jianqiang Xu carried out the cloning, TrxR preparation and most biochemical analyses. Qing Cheng performed the treatment of enzyme and preparations for mass spectrometry. Jianqiang Xu and Elias Arnér analyzed data and wrote the manuscript.
Funding
This study was supported by Grants from the Swedish Cancer Society, the Swedish Research Council (Medicine) and the Karolinska Institutet. The mass spectrometry analyses were supported by the Science for Life Laboratory Mass Spectrometry Based Proteomics Facility in Uppsala. We also thank the partial support from the Chinese Fundamental Research Funds for the Central Universities (DUT14RC(3)145 & DUT15RC(3)052).
Acknowledgments
The authors wish to thank Irina Pader, Marcus Cebula, Katarina Johansson and Margareta Ramström for assistance and good discussions, and Arne Holmgren for protein reagents, all at Karolinska Institutet, Stockholm, Sweden, except M.R. who is at Science for Life Laboratory, Uppsala, Sweden.
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Thioredoxin reductase selenoproteins from different organisms as potential drug targets for treatment of human diseases
2022, Free Radical Biology and MedicineCitation Excerpt :When these proteins are formed intracellularly, they can promote cell death in certain cellular contexts, likely triggered by an excessive oxidative stress [78]. The reduction of most TrxR substrates, including that of the active site disulfide motifs of Trx-fold proteins, is strictly dependent on an intact Sec in the active site, but some low molecular weight non-disulfide substrates such as 5-hydroxy-1,4-naphthoquinone (juglone, walnut toxin) are notable exceptions to this Sec dependence [79–82]. When coupled with juglone, a SecTRAP can propagate a futile redox cycle leading to excessive formation of H2O2 [78,82].
Mitochondrial thioredoxin system is required for enhanced stress resistance and extended longevity in long-lived mitochondrial mutants
2022, Redox BiologyCitation Excerpt :A similar redox cycling mechanism is involved for juglone, which also induces oxidative stress through generation of superoxide. It has been shown that thioredoxin reductase can participate in redox cycling with juglone to generate superoxide and that disruption of thioredoxin reductase reduces superoxide generation from juglone [67]. If thioredoxin reductase is also involved in the generation of superoxide from paraquat, then disruption of trxr-2 and trxr-1 may be increasing the survival of wild type worms on paraquat by decreasing superoxide generation.