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Human thioredoxin reductase (TrxR) is a selenoprotein with a central role in cellular redox homeostasis, utilizing a highly reactive and solvent-exposed selenocysteine (Sec) residue in its active site. Pharmacological modulation of TrxR can be obtained with several classes of small compounds showing different mechanisms of action, but most often dependent upon interactions with its Sec residue. The clinical implications of TrxR modulation as mediated by small compounds have been studied in diverse diseases, from rheumatoid arthritis and ischemia to cancer and parasitic infections. The possible involvement of TrxR in these diseases was in some cases serendipitously discovered, by finding that existing clinically used drugs are also TrxR inhibitors. Inhibiting isoforms of human TrxR is, however, not the only strategy for human disease treatment, as some pathogenic parasites also depend upon Sec-containing TrxR variants, including S. mansoni, B. malayi or O. volvulus. Inhibiting parasite TrxR has been shown to selectively kill parasites and can thus become a promising treatment strategy, especially in the context of quickly emerging resistance towards other drugs. Here we have summarized the basis for the targeting of selenoprotein TrxR variants with small molecules for therapeutic purposes in different human disease contexts. We discuss how Sec engagement appears to be an indispensable part of treatment efficacy and how some therapeutically promising compounds have been evaluated in preclinical or clinical studies. Several research questions remain before a wider application of selenoprotein TrxR inhibition as a first-line treatment strategy might be developed. These include further mechanistic studies of downstream effects that may mediate treatment efficacy, identification of isoform-specific enzyme inhibition patterns for some given therapeutic compounds, and the further elucidation of cell-specific effects in disease contexts such as in the tumor microenvironment or in host-parasite interactions, and which of these effects may be dependent upon the specific targeting of Sec in distinct TrxR isoforms.

Cellular redox networks power a multitude of cellular processes, and are often dysregulated in pathologies including cancer and inflammatory diseases. Quantifying the turnover of the key players in redox homeostasis is crucial for understanding their physiological dynamics and for targeting them in pathologies. However, suitably selective probes for assessing specific redox enzyme activities in cells are lacking. We rationally developed the first chemical probes targeting the mammalian selenoprotein thioredoxin reductase (TrxR) while fully resisting other cellular thiols and oxidoreductases. We used a cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger, oriented to harness the chemistry of TrxR's unique selenolthiol active site, and integrated it into modular probes releasing arbitrary cargos upon reduction. The probes showed remarkable selenocysteine-dependent sensitivity to cytosolic TrxR1, against a panel of oxidoreductases. Lead probe RX1 also had excellent TrxR1-selective performance in cells, as cross-validated by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and TrxRselective chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors. This indicated that tempered SNAr electrophiles may be a more favorable drug class than classically-used electrophiles. The RX1 design is thus a robust, cellularly validated, high-performance modular system for mammalian TrxR1. This sets the stage for in vivo imaging TrxR1 activity in health and disease, and can also drive and reorient TrxR1-inhibitor drug design. The thermodynamic and kinetic considerations behind RX1's selectivity also outline paths towards rationally-designed probes for other key players in redox biology.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Selenoproteins are translated via animal domain-specific elongation machineries that redefine dedicated UGA opal codons from termination of translation to selenocysteine (Sec) insertion, utilizing specific tRNA species and Sec-specific elongation factors. This has made recombinant production of mammalian selenoproteins in E. coli technically challenging but recently we developed a methodology that enables such production, using recoding of UAG for Sec in an RF1-deficient host strain. Here we used that approach for production of the human glutathione peroxidases 1, 2 and 4 (GPX1, GPX2 and GPX4), with all these three enzymes being important antioxidant selenoproteins. Among these, GPX4 is the sole embryonically essential enzyme, and is also known to be essential for spermatogenesis as well as protection from cell death through ferroptosis. Enzyme kinetics, ICP-MS and mass spectrometry analyses of the purified recombinant proteins were used to characterize selenoprotein characteristics and their Sec contents. This revealed a unique phenomenon of one-codon skipping, resulting in a lack of a single amino acid at the position corresponding to the selenocysteine (Sec) residue, in about 30% of the recombinant GPX isoenzyme products. We furthermore confirmed the previously described UAG suppression with Lys or Gln as well as a minor suppression with Tyr, together resulting in about 20% Sec contents in the full-length proteins. No additional frameshifts or translational errors were detected. We subsequently found that Sec-containing GPX4 could be further purified over a bromosulfophthalein-column, yielding purified recombinant GPX4 with close to complete Sec contents. This production method for homogenously purified GPX4 should help to further advance the studies of this important selenoprotein.

The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1), and to some extent mitochondrial TrxR2 (TXNRD2), can be inhibited by a wide range of electrophilic compounds. Many such compounds also yield cytotoxicity toward cancer cells in culture or in mouse models, and most compounds are likely to irreversibly modify the easily accessible selenocysteine residue in TrxR1, thereby inhibiting its normal activity to reduce cytosolic thioredoxin (Trx1, TXN) and other substrates of the enzyme. This leads to an oxidative challenge. In some cases, the inhibited forms of TrxR1 are not catalytically inert and are instead converted to prooxidant NADPH oxidases, named SecTRAPs, thus further aggravating the oxidative stress, particularly in cells expressing higher levels of the enzyme. In this review, the possible molecular and cellular consequences of these effects are discussed in relation to cancer therapy, with a focus on outstanding questions that should be addressed if targeted TrxR1 inhibition is to be further developed for therapeutic use.

Selenocysteine (Sec), the sulfur-to-selenium substituted variant of cysteine (Cys), is the defining entity of selenoproteins. These are naturally expressed in many diverse organisms and constitute a unique class of proteins. As a result of the physicochemical characteristics of selenium when compared with sulfur, Sec is typically more reactive than Cys while participating in similar reactions, and there are also some qualitative differences in the reactivities between the two amino acids. This minireview discusses the types of modifications of Sec in selenoproteins that have thus far been experimentally validated. These modifications include direct covalent binding through the Se atom of Sec to other chalcogen atoms (S, O and Se) as present in redox active molecular motifs, derivatization of Sec via the direct covalent binding to non-chalcogen elements (Ni, Mb, N, Au and C), and the loss of Se from Sec resulting in formation of dehydroalanine. To understand the nature of these Sec modifications is crucial for an understanding of selenoprotein reactivities in biological, physiological and pathophysiological contexts.

Cellular antioxidant systems control the levels of hydrogen peroxide (H2O2) within cells. Multiple theoretical models exist that predict the diffusion properties of H2O2 depending on the rate of H2O2 generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H2O2 gradients. The relative role of thioredoxin (Trx) and glutathione systems in H2O2 pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H2O2 gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H2O2 and using the H2O2 biosensor HyPer to analyze the resulting H2O2 distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H2O2 gradients. Antioxid. Redox Signal. 31, 664-670.

Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems. Here, using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H(2)O(2) to form the more reactive peroxymonocarbonate, potently facilitates H(2)O(2)-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH. The cellular experiments revealed that intracellular bicarbonate proportionally dictates total protein phosphotyrosine levels obtained after stimulation with epidermal growth factor (EGF) and that bicarbonate levels directly correlate with the extent of PTP1B oxidation. In fact, EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate. These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid–base balance.

Auranofin is a gold (1)-containing compound used for the treatment of rheumatic arthritis. Auranofin has anticancer activity in animal models and is approved for clinical trials for lung and ovarian carcinomas. Both the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase (TrxR) are well documented targets of auranofin. Auranofin was recently reported to also inhibit proteasome activity at the level of the proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14. We here set out to re-examine the molecular mechanism underlying auranofin cytotoxicity towards cultured cancer cells. The effects of auranofin on the proteasome were examined in cells and in vitro, effects on DUB activity were assessed using different substrates. The cellular response to auranofin was compared to that of the 20S proteasome inhibitor bortezomib and the 19S DUB inhibitor b-AP15 using proteomics. Auranofin was found to inhibit mitochondrial activity and to an induce oxidative stress response at IC50 doses. At 2-3-fold higher doses, auranofin inhibits proteasome processing in cells. At such supra-pharmacological concentrations USP14 activity was inhibited. Analysis of protein expression profiles in drug-exposed tumor cells showed that auranofin induces a response distinct from that of the 20S proteasome inhibitor bortezomib and the DUB inhibitor b-AP15, both of which induced similar responses. Our results support the notion that the primary mechanism of action of auranofin is TrxR inhibition and suggest that proteasome DUB inhibition is an off-target effect. Whether proteasome inhibition will contribute to the antineoplastic effect of auranofin in treated patients is unclear but remains a possibility. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Funding Agencies|Swedish Cancer Society; Radiumhemmets Forskningsfonder; Vetenskapsradet; Barncancerfonden; Knut and Alice Wallenbergs Foundation

Specialised cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist nonspecific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols, yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx-TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.

The cyclic five-membered disulfide 1,2-dithiolane has been used as the key element in numerous chemical biology probes. Contradictory views of this disulfide motif populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We show that 1,2-dithiolanes are nonspecifically reduced by a broad range of thiol reductants and redox-active proteins, and that their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and probe designs treating 1,2-dithiolanes as TrxR-selective substrates should be treated with caution and previous interpretations may need careful re-evaluation. Considering ring-opening polymerisation, and stringently interpreting assays involving the thiophilic gold-based inhibitor auranofin, are critical to assess 1,2-dithiolane’s true behaviour. We present an approach to control against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient redox probe research in the future.

The article by Fan et al. inaccurately states that ProPerDP is inadequate for protein persulfide detection., The recent report by Fan et al. alleged that the ProPerDP method is inadequate for the detection of protein persulfidation. Upon careful evaluation of their work, we conclude that the claim made by Fan et al. is not supported by their data, rather founded in methodological shortcomings. It is understood that the ProPerDP method generates a mixture of cysteine-containing and non–cysteine-containing peptides. Instead, Fan et al. suggested that the detection of non–cysteine-containing peptides indicates nonspecific alkylation at noncysteine residues. However, if true, then such peptides would not be released by reduction and therefore not appear as products in the reported workflow. Moreover, the authors’ biological assessment of ProPerDP using Escherichia coli mutants was based on assumptions that have not been confirmed by other methods. We conclude that Fan et al. did not rigorously assess the method and that ProPerDP remains a reliable approach for analyses of protein per/polysulfidation.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general., Graphical abstract Image 1

Thioredoxin system–mediated persulfidation of Cys residues controls protein function and protects them from oxidative stress., Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug resistance parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1 – 5 are active against all major species and development stages. Ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1 – 5, 7, and 8 displayed schistosomicidal activity even after only one-hour incubation with the worms. Compounds 1 – 4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild type and mutant TGR proteins. Compounds 4 – 6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

Redox regulation of STAT3 is targeted by direct inhibition of TrxR1, illuminating a promising link for therapeutic development., Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

Quinones are common in nature, and often cytotoxic. Their proposed toxicity mechanisms involve redox cycling with radical generation, and/or reactions with nucleophiles, such as protein cysteine (Cys) residues, forming adducts via Michael addition reactions. The selenenyl anion of selenocysteine (Sec) is a stronger nucleophile, more prevalent at physiological pH, and more reactive than the corresponding thiolate anion of Cys. We therefore hypothesized that Sec residues should be readily modified by quinones and with potential consequences for the structure and function of selenoproteins. Here, we report data on the interaction of p-benzoquinone (BQ) with the selenoprotein thioredoxin reductase-1 (TrxR1), which exposes an accessible Sec residue upon physiological reduction by NADPH. Our results reveal that BQ targets NADPH-reduced TrxR1 and inhibits its activity using 5,5′-dithiobis(2-nitrobenzoic acid) or juglone as model substrates, consistent with the targeting of both the Cys and Sec residues of TrxR1. In the absence of NADPH, BQ modified the non-catalytic Cys residues, leading to subunit crosslinking, mainly through disulfides, which also resulted in some loss of activity. This crosslinking was time-dependent and independent of the Sec residue. Addition of NADPH after BQ pre-treatment could resolve the disulfide-linked crosslinking. TrxR activity loss was also observed upon incubation of J774A.1 cells or cell lysates with BQ. These data suggest that BQ readily targets TrxR1, albeit in a rather complex manner, which results in structural changes and loss of enzyme activity. We suggest that TrxR1 targeting can explain some of the cytotoxicity of BQ, and potentially also that of other quinone compounds., Graphical abstract Image 1

Over the past seven decades, research on autotrophic and heterotrophic model organisms has defined how the flow of electrons (“reducing power”) from high-energy inorganic sources, through biological systems, to low-energy inorganic products like water, powers all of Life’s processes. Universally, an initial major biological recipient of these electrons is nicotinamide adenine dinucleotide-phosphate, which thereby transits from an oxidized state (NADP(+)) to a reduced state (NADPH). A portion of this reducing power is then distributed via the cellular NADPH-dependent disulfide reductase systems as sequential reductions of disulfide bonds. Along the disulfide reduction pathways, some enzymes have active sites that use the selenium-containing amino acid, selenocysteine, in place of the common but less reactive sulfur-containing cysteine. In particular, the mammalian/metazoan thioredoxin systems are usually selenium-dependent as, across metazoan phyla, most thioredoxin reductases are selenoproteins. Among the roles of the NADPH-dependent disulfide reductase systems, the most universal is that they provide the reducing power for the production of DNA precursors by ribonucleotide reductase (RNR). Some studies, however, have uncovered examples of NADPH-independent disulfide reductase systems that can also support RNR. These systems are summarized here and their implications are discussed.

Anticancer drugs that target cellular antioxidant systems have recently attracted much attention. Auranofin (AF) is currently evaluated in several clinical trials as an anticancer agent that targets the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase, TXNRD1 and TXNRD2. Recently, two novel TXNRD1 inhibitors (TRi-1 and TRi-2) have been developed that showed anticancer efficacy comparable to AF, but with lower mitochondrial toxicity. However, the cellular action mechanisms of these drugs have not yet been thoroughly studied. Here we used several proteomics approaches to determine the effects of AF, TRi-1 and TRi-2 when used at IC50 concentrations with the mouse B16 melanoma and LLC lung adenocarcinoma cells, as these are often used for preclinical mouse models in evaluation of anticancer drugs. The results demonstrate that TRi-1 and TRi-2 are more specific TXNRD1 inhibitors than AF and reveal additional AF-specific effects on the cellular proteome. Interestingly, AF triggered stronger Nrf2-driven antioxidant responses than the other two compounds. Furthermore, AF affected several additional proteins, including GSK3A, GSK3B, MCMBP and EEFSEC, implicating additional effects on glycogen metabolism, cellular differentiation, inflammatory pathways, DNA replication and selenoprotein synthesis processes. Our proteomics data provide a resource for researchers interested in the multidimensional analysis of proteome changes associated with oxidative stress in general, and the effects of TXNRD1 inhibitors and AF protein targets in particular., Graphical abstract Image 1, Highlights • Auranofin is an inhibitor of thioredoxin reductase currently in clinical trials against cancer. • TRi-1 and TRi-2 are recently developed alternative inhibitors of thioredoxin reductase with anticancer effects. • Comprehensive proteomics reveal additional targets of Auranofin beyond thioredoxin reductase. • TRi-2 is a more specific thioredoxin reductase inhibitor than Auranofin but also targets aldo-keto reductases. • TRi-1 is the most specific thioredoxin reductase inhibitor of these three compounds.

SUMMARY Energetic nutrients are oxidized to sustain high intra-cellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity., In Brief All cells require cytosolic disulfide-reducing power fueled by intracellular NADPH or, in mammals, alternatively generated by catabolism of the essential amino acid methionine. Prigge et al. show that liver homeostasis under cytosolic disulfide reductase paucity involves noncanonical trafficking of reducing power and a hyperproliferation-based organ survival strategy.

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family- all siblings of the index patient were homozygous and the parents heterozygous-were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by Se-75 labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34 +/- 3% and 16 +/- 8% of controls, respectively). In fibroblasts, we detected reduced Se-75-labeling of the enzyme (41 +/- 3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered k(cat)/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and Se-75 labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.

Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. In Escherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to a Sec insertion sequence (SECIS) element in the selenoprotein-encoding mRNA and competes with UGA-directed translational termination. Here, we found that Sec can also be efficiently incorporated at a predefined UAG amber codon, thereby competing with RF1 rather than RF2. Subsequently, utilizing the RF1-depleted E. coli strain C321.ΔA, we could produce mammalian selenoprotein thioredoxin reductases with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such a system. Human glutathione peroxidase 1 could thereby also be produced, and we could confirm a previously proposed catalytic tetrad in this selenoprotein. We believe that the versatility of this new UAG-directed production methodology should enable many further studies of diverse selenoproteins.

Many transcription factors with importance in health and disease are redox regulated. However, how their activities may be intertwined in responses to redox-perturbing stimuli is poorly understood. To enable in-depth characterization of this aspect, we here developed a methodology for simultaneous determination of nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation at single-cell resolution, using a new tool named pTRAF (plasmid for transcription factor reporter activation based upon fluorescence). The pTRAF allowed determination of Nrf2, HIF, and NF-κB activities in a high-resolution and high-throughput manner, and we here assessed how redox therapeutics affected the activities of these transcription factors in human embryonic kidney cells (HEK293).Cross talk was detected between the three signaling pathways upon some types of redox therapeutics, also by using inducers typically considered specific for Nrf2, such as sulforaphane or auranofin, hypoxia for HIF activation, or tumor necrosis factor alpha (TNFα) for NF-κB stimulation. Doxorubicin, at low nontoxic doses, potentiated TNFα-induced activation of NF-κB and HIF, without effects in stand-alone treatment. Stochastic activation patterns in cell cultures were also considerable upon challenges with several redox stimuli.A novel strategy was here used to study simultaneous activation of Nrf2, HIF, and NF-κB in single cells. The method can also be adapted for studies of other transcription factors.The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-κB are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells. Antioxid. Redox Signal. 26, 229-246.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (similar to 20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups. Funding Agencies|Cancerfonden; Vetenskapsradet; Radiumhemmets forskningsfonder; Knut och Alice Wallenbergs Stiftelse; Barncancerfonden

Persulfide or polysulfide formation on Cys residues is emerging as an abundant protein posttranslational modification, with important regulatory functions. However, as many other Cys oxidative modifications, per- and polysulfides are relatively labile, dynamically interchanging species, which makes their intracellular detections challenging. Here we report our recently developed highly selective method, Protein Persulfide Detection Protocol (ProPerDP), which can detect protein per- and polysulfide species in isolated protein systems, in blood plasma, or in cells and tissue samples. The method is easy to use and relatively inexpensive and requires only readily commercially available reagents. The biggest advantage of ProPerDP compared to other previously published persulfide detecting methods is the fact that in this protocol, all thiol and persulfide species are appropriately alkylated before any cell lysis step. This greatly reduces the potential of detecting lysis-induced oxidation-driven artifact persulfide formation.

Protein tyrosine phosphatases (PTPs) can be regulated by several redox-dependent mechanisms and control growth factor-activated receptor tyrosine kinase phosphorylation cascades. Reversible oxidation of PTPs is counteracted by reductive enzymes, including thioredoxin (Trx) and Trx-related protein of 14 kDa (TRP14), keeping PTPs in their reduced active states. Different modes of oxidative inactivation of PTPs concomitant with assessment of activating reduction have been little studied in direct comparative analyses. Determining PTP1B activities, we here compared the potency of inactivation by bicarbonate-assisted oxidation using H2O2 with that of polysulfide-mediated inactivation. Inactivation of pure PTP1B was about three times more efficient with polysulfides as compared to the combination of bicarbonate and H2O2. Bicarbonate alone had no effect on PTP1B, neither with nor without a combination with polysulfides, thus strengthening the notion that bicarbonate-assisted H2O2-mediated inactivation of PTP1B involves formation of peroxymonocarbonate. Furthermore, PTP1B was potently protected from polysulfide-mediated inactivation by either TRP14 or Trx1, in contrast to the inactivation by bicarbonate and H2O2. Comparing reductive activation of polysulfide-inactivated PTP1B with that of bicarbonate- and H2O2-treated enzyme, we found Trx1 to be more potent in reactivation than TRP14. Altogether we conclude that inactivation of PTP1B by polysulfides displays striking qualitative differences compared to that by H2O2 together with bicarbonate, also with regard to maintenance of PTP1B activity by either Trx1 or TRP14.

Mammalian thioredoxin 1 (Trx1) and the selenoprotein Trx reductase 1 (TrxR1) are key cellular enzymes that function coordinately in thiol-based redox regulation and signaling. Recent studies have revealed that the Trx1/TrxR1 system has an S-nitrosothiol reductase (denitrosylase) activity through which it can regulate nitric oxide-related cellular processes. In this study we revealed that TrxR1 is itself susceptible to nitrosylation, characterized the underlying mechanism, and explored its functional significance. We found that nitrosothiol or nitric oxide donating agents rapidly and effectively inhibited the activity of recombinant or endogenous TrxR1. In particular, the NADPH-reduced TrxR1 was partially and reversibly inhibited upon exposure to low concentrations (

International audience; Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene) indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene) indolin-2-one], bear a nitrogen-linked alpha,beta-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate “paradoxical” outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of “antioxidant” nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that “paradoxical” roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways.

Background Toxoflavin (1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione; xanthothricin) is a well-known natural toxin of the pyrimidinetriazinedione type that redox cycles with oxygen under reducing conditions. In mammalian systems, toxoflavin is an inhibitor of Wnt signaling as well as of SIRT1 and SIRT2 activities, but other molecular targets in mammalian cells have been scarcely studied. Interestingly, in a library of nearly 400,000 compounds (PubChem assay ID 588456), toxoflavin was identified as one out of only 56 potential substrates of the mammalian selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1). This activity was here examined in further detail. Methods Kinetic parameters in interactions of toxoflavin with rat or human TrxR isoenzymes were determined and compared with those of juglone (5-Hydroxy-1,4-naphthoquinone; walnut toxin) and 9,10-phenanthrene quinone. Selenocysteine dependence was examined using Sec-to-Cys and Sec-to-Ser substituted variants of recombinant rat TrxR1. Results Toxoflavin was confirmed as an efficient substrate for TrxR. Rat and human cytosolic TrxR1 supported NADPH-dependent redox cycling coupled to toxoflavin reduction, accompanied by H 2 O 2 production under aerobic conditions. Apparent kinetic parameters for the initial rates of reduction showed that rat TrxR1 displayed higher apparent turnover ( k cat = 1700 ± 330 min −1 ) than human TrxR1 ( k cat = 1100 ± 82 min −1 ) but also a higher Km ( K m = 24 ± 4.3 μM for human TrxR1 versus K m = 54 ± 18 μM for rat TrxR1). Human TrxR2 (TXNRD2) was less efficient in reduction of toxoflavin ( K m = 280 ± 110 μM and k cat = 740 ± 240 min −1 ). The activity was absolutely dependent upon selenocysteine (Sec). Toxoflavin was also a subversive substrate indirectly inhibiting reduction of other substrates of TrxR1. Conclusions Our results identify toxoflavin as an efficient redox cycling substrate of mammalian TrxR enzymes, in a strict Sec-dependent manner. General significance Тhe interactions of toxoflavin with mammalian TrxR isoenzymes can help to explain parts of the molecular mechanisms giving rise to the well-known toxicity as well as pro-oxidant properties of this toxin.

Selenoproteins are rare proteins among all kingdoms of life containing the 21 st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4 cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided. The trace element selenium protects a critical population of interneurons from ferroptotic cell death.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Mammalian thioredoxin reductases (TrxRs) are selenocysteine-containing proteins (selenoproteins) that propel a large number of functions through reduction of several substrates including the active site disulfide of thioredoxins (Trxs). Well-known enzymatic systems that in turn are supported by Trxs and TrxRs include deoxyribonucleotide synthesis through ribonucleotide reductase, antioxidant defense through peroxiredoxins and methionine sulfoxide reductases, and redox modulation of a number of transcription factors. Although these functions may be essential for cells due to crucial roles in maintenance of cell viability and proliferation, findings during the last decade reveal that mammals have major redundancy in their cellular reductive systems. The synthesis of glutathione (GSH) and reductive functions of GSH-dependent pathways typically act in parallel with Trx-dependent pathways, with only one of these systems often being sufficient to support viability. Importantly, this does not imply that a modulation of the Trx system will remain without consequences, even when GSH-dependent pathways remain functional. As suggested by several recent findings, the Trx system in general and the TrxRs in particular, function as key regulators of signaling pathways. In this review article we will discuss findings that collectively suggest that modulation in mammalian systems of cytosolic TrxR1 (TXNRD1) or mitochondrial TrxR2 (TXNRD2) influence cell patterning and cellular stress responses. Effects of lower activities include increased adipogenesis, insulin responsiveness, glycogen accumulation, hyperproliferation, and distorted embryonic development, while increased activities correlate with decreased proliferation and extended lifespan, as well as worse cancer prognosis. The molecular mechanisms that underlie these diverse effects, involving regulation of protein phosphorylation cascades and of key transcription factors that guide cellular differentiation pathways, will be discussed. We conclude that the selenium-dependent oxidoreductases TrxR1 and TrxR2 should be considered as key components of signaling pathways that control cell differentiation and cellular stress responses.

Thioredoxin reductases are important oxidoreductases that keep the active site disulfide/dithiol motif of thioredoxins reduced using NADPH, thereby supporting many thioredoxin-dependent reductive pathways in cells. Mammalian thioredoxin reductases are selenoproteins that have several additional substrates beyond thioredoxins. This chapter first lists several different assays for measurement of thioredoxin reductase activities, before giving a protocol for a selective evaluation of these activities that can be used in either crude cell lysates as well as with purified enzymes. The same assay can also be easily adopted for the determination of thioredoxin activities.

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2. Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.

Background: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems.Methods and results: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3 h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keapl. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and, upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-KB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function.Conclusion and significance: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD.General significance: The newly found mechanism behind the long-term protection of rutin against cardiovas-cular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Selenoproteins, present in various organisms, including man, are rare proteins containing the 21st amino acid selenocysteine. Since selenocysteine and cysteine differ only by the substitution of selenium to sulfur, the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins exist as Cys-containing homologs in varying organisms. It has been proposed that the main advantage of selenocysteine is the inherently higher nucleophilicity of selenium allowing a higher enzymatic activity compared to thiol-based catalysis yet this has been a matter of controversy. Here we show that a selenocysteine to cysteine substitution in glutathione peroxidase 4 (GPX4), an essential selenoprotein in mammals, unexpectedly allows proper embryogenesis in mice. Yet, the loss of parvalbumin-positive interneurons causes early death of animals and development of severe epileptic seizures, demonstrating that this specific type of interneurons exclusively depends on selenolate-based catalysis in GPX4. Mechanistic studies provided evidence that unlike wildtype GPX4 the GPX4-cys variant is inherently sensitive to overoxidation by hydroperoxides. Specifically, hydroperoxides were able to trigger ferroptosis in GPX4-cys mutants but not in wildtype GPX4. Remarkably, a cellular system devoid of any selenoproteins could be generated in the GPX4-cys background, revealing that selenoprotein expression is not required for cell viability when partial GPX4 activity is provided. Conclusively, this study presents a new perspective to the biological role of selenium in mammals.

Plants provide dietary selenium (Se) for higher trophic levels including livestock and humans that depend on Se for their survival, but may also suffer Se toxicity upon excessive ingestion. Therefore, the fate of Se in plants and algae is relevant for mammals as well as for Se cycling in ecosystems. Conversely, the fate of Se in higher trophic levels is relevant for Se cycling back to soil and plants. This chapter focuses on the fate of dietary Se in humans and other animals, and health issues related to Se status. Selenium is an essential trace element for all mammals, implying that mammals develop disease upon Se deficiency. This is explained by the fact that certain selenoproteins, i.e. proteins containing the rare co-translationally inserted amino acid selenocysteine, have essential functions for physiology and health. Humans have 25 selenoprotein genes, about 0.1% of all protein-encoding genes. Among these, two thioredoxin reductases and one isoenzyme of glutathione peroxidase are essential for embryogenesis. For maintaining health and minimizing disease risks the human recommended daily intake is at least 50 μg Se per day. The majority of nutritional Se is provided in the form of two amino acids, selenocysteine or selenomethionine, and their derivatives, mainly in form of ingested proteins, as well as low molecular weight selenocompounds or rarely, in form of inorganic Se salts. If chronically ingested at high levels, above approximately 1 mg per day for an adult human, Se may become toxic. In this chapter, we give a brief overview of the main nutritional sources of Se for mammals, mammalian pathways of Se metabolism and excretion, and the biochemical and physiological functions that are sustained by mammalian selenoproteins. Established health risks of Se deficiency along with rare cases of inherited defects in selenoprotein biosynthesis will complement the picture on its essentiality.

The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, encoded in human by TXNRD1) is implied to have several different roles in relation to cancer. Its physiologic functions may protect normal cells from carcinogenesis, but may also promote cancer progression if carcinogenesis nonetheless occurs. With distinct links to Nrf2 signaling, ribonucleotide reductase-dependent production of deoxyribonucleotides and its support of several antioxidant systems counteracting oxidative stress, the metabolic pathways regulated, and affected by TrxR1, are altogether of crucial importance in cancer. These pathways and causal relationships are at the same time highly intricate. In spite of the complexity in the cellular redox networks, several observations discussed in this chapter suggest that specific targeting of TrxR1 may be promising as a mechanistic principle for anticancer therapy.

Background Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low. Methods Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl4). Results TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT. Conclusions Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury. General significance The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.

Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.