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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.

Dynamically driven cellular redox networks power a broad range of physiological cellular processes, and additionally are often dysregulated in various pathologies including cancer and inflammatory diseases. Therefore it is vital to be able to image and to respond to the turnover of the key players in redox homeostasis, to understand their physiological dynamics and to target pathological conditions. However, selective modular probes for assessing specific redox enzyme activities in cells are lacking. Here we report the development of cargo-releasing chemical probes that target the mammalian selenoprotein thioredoxin reductase (TrxR) while being fully resistant to thiol reductants in cells, such as the monothiol glutathione (GSH). We used a rationally oriented cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger that matches the chemistry of the unique TrxR active site, and integrated this reducible trigger into modular probes that release arbitrary cargos upon reduction. The probes' redox biochemistry was evaluated over a panel of thiol-type oxidoreductases, particularly showing remarkable, selenocysteine-dependent sensitivity of the "RX1" probe design to cytosolic TrxR1, with little response to mitochondrial TrxR2. The probe was cross-validated in cells by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and use of TrxR-selective chemical inhibitors, showing excellent TrxR1-dependent cellular performance. The RX1 design is therefore a robust, cellularly-validated, modular probe system for mammalian TrxR1. This sets the stage for in vivo imaging of TrxR1 activity in health and disease; and the thermodynamic and kinetic considerations behind its selectivity mechanism represent a significant advance towards rationally-designed probes for other key players in redox biology.

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.

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.

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery., The global identification of enzyme substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover enzyme substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase substrates.

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.

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.

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

The predominantly cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, encoded in human by the TXNRD1 gene) is a key enzyme for protection against oxidative stress and support of many reductive pathways in cells. For several decades, the enzyme has also been studied as a potential drug target for cancer therapy. The notion that TrxR1 may represent a promising target for anticancer therapy was further strengthened in recent years, with several clinical trials using inhibitors of the enzyme currently ongoing and with many novel experimental inhibitors being developed. This chapter will discuss the underlying biochemistry, physiological roles, and functional pathways of TrxR1 that can provide a rationale for targeting this enzyme for cancer therapy.

The mammalian thioredoxin system is driven by NADPH through the activities of isoforms of the selenoprotein thioredoxin reductase (TXNRD, TrxR), which in turn help to keep thioredoxins (TXN, Trx) and further downstream targets reduced. Due to a wide range of functions in antioxidant defense, cell proliferation, and redox signaling, strong cellular aberrations are seen upon the targeting of TrxR enzymes by inhibitors. However, such inhibition can nonetheless have rather unexpected consequences. Accumulating data suggest that inhibition of TrxR in normal cells typically yields a paradoxical effect of increased antioxidant defense, with metabolic pathway reprogramming, increased cellular proliferation, and altered cellular differentiation patterns. Conversely, inhibition of TrxR in cancer cells can yield excessive levels of reactive oxygen species (ROS) resulting in cell death and thus anticancer efficacy. The observed increases in antioxidant capacity upon inhibition of TrxR in normal cells are in part dependent upon activation of the Nrf2 transcription factor, while exaggerated ROS levels in cancer cells can be explained by a non-oncogene addiction of cancer cells to TrxR1 due to their increased endogenous production of ROS. These separate consequences of TrxR inhibition can be utilized therapeutically. Importantly, however, a thorough knowledge of the molecular mechanisms underlying effects triggered by TrxR inhibition is crucial for the understanding of therapy outcomes after use of such inhibitors. The mammalian thioredoxin system is driven by thioredoxin reductases (TXNRD, TrxR), which keeps thioredoxins (TXN, Trx) and further downstream targets reduced. In normal cells, inhibition of TrxR yields a paradoxical effect of increased antioxidant defense upon activation of the Nrf2 transcription factor. In cancer cells, however, inhibition of TrxR yields excessive reactive oxygen species (ROS) levels resulting in cell death and thus anticancer efficacy, which can be explained by a non-oncogene addiction of cancer cells to TrxR1 due to their increased endogenous production of ROS. These separate consequences of TrxR inhibition can be utilized therapeutically.

Thioredoxin-related protein of 14 kDa (TRP14; also named TXNDC17 for thioredoxin domain-containing protein 17) is a highly conserved and ubiquitously expressed oxidoreductase. It is expressed in parallel with thioredoxin 1 (Trx1, TXN; TXN1), an efficient substrate for the mammalian cytosolic selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1). However, TRP14, in sharp contrast to Trx1, cannot support the activities of ribonucleotide reductase, peroxiredoxins or methionine sulfoxide reductases, thus is unable to directly support cell proliferation or antioxidant defence through these pathways. However, TRP14 has been shown to efficiently reduce l-cystine, which thereby indirectly supports glutathione synthesis. TRP14 can also suppress NF-κB signalling, is functionally linked to STAT3 signalling, and can directly reactivate oxidized protein-tyrosine phosphatase PTP1B. Furthermore, TRP14 can efficiently reduce persulfidated or nitrosylated cysteine residues in many proteins, thereby having the capacity to modulate signalling through hydrogen sulfide or NO. Additional bioinformatics analyses and observations suggest further roles for TRP14; therefore, further studies of its functions are warranted. Collectively, the results available suggest that TRP14 is a member of the thioredoxin system dedicated to the control of cellular redox signalling pathways. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

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.

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.

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.

Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

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.

Expression of selenoproteins necessitates a process of decoding of a UGA codon from termination of translation to insertion of selenocysteine. The mechanisms of this process pose major challenges with regards to recombinant selenoprotein production in E. coli, which however can be overcome especially if the Sec residue is located close to the C-terminal end, as is the case for several naturally found selenoproteins. This chapter summarizes a method to achieve such a production.

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.

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.

Thioredoxin related protein of 14 kDa is a cytosolic protein of the thioredoxin fold family expressed in all tissues and an efficient substrate of the selenoprotein thioredoxin reductase 1. TRP14 cannot support classical Trx1 substrates such as peroxiredoxins, methionine sulfoxide reductases or ribonucleotide reductase, but affects NFκB signaling. However, it is efficient in reducing L-cystine and support S-denitrosylation reactions, reduce protein persulfides or reactivate oxidized protein tyrosine phosphatase PTP1B. These and other observations suggest that TRP14 may have dedicated roles in cellular signaling pathways, which was studied here. Using our previously developed pTRAF, we assessed activation patterns of the transcription factors Nrf2, NFκB and HIF in HEK293 cells with or without modulated TRP14 levels. Our preliminary results suggest that TRP14 is an unusual member of the Trx system, in that TRP14 may act as a repressor of NRF2, modulate HIF activation under hypoxia as well as acting as a weak repressor of NFκB activation. Further analyses are needed to fully evaluate the pTRAF readout also including additional methodologies Our results point towards the role of TRP14 as a dedicated fine-tuning modulator of redox regulated signaling pathways.

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.

S-nitrosothiols (SNOs), formed by nitric oxide (NO)-mediated S-nitrosylation, and hydrogen peroxide (H2O2), a prominent reactive oxygen species, are implicated in diverse physiological and pathological processes. Recent research has shown that the cellular action and metabolism of SNOs and H2O2 involve overlapping, thiol-based mechanisms, but how these reactive species may affect each other's fate and function is not well understood. In this study we investigated how NO/SNO may affect the redox cycle of mammalian peroxiredoxin-1 (Prx1), a representative of the 2-Cys Prxs, a group of thioredoxin (Trx)-dependent peroxidases. We found that, both in a cell-free system and in cells, NO/SNO donors such as S-nitrosocysteine and S-nitrosoglutathione readily induced the S-nitrosylation of Prx1, causing structural and functional alterations. In particular, nitrosylation promoted disulfide formation involving the pair of catalytic cysteines (Cys-52 and Cys-173) and disrupted the oligomeric structure of Prx1, leading to loss of peroxidase activity. A highly potent inhibition of the peroxidase catalytic reaction by NO/SNO was seen in assays employing the coupled Prx-Trx system. In this setting, S-nitrosocysteine (10 μM) effectively blocked the Trx-mediated regeneration of oxidized Prx1. This effect appeared to be due to both competition between S-nitrosocysteine and Prx1 for the Trx system and direct modulation by S-nitrosocysteine of Trx reductase activity. Our findings that NO/SNO target both Prx and Trx reductase may have implications for understanding the impact of nitrosylation on cellular redox homeostasis.

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing redox-active enzyme that is thought to be important during carcinogenesis. We have recently shown that treatment with statins, HMGCoA reductase inhibitors, reduces the levels of TrxR1 in liver of both rat and human. The reduced TrxR1 levels were correlated with inhibited hepatocarcinogenesis in a rat model. The aim of the present study was to investigate if statins affect the activity of the human TXNRD1 core promoter, which guides expression of TrxR1, and if the effects by statins on TrxR1 expression in liver could be reproduced in a cellular model system. We found that simvastatin and fluvastatin decreased cellular TrxR activity in cultured human liver-derived HepG2 cells with approximately 40% (p

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold (k(cat)/K(m) increased from 105 min(-1) μM(-1) to 1331 min(-1) μM(-1) for juglone in the presence of 50 μM PQQ, mainly through a lowered apparent K(m) for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.

The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1) is a key enzyme for redox control of cell function and antioxidant capacity. Many cancer cells have high levels of TrxR1 as a means of surviving their increased endogenous oxidative stress. This in turn has led to the hypothesis that TrxR1 may be an anticancer drug target, especially as the enzyme is dispensable for normal adult cells. Interestingly TrxR1 is also inhibited by several anticancer drugs in clinical use, including cisplatin, chlorambucil, nitrosoureas and melphalan. It is also a well-known target of gold compounds such as auranofin, currently in clinical trials for anticancer treatment. It has not been known, however, whether inhibition of TrxR1 is a mechanism leading to some of the anticancer effects seen upon use of some of these compounds, or whether this enzyme inhibition is merely a correlative event due to a high inherent reactivity of TrxR1. Novel findings with more specific inhibitors however suggest that TrxR1 can indeed be an authentic anticancer drug target, the inhibition of which yields anticancer efficacy in mouse models without overt toxicity to normal cells and tissues.

Disulfide stress has been associated with inflammation and characterized by an increase in cystine levels and protein cysteinylation. Furthermore, it was recently discovered that thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) exhibits efficient cystine reductase activity. The aim of our research was to elucidate if TRP14 is also able to reduce cysteinylated proteins in mammalian cells. Thus, protein cysteinylation was assessed in control and TRP14 knockdown cells in vitro through their pre-treatment with 25 µg/ml cycloheximide for 30 min and incubation with 250 µM biotinylated cysteine for 1 h. Moreover, such TRP14 knockdown cell lysates were tested as cysteinylated substrates for TRP14 activity through their incubation with 20 µM human TRP14, 1 uM thioredoxin reductase 1 (TrxR1) and 1 mM NADPH for 15 min at 25oC. TRP14 knockdown cells showed more protein cysteinylation than control cells. Human TRP14, in a reaction together with TrxR1 and NADPH, could also reduce cysteinylated protein substrates in cell lysates. In conclusion, TRP14 is able to directly reduce protein cysteinylation motifs. The potential in vivo significance of this enzymatic activity awaits further clarification.

Cancer cells have higher basal levels of reactive oxygen species (ROS) production, and altered expression of antioxidant enzymes, when compared to normal cells. At high levels, ROS can cause substantial cellular damage, often leading to cell death, and many cancer therapeutics function through targeting these imbalanced redox systems. Glutathione peroxidases (GPx) protect cells from increased levels of ROS and oxidative stress by coupling the reduction of hydrogen and organic peroxides with the oxidization of glutathione (GSH) to glutathione disulfide (GSSG) [1]. Because of the role of glutathione peroxidases in controlling ROS levels, reducing oxidative stress, and regulating cell death pathways, we are interested in identifying inhibitors of GPx1 and GPx4. Moreover, GPx4 has recently been identified as a unique and important regulator of non-apoptotic cell death pathway, ferroptosis [2]. Using a novel method of recombinant GPx expression [3] we are developing an assay amenable to high-throughput screening of small molecules targeting GPx, and will characterize the identified inhibitors as potential chemotherapeutics for treating Glioblastoma Multiforme.

The defining entity of a selenoprotein is the inclusion of at least one selenocysteine (Sec) residue in its sequence. Sec, the 21st naturally occurring genetically encoded amino acid, differs from its significantly more common structural analog cysteine (Cys) by the identity of a single atom: Sec contains selenium instead of the sulfur found in Cys. Selenium clearly has unique chemical properties that differ from sulfur, but more striking are perhaps the similarities between the two elements. Selenium was discovered by Jöns Jacob Berzelius, a renowned Swedish scientist instrumental in establishing the institution that would become Karolinska Institutet. Written at the occasion of the bicentennial anniversary of Karolinska Institutet, this mini review focuses on the unique selenium-derived properties that may potentially arise in a protein upon the inclusion of Sec in place of Cys. With 25 human genes encoding selenoproteins and in total several thousand selenoproteins yet described in nature, it seems likely that the presence of that single selenium atom of Sec should convey some specific feature, thereby explaining the existence of selenoproteins in spite of demanding and energetically costly Sec-specific synthesis machineries. Nonetheless, most, if not all, of the currently known selenoproteins are also found as Cys-containing non-selenoprotein orthologues in other organisms, wherefore any potentially unique properties of selenoproteins are yet a matter of debate. The pK(a) of free Sec (approximately 5.2) being significantly lower than that of free Cys (approximately 8.5) has often been proposed as one of the unique features of Sec. However, as discussed herein, this pK(a) difference between Sec and Cys can hardly provide an evolutionary pressure for maintenance of selenoproteins. Moreover, the typically 10- to 100-fold lower enzymatic efficiencies of Sec-to-Cys mutants of selenoprotein oxidoreductases, are also weak arguments for the overall existence of selenoproteins. Here, it is however emphasized that the inherent high nucleophilicity of Sec and thereby its higher chemical reaction rate with electrophiles, as compared to Cys, seems to be a truly unique property of Sec that cannot easily be mimicked by the basicity of Cys, even within the microenvironment of a protein. The chemical rate enhancement obtained with Sec can have other consequences than those arising from a low redox potential of some Cys-dependent proteins, typically aiming at maintaining redox equilibria. Another unique aspect of Sec compared to Cys seems to be its efficient potency to support one-electron transfer reactions, which, however, has not yet been unequivocally shown as a Sec-dependent step during the natural catalysis of any known selenoprotein enzyme.

Mammalian thioredoxin reductase 1 (TrxR1) is a selenoprotein that contains a selenocysteine (Sec) residue at the penultimate C-terminal position. When rat TrxR1 is expressed recombinantly in Escherichia coli, partial truncation at the Sec-encoding UGA gives rise to additional protein species that lack Sec. Phenylarsine oxide (PAO) Sepharose can subsequently be used to enrich the Sec-containing protein and yield activity corresponding to that of native enzyme. Herein we extensively purified recombinant rat TrxR1 over PAO Sepharose, which gave an enzyme with about 53 U/mg specific activity. Surprisingly, only about 65% of the subunits of this TrxR1 preparation contained Sec, whereas about 35% were protein products derived from UGA truncation. Further analyses revealed a theoretical maximal specific activity of 70-80 U/mg for the homodimeric enzyme with full Sec content, i.e., significantly higher than that reported for native TrxR1. We also discovered the formation of highly stable noncovalently linked tetrameric forms of TrxR1, having full FAD content but about half the specific activity in relation to the selenium content compared to the dimeric protein. The characterization of these different forms of recombinant TrxR1 revealed that inherent turnover capacity of the enzyme must be revised, that multimeric states of the protein may be formed, and that the yield of bacterial selenoprotein production may be lower than earlier reported. The biological significance of the hitherto unsurpassed high specific activity of the enzyme involves the capacity to support a higher turnover in vivo than previously believed. The tetrameric forms of the protein could represent hitherto unknown regulatory states of the enzyme.

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli , catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P BAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P BAD -promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.

Significance: All cells must maintain a balance between oxidants and reductants, while allowing for fluctuations in redox states triggered by signaling, altered metabolic flow, or extracellular stimuli. Furthermore, they must be able to rapidly sense and react to various challenges that would disrupt the redox homeostasis. Recent Advances: Many studies have identified Keap1 as a key sensor for oxidative or electrophilic stress, with modification of Keap1 by oxidation or electrophiles triggering Nrf2-mediated transcriptional induction of enzymes supporting reductive and detoxification pathways. However, additional mechanisms for Nrf2 regulation are likely to exist upstream of, or in parallel with, Keap1. Critical Issues: Here, we propose that the mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a potent regulator of Nrf2. A high chemical reactivity of TrxR1 and its vital role for the thioredoxin (Trx) system distinguishes TrxR1 as a prime target for electrophilic challenges. Chemical modification of the selenocysteine (Sec) in TrxR1 by electrophiles leads to rapid inhibition of thioredoxin disulfide reductase activity, often combined with induction of NADPH oxidase activity of the derivatized enzyme, thereby affecting many downstream redox pathways. The notion of TrxR1 as a regulator of Nrf2 is supported by many publications on effects in human cells of selenium deficiency, oxidative stress or electrophile exposure, as well as the phenotypes of genetic mouse models. Future Directions: Investigation of the role of TrxR1 as a regulator of Nrf2 activation will facilitate further studies of redox control in diverse cells and tissues of mammals, and possibly also in animals of other classes. Antioxid. Redox Signal. 23, 823–853.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Selenocysteine (Sec; U in one-letter code) is the twenty-first naturally occurring amino acid, with a selenium atom that gives this cysteine (Cys) homolog unique biochemical properties, including a high nucleophilicity and significant reactivity with electrophilic agents. This can be used in biotechnological Sec-dependent applications. Here, we describe how Sec can be introduced into a carboxy-terminal tetrapeptide motif (-Gly-Cys-Sec-Gly-COOH, known as a Sel-tag) for recombinant proteins by tailoring the encoding gene to become compatible with the Escherichia coli selenoprotein synthesis machinery. We also describe how the Sel-tag can be used as a basis for efficient one-step protein purification, rapid Sec-targeting protein labeling with electrophilic compounds, or radiolabeling with the positron emitter 11C.