Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis.

Selenoproteins are rare proteins among all kingdoms of life containing the 21st 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 Gpx4cys/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.

Redox active motifs in selenoproteins.

Selenoproteins use the rare amino acid selenocysteine (Sec) to act as the first line of defense against oxidants, which are linked to aging, cancer, and neurodegenerative diseases. Many selenoproteins are oxidoreductases in which the reactive Sec is connected to a neighboring Cys and able to form a ring. These Sec-containing redox motifs govern much of the reactivity of selenoproteins. To study their fundamental properties, we have used (77)Se NMR spectroscopy in concert with theoretical calculations to determine the conformational preferences and mobility of representative motifs. This use of (77)Se as a probe enables the direct recording of the properties of Sec as its environment is systematically changed. We find that all motifs have several ring conformations in their oxidized state. These ring structures are most likely stabilized by weak, nonbonding interactions between the selenium and the amide carbon. To examine how the presence of selenium and ring geometric strain governs the motifs' reactivity, we measured the redox potentials of Sec-containing motifs and their corresponding Cys-only variants. The comparisons reveal that for C-terminal motifs the redox potentials increased between 20-25 mV when the selenenylsulfide bond was changed to a disulfide bond. Changes of similar magnitude arose when we varied ring size or the motifs' flanking residues. This suggests that the presence of Sec is not tied to unusually low redox potentials. The unique roles of selenoproteins in human health and their chemical reactivities may therefore not necessarily be explained by lower redox potentials, as has often been claimed.

Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase.

Thiophosphate (SPO(3)) was recently shown to promote cysteine insertion at Sec (selenocysteine)-encoding UGA codons during selenoprotein synthesis. We reported previously that irreversible targeting by cDDP [cis-diamminedichloroplatinum(II) or cisplatin] of the Sec residue in TrxR1 (thioredoxin reductase 1) contributes to cDDP cytotoxicity. This effect could possibly be attenuated in cells expressing less reactive Sec-to-cysteine-substituted TrxR1 variants, or pronounced in cells with higher levels of Sec-containing TrxR1. To test this, we supplemented cells with either SPO(3) or selenium and subsequently determined total as well as specific activities of cellular TrxR1, together with extent of drug-induced cell death. We found that cDDP became less cytotoxic after incubation of A549 or HCT116 cells with lower SPO(3) concentrations (100-300 µM), whereas higher SPO(3) (>300 µM) had pronounced direct cytotoxicity. NIH 3T3 cells showed low basal TrxR1 activity and high susceptibility to SPO(3) cytotoxicity, or to glutathione depletion. Supplementing NIH 3T3 cells with selenite, however, gave increased cellular TrxR1 activity with concomitantly decreased dependence on glutathione, whereas the susceptibility to cDDP increased. The results suggest molecular mechanisms by which the selenium status of cells can affect their glutathione dependence while modulating the cytotoxicity of drugs that target TrxR1.

Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase.

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

Biochemical discrimination between selenium and sulfur 2: mechanistic investigation of the selenium specificity of human selenocysteine lyase.

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom.The crystal structure of hSCL was recently determined and gain-of-function protein variants that also could accept Cys as substrate were identified. To obtain mechanistic insight into the chemical basis for its substrate discrimination, we here report time-resolved spectroscopic studies comparing the reactions of the Sec-specific wild-type hSCL and the gain-of-function D146K/H389T variant, when given Cys as a substrate. The data are interpreted in light of other studies of SCL/CD enzymes and offer mechanistic insight into the function of the wild-type enzyme. Based on these results and previously available data we propose a reaction mechanism whereby the Sec over Cys specificity is achieved using a combination of chemical and physico-mechanical control mechanisms.

The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.

Focus on mammalian thioredoxin reductases--important selenoproteins with versatile functions.

Thioredoxin systems, involving redox active thioredoxins and thioredoxin reductases, sustain a number of important thioredoxin-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense and redox-regulated signaling cascades. Mammalian thioredoxin reductases are selenium-containing flavoprotein oxidoreductases, dependent upon a selenocysteine residue for reduction of the active site disulfide in thioredoxins. Their activity is required for normal thioredoxin function. The mammalian thioredoxin reductases also display surprisingly multifaceted properties and functions beyond thioredoxin reduction. Expressed from three separate genes (in human named TXNRD1, TXNRD2 and TXNRD3), the thioredoxin reductases can each reduce a number of different types of substrates in different cellular compartments. Their expression patterns involve intriguingly complex transcriptional mechanisms resulting in several splice variants, encoding a number of protein variants likely to have specialized functions in a cell- and tissue-type restricted manner. The thioredoxin reductases are also targeted by a number of drugs and compounds having an impact on cell function and promoting oxidative stress, some of which are used in treatment of rheumatoid arthritis, cancer or other diseases. However, potential specific or essential roles for different forms of human or mouse thioredoxin reductases in health or disease are still rather unclear, although it is known that at least the murine Txnrd1 and Txnrd2 genes are essential for normal development during embryogenesis. This review is a survey of current knowledge of mammalian thioredoxin reductase function and expression, with a focus on human and mouse and a discussion of the striking complexity of these proteins. Several yet open questions regarding their regulation and roles in different cells or tissues are emphasized. It is concluded that the intriguingly complex regulation and function of mammalian thioredoxin reductases within the cellular context and in intact mammals strongly suggests that their functions are highly fi ne-tuned with the many pathways involving thioredoxins and thioredoxin-related proteins. These selenoproteins furthermore propagate many functions beyond a reduction of thioredoxins. Aberrant regulation of thioredoxin reductases, or a particular dependence upon these enzymes in diseased cells, may underlie their presumed therapeutic importance as enzymatic targets using electrophilic drugs. These reductases are also likely to mediate several of the effects on health and disease that are linked to different levels of nutritional selenium intake. The thioredoxin reductases and their splice variants may be pivotal components of diverse cellular signaling pathways, having importance in several redox-related aspects of health and disease. Clearly, a detailed understanding of mammalian thioredoxin reductases is necessary for a full comprehension of the thioredoxin system and of selenium dependent processes in mammals.

Highly active dimeric and low-activity tetrameric forms of selenium-containing rat thioredoxin reductase 1.

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.

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation.

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.

Active sites of thioredoxin reductases: why selenoproteins?

Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

Rapid induction of cell death by selenium-compromised thioredoxin reductase 1 but not by the fully active enzyme containing selenocysteine.

Mammalian thioredoxin reductases are selenoproteins. For native catalytic activity, these enzymes utilize a C-terminal -Gly-Cys-Sec-Gly-COOH sequence (where Sec is selenocysteine) forming a redox active selenenylsulfide/selenolthiol motif. A range of cellular systems depend upon or are regulated by thioredoxin reductase and its major protein substrate thioredoxin, including apoptosis signal-regulating kinase 1, peroxiredoxins, methionine sulfoxide reductase, and several transcription factors. Cytosolic thioredoxin reductase 1 (TrxR1) is moreover inhibited by various electrophilic anticancer compounds. TrxR1 is hence generally considered to promote cell viability. However, several recent studies have suggested that TrxR1 may promote apoptosis, and the enzyme was identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12). Transient transfection with GRIM-12/TrxR1 was also shown to directly induce cell death. To further analyze such effects, we have here employed lipid-mediated delivery of recombinant TrxR1 preparations into human A549 cells, thereby bypassing selenoprotein translation to facilitate assessment of the protein-related effects on cell viability. We found that selenium-deficient TrxR1, having a two-amino acid-truncated C-terminal -Gly-Cys-COOH motif, rapidly induced cell death (38 +/- 29% apoptotic cells after 4 h; p < 0.005 compared with controls). Cell death induction was also promoted by selenium-compromised TrxR1 derivatized with either cis-diamminedichloroplatinum (II) (cisplatin) or dinitrophenyl moieties but not by the structurally related non-selenoprotein glutathione reductase. In contrast, TrxR1 with intact selenocysteine could not promote cell death. The direct cellular effects of selenium-compromised forms of TrxR1 may be important for the pathophysiology of selenium deficiency as well as for the efficacy of antiproliferative drugs targeting the selenocysteine moiety of this enzyme.