Selenoprotein Gene Nomenclature.

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.

Selenium and redox signaling.

Selenium compounds that contain selenol functions or can be metabolized to selenols are toxic via superoxide and H2O2 generation, when ingested at dosages beyond requirement. At supra-nutritional dosages various forms of programmed cell death are observed. At physiological intakes, selenium exerts its function as constituent of selenoproteins, which overwhelmingly are oxidoreductases. Out of those, the glutathione peroxidases counteract hydroperoxide-stimulated signaling cascades comprising inflammation triggered by cytokines or lipid mediators, insulin signaling and different forms of programmed cell death. Similar events are exerted by peroxiredoxins, which functionally depend on the selenoproteins of the thioredoxin reductase family. The thiol peroxidases of both families can, however, also act as sensors for hydroperoxides, thereby initiating signaling cascades. Although the interaction of selenoproteins with signaling events has been established by genetic techniques, the in vivo relevance of these findings is still hard to delineate for several reasons: The biosynthesis of individual selenoproteins responds differently to variations of selenium intakes; selenium is preferentially delivered to privileged tissues via inter-organ trafficking and receptor-mediated uptake, and only half of the selenoproteins known by sequence have been functionally characterized. The fragmentary insights do not allow any uncritical use of selenium for optimizing human health.

Helmut Sies and the compartmentation of hydroperoxide metabolism.

The early work of Helmut Sies on mammalian hydroperoxide metabolism is reviewed with particular emphasis on the in situ function of catalase and glutathione peroxidase1. Starting out from a catalase-dominated thinking in the middle of the last century, Sies first demonstrated, by whole organ spectroscopy, that H2O2 is generated in rat liver and metabolized by catalase. In a joined effort with the author's group, he then worked out that glutathione peroxidase can kinetically compete with catalase in hydroperoxide metabolism in situ. In compartmentalized cells, however, the "competition" of the two enzymes turned out to be a mutual complementation because of their different subcellular location. The studies for the first time documented that the metabolism of freely diffusible hydroperoxides is compartmentalized and, thus, paved the way to a better understanding of oxidant challenges and redox regulation. The article, garnished with personal memories, is meant as a nostalgic journey though ancient times of biochemistry with their changing fashions and paradigms, revealing the roots of topical perspectives and controversies in redox biology.

The fairytale of the GSSG/GSH redox potential.

BACKGROUND: The term GSSG/GSH redox potential is frequently used to explain redox regulation and other biological processes. SCOPE OF REVIEW: The relevance of the GSSG/GSH redox potential as driving force of biological processes is critically discussed. It is recalled that the concentration ratio of GSSG and GSH reflects little else than a steady state, which overwhelmingly results from fast enzymatic processes utilizing, degrading or regenerating GSH. MAJOR CONCLUSIONS: A biological GSSG/GSH redox potential, as calculated by the Nernst equation, is a deduced electrochemical parameter based on direct measurements of GSH and GSSG that are often complicated by poorly substantiated assumptions. It is considered irrelevant to the steering of any biological process. GSH-utilizing enzymes depend on the concentration of GSH, not on [GSH](2), as is predicted by the Nernst equation, and are typically not affected by GSSG. Regulatory processes involving oxidants and GSH are considered to make use of mechanistic principles known for thiol peroxidases which catalyze the oxidation of hydroperoxides by GSH by means of an enzyme substitution mechanism involving only bimolecular reaction steps. GENERAL SIGNIFICANCE: The negligibly small rate constants of related spontaneous reactions as compared with enzyme-catalyzed ones underscore the superiority of kinetic parameters over electrochemical or thermodynamic ones for an in-depth understanding of GSH-dependent biological phenomena. At best, the GSSG/GSH potential might be useful as an analytical tool to disclose disturbances in redox metabolism. This article is part of a Special Issue entitled Cellular Functions of Glutathione.

The trypanothione system and its implications in the therapy of trypanosomatid diseases.

Biosynthesis and the use of trypanothione, a redox metabolite of parasitic trypanosomatids, are reviewed here with special emphasis on the development of trypanocidal drugs. This metabolic system is unique to and essential for the protozoal parasites. Selective inhibition of key elements of trypanothione metabolism, therefore, promises eradication of the parasites without affecting the host. Considering the metabolic importance and drugability of system components, inhibition of the enzymes for regeneration and de novo synthesis of trypanothione is rated as the most promising approach, while related peroxidases and redoxins are disregarded as targets because of limited chances to achieve selective inhibition. The organizational need to exploit the accumulating knowledge of trypanosomatid metabolism for medical practice is briefly addressed.

The glutathione peroxidase family: Discoveries and mechanism.

The discoveries leading to our present understanding of the glutathione peroxidases (GPxs) are recalled. The cytosolic GPx, now GPx1, was first described by Mills in 1957 and claimed to depend on selenium by Rotruck et al., in 1972. With the determination of a stoichiometry of one selenium per subunit, GPx1 was established as the first selenoenzyme of vertebrates. In the meantime, the GPxs have grown up to a huge family of enzymes that prevent free radical formation from hydroperoxides and, thus, are antioxidant enzymes, but they are also involved in regulatory processes or synthetic functions. The kinetic mechanism of the selenium-containing GPxs is unusual in neither showing a defined KM nor any substrate saturation. More recently, the reaction mechanism has been investigated by the density functional theory and nuclear magnetic resonance of model compounds mimicking the reaction cycle. The resulting concept sees a selenolate oxidized to a selenenic acid. This very fast reaction results from a concerted dual attack on the hydroperoxide bond, a nucleophilic one by the selenolate and an electrophilic one by a proton that is unstably bound in the reaction center. Postulated intermediates have been identified either in the native enzymes or in model compounds.

A fast virtual screening approach to identify structurally diverse inhibitors of trypanothione reductase.

Trypanothione reductase (TryR) is one of the favorite targets for those designing drugs for the treatment of Chagas disease. We present the application of a fast virtual screening approach for designing hit compounds active against TryR. Our protocol combines information derived from structurally known inhibitors and from the TryR receptor structure. Five structurally diverse hit compounds active against TryR and holding promise for the treatment of Chagas disease are reported.

Selenium-Catalyzed Reduction of Hydroperoxides in Chemistry and Biology.

Among the chalcogens, selenium is the key element for catalyzed H2O2 reduction. In organic synthesis, catalytic amounts of organo mono- and di-selenides are largely used in different classes of oxidations, in which H2O2 alone is poorly efficient. Biological hydroperoxide metabolism is dominated by peroxidases and thioredoxin reductases, which balance hydroperoxide challenge and contribute to redox regulation. When their selenocysteine is replaced by cysteine, the cellular antioxidant defense system is impaired. Finally, classes of organoselenides have been synthesized with the aim of mimicking the biological strategy of glutathione peroxidases, but their therapeutic application has so far been limited. Moreover, their therapeutic use may be doubted, because H2O2 is not only toxic but also serves as an important messenger. Therefore, over-optimization of H2O2 reduction may lead to unexpected disturbances of metabolic regulation. Common to all these systems is the nucleophilic attack of selenium to one oxygen of the peroxide bond promoting its disruption. In this contribution, we revisit selected examples from chemistry and biology, and, by using results from accurate quantum mechanical modelling, we provide an accurate unified picture of selenium's capacity of reducing hydroperoxides. There is clear evidence that the selenoenzymes remain superior in terms of catalytic efficiency.

Proton Transfer and SN 2 Reactions as Steps of Fast Selenol and Thiol Oxidation in Proteins: A Model Molecular Study Based on GPx.

Invited for this month's cover are collaborating groups from Università degli Studi di Padova, Vrije Universiteit Amsterdam, and Universidad de la República Uruguay. The cover picture shows two lorries along the road directed to the destination 'H2 O2 reduction', and the selenol (SeH) lorry is faster than the thiol (SH) lorry. This cartoon represents the situation of glutathione peroxidase (GPx), in which the presence of selenium rather than sulfur warrants a significantly faster hydroperoxide reduction along the same mechanistic path. Read the full text of the article at 10.1002/cplu.202000660.

Proton Transfer and SN 2 Reactions as Steps of Fast Selenol and Thiol Oxidation in Proteins: A Model Molecular Study Based on GPx.

The so-called peroxidatic cysteines and selenocysteines in proteins reduce hydroperoxides through a dual attack to the peroxide bond in a two-step mechanism. First, a proton dislocation from the thiol/selenol to a close residue of the enzymatic pocket occurs. Then, a nucleophilic attack of the anionic cysteine/selenocysteine to one O atom takes place, while the proton is shuttled back to the second O atom, promoting the formation of a water molecule. In this computational study, we use a molecular model of GPx to demonstrate that the enzymatic environment significantly lowers the barrier of the latter SN 2 step. Particularly, in our Se-based model the energy barriers for the two steps are 29.82 and 2.83 kcal mol-1 , both higher than the corresponding barriers computed in the enzymatic cluster, i. e., 21.60 and null, respectively. Our results, obtained at SMD-B3LYP-D3(BJ)/6-311+G(d,p), cc-pVTZ//B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ level of theory, show that the mechanistic details can be well reproduced using an oversimplified model, but the energetics is definitively more favorable in the GPx active site. In addition, we pinpoint the role of the chalcogen in the peroxide reduction process, rooting the advantages of the presence of selenium in its acidic and nucleophilic properties.

The labour pains of biochemical selenology: the history of selenoprotein biosynthesis.

The serendipitous discoveries leading to the present knowledge on selenium's role in biology are reviewed. Detected in 1818 as by-product of sulphuric acid production, selenium first attracted medical attention as an industrial hazard. In parallel selenium intoxication was recognized as cause of life stock diseases. Reports on teratogenic effects and carcinogenicity of selenium followed since the middle of the past century. In 1954 first hints towards specific biological functions of selenium were contributed from microbiology, and its essentiality for mammalian life was discovered in 1957. Independent and unrelated studies led to the identification of selenium as an integral constituent of one mammalian and two bacterial enzymes in the early 70ies followed by the identification of selenocysteine in these proteins. In the 80ies, independent sequencing of selenoproteins and cloned DNAs revealed that the selenocysteine of selenoproteins is encoded by the termination codon TGA (UGA). Recoding of TGA as selenocysteine codon by secondary mRNA structures was first elucidated by molecular genetics in bacteria and later in mammals. During the 90ies, finally, the basic principles of selenoprotein synthesis were worked out by molecular biology tools. The article closes with spotlight comments on proven and potential biomedical benefits of selenium and related research deficits.

Catalytic mechanisms and specificities of glutathione peroxidases: variations of a basic scheme.

Kinetics and molecular mechanisms of GPx-type enzymes are reviewed with emphasis on structural features relevant to efficiency and specificity. In Sec-GPxs the reaction takes place at a single redox centre with selenocysteine as redox-active residue (peroxidatic Sec, U(P)). In contrast, most of the non-vertebrate GPx have the U(P) replaced by a cysteine (peroxidatic Cys, C(P)) and work with a second redox centre that contains a resolving cysteine (C(R)). While the former type of enzymes is more or less specific for GSH, the latter are reduced by "redoxins". The common denominator of the GPx family is the first redox centre comprising the (seleno)cysteine, tryptophan, asparagine and glutamine. In this architectural context the rate of hydroperoxide reduction by U(P) or C(P), respectively, is enhanced by several orders of magnitude compared to that of free selenolate or thiolate. Mammalian GPx-1 dominates H(2)O(2) metabolism, whereas the domain of GPx-4 is the reduction of lipid hydroperoxides with important consequences such as counteracting 12/15-lipoxygenase-induced apoptosis and regulation of inflammatory responses. Beyond, the degenerate GSH specificity of GPx-4 allows selenylation and oxidation to disulfides of protein thiols. Heterodimer formation of yeast GPx with a transcription factor is discussed as paradigm of a redox sensing that might also be valid in vertebrates.

Looking Back at the Early Stages of Redox Biology.

The beginnings of redox biology are recalled with special emphasis on formation, metabolism and function of reactive oxygen and nitrogen species in mammalian systems. The review covers the early history of heme peroxidases and the metabolism of hydrogen peroxide, the discovery of selenium as integral part of glutathione peroxidases, which expanded the scope of the field to other hydroperoxides including lipid hydroperoxides, the discovery of superoxide dismutases and superoxide radicals in biological systems and their role in host defense, tissue damage, metabolic regulation and signaling, the identification of the endothelial-derived relaxing factor as the nitrogen monoxide radical (more commonly named nitric oxide) and its physiological and pathological implications. The article highlights the perception of hydrogen peroxide and other hydroperoxides as signaling molecules, which marks the beginning of the flourishing fields of redox regulation and redox signaling. Final comments describe the development of the redox language. In the 18th and 19th century, it was highly individualized and hard to translate into modern terminology. In the 20th century, the redox language co-developed with the chemical terminology and became clearer. More recently, the introduction and inflationary use of poorly defined terms has unfortunately impaired the understanding of redox events in biological systems.

Regulatory Phenomena in the Glutathione Peroxidase Superfamily.

Significance: The selenium-containing Glutathione peroxidases (GPxs)1-4 protect against oxidative challenge, inhibit inflammation and oxidant-induced regulated cell death. Recent Advances: GPx1 and GPx4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides. GPx2 regulates the balance between regeneration and apoptotic cell shedding in the intestine. It inhibits inflammation-induced carcinogenesis in the gut but promotes growth of established cancers. GPx3 deficiency facilitates platelet aggregation likely via disinhibition of thromboxane biosynthesis. It is also considered a tumor suppressor. GPx4 is expressed in three different forms. The cytosolic form proved to inhibit interleukin-1-driven nuclear factor κB activation and leukotriene biosynthesis. Moreover, it is a key regulator of ferroptosis, because it reduces hydroperoxy groups of complex lipids and silences lipoxygenases. By alternate substrate use, the nuclear form contributes to chromatin compaction. Mitochondrial GPx4 forms the mitochondrial sheath of spermatozoa and, thus, guarantees male fertility. Out of the less characterized GPxs, the cysteine-containing GPx7 and GPx8 are unique in contributing to oxidative protein folding in the endoplasmic reticulum by reacting with protein isomerase as an alternate substrate. A yeast 2-Cysteine glutathione peroxidase equipped with CP and CR was reported to sense H2O2 for inducing an adaptive response. Critical Issues: Most of the findings compiled are derived from tissue culture and/or animal studies only. Their impact on human physiology is sometimes questionable. Future Directions: The expression of individual GPxs and GPx-dependent regulatory phenomena are to be further investigated, in particular in respect to human health.

Insights into the redox biology of Trypanosoma cruzi: Trypanothione metabolism and oxidant detoxification.

Trypanosoma cruzi is the etiologic agent of Chagas' disease, an infection that affects several million people in Latin America. With no immediate prospect of a vaccine and problems associated with current chemotherapies, the development of new treatments is an urgent priority. Several aspects of the redox metabolism of this parasite differ enough from those in the mammalian host to be considered targets for drug development. Here, we review the information about a trypanosomatid-specific molecule centrally involved in redox metabolism, the dithiol trypanothione, and the main effectors of cellular antioxidant defense. We focus mainly on data from T. cruzi, making comparisons with other trypanosomatids whenever possible. In these parasites trypanothione participates in crucial thiol-disulfide exchange reactions and serves as electron donor in different metabolic pathways, from synthesis of DNA precursors to oxidant detoxification. Interestingly, the levels of several enzymes involved in trypanothione metabolism and oxidant detoxification increase during the transformation of T. cruzi to its mammalian-infective form and the overexpression of some of them has been associated with increased resistance to macrophage-dependent oxidative killing. Together, the evidence suggests a central role of the trypanothione-dependent antioxidant systems in the infection process.

The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases.

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.

Depletion of the thioredoxin homologue tryparedoxin impairs antioxidative defence in African trypanosomes.

In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3-7-fold higher in the parasite form infecting mammals (50-100 microM) than in the form hosted by the tsetse fly (7-34 microM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 muM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2-3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.

Introduction. History of the peroxiredoxins and topical perspectives.

We have surveyed the early biochemical, structural and enzymatic studies on the peroxiredoxin family, within the broad context of the other chapters included within this book. Both the antioxidant defence and peroxide-linked cell signalling roles of the peroxiredoxins are introduced. The possible membrane-association of certain peroxiredoxins is assessed and the structural characterization of the peroxiredoxins by electron microscopy is given some emphasis here. The important contribution of X-ray crystallographic studies to the understanding of peroxiredoxin structure is given due attention. Finally, some medical perpectives are introduced, with emphasis upon the understanding of the microbial peroxiredoxins as possible future drug targets.

Kinetics of peroxiredoxins and their role in the decomposition of peroxynitrite.

Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.