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.

Gold-Promoted Biocompatible Selenium Arylation of Small Molecules, Peptides and Proteins.

A low pKa (5.2), high polarizable volume (3.8 Å), and proneness to oxidation under ambient conditions make selenocysteine (Sec, U) a unique, natural reactive handle present in most organisms across all domains of life. Sec modification still has untapped potential for site-selective protein modification and probing. Herein we demonstrate the use of a cyclometalated gold(III) compound, [Au(bnpy)Cl2 ], in the arylation of diselenides of biological significance, with a scope covering small molecule models, peptides, and proteins using a combination of multinuclear NMR (including 77 Se NMR), and LC-MS. Diphenyl diselenide (Ph-Se)2 and selenocystine, (Sec)2 , were used for reaction optimization. This approach allowed us to demonstrate that an excess of diselenide (Au/Se-Se) and an increasing water percentage in the reaction media enhance both the conversion and kinetics of the C-Se coupling reaction, a combination that makes the reaction biocompatible. The C-Se coupling reaction was also shown to happen for the diselenide analogue of the cyclic peptide vasopressin ((Se-Se)-AVP), and the Bos taurus glutathione peroxidase (GPx1) enzyme in ammonium acetate (2 mM, pH=7.0). The reaction mechanism, studied by DFT revealed a redox-based mechanism where the C-Se coupling is enabled by the reductive elimination of the cyclometalated Au(III) species into Au(I).

Electrochemical Modification of Polypeptides at Selenocysteine.

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.

Highly Electrophilic Intermediates in the Bypass Mechanism of Glutathione Peroxidase: Synthesis, Reactivity, and Structures of Selenocysteine-Derived Cyclic Selenenyl Amides.

Selenocysteine (Sec)-derived cyclic selenenyl amides, formed by the intramolecular cyclization of Sec selenenic acids (Sec-SeOHs), have been postulated to function as protective forms in the bypass mechanism of glutathione peroxidase (GPx). However, their chemical properties have not been experimentally elucidated in proteins or small-molecule systems. Recently, we reported the first nuclear magnetic resonance observation of Sec-SeOHs and their cyclization to the corresponding cyclic selenenyl amides by using selenopeptide model systems incorporated in a molecular cradle. Herein, we elucidate the structures and reactivities of Sec-derived cyclic selenenyl amides. The crystal structures and reactions toward a cysteine thiol or a 1,3-diketone-type chemical probe indicated the highly electrophilic character of cyclic selenenyl amides. This suggests that they can serve not only as protective forms to suppress the inactivation of Sec-SeOHs in GPx but also as highly electrophilic intermediates in the reactions of selenoproteins.

Selenium in Peptide Chemistry.

In recent years, researchers have been exploring the potential of incorporating selenium into peptides, as this element possesses unique properties that can enhance the reactivity of these compounds. Selenium is a non-metallic element that has a similar electronic configuration to sulfur. However, due to its larger atomic size and lower electronegativity, it is more nucleophilic than sulfur. This property makes selenium more reactive toward electrophiles. One of the most significant differences between selenium and sulfur is the dissociation of the Se-H bond. The Se-H bond is more easily dissociated than the S-H bond, leading to higher acidity of selenocysteine (Sec) compared to cysteine (Cys). This difference in acidity can be exploited to selectively modify the reactivity of peptides containing Sec. Furthermore, Se-H bonds in selenium-containing peptides are more susceptible to oxidation than their sulfur analogs. This property can be used to selectively modify the peptides by introducing new functional groups, such as disulfide bonds, which are important for protein folding and stability. These unique properties of selenium-containing peptides have found numerous applications in the field of chemical biology. For instance, selenium-containing peptides have been used in native chemical ligation (NCL). In addition, the reactivity of Sec can be harnessed to create cyclic and stapled peptides. Other chemical modifications, such as oxidation, reduction, and photochemical reactions, have also been applied to selenium-containing peptides to create novel molecules with unique biological properties.

Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases.

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

Concomitant selenoenzyme inhibitor exposures as etiologic contributors to disease: Implications for preventative medicine.

The physiological activities of selenium (Se) occur through enzymes that incorporate selenocysteine (Sec), a rare but important amino acid. The human genome includes 25 genes coding for Sec that employ it to catalyze challenging reactions. Selenoenzymes control thyroid hormones, calcium activities, immune responses, and perform other vital roles, but most are devoted to preventing and reversing oxidative damage. As the most potent intracellular nucleophile (pKa 5.2), Sec is vulnerable to binding by metallic and organic soft electrophiles (E*). These electron poor reactants initially form covalent bonds with nucleophiles such as cysteine (Cys) whose thiol (pKa 8.3) forms adducts which function as suicide substrates for selenoenzymes. These adducts orient E* to interact with Sec and since Se has a higher affinity for E* than sulfur, the E* transfers to Sec and irreversibly inhibits the enzyme's activity. Organic electrophiles have lower Se-binding affinities than metallic E*, but exposure sources are more abundant. Individuals with poor Se status are more vulnerable to the toxic effects of high E* exposures. The relative E*:Se stoichiometries remain undefined, but the aggregate effects of multiple E* exposures are predicted to be additive and possibly synergistic under certain conditions. The potential for the combined Se-binding effects of common pharmaceutical, dietary, or environmental E* require study, but even temporary loss of selenoenzyme activities would accentuate oxidative damage to tissues. As various degenerative diseases are associated with accumulating DNA damage, defining the effects of complementary E* exposures on selenoenzyme activities may enhance the ability of preventative medicine to support healthy aging.

Chemical synthesis of per-selenocysteine human epidermal growth factor.

Human seleno-epidermal growth factor (seleno-EGF), a 53-residue peptide where all six cysteine residues of the parent human EGF sequence were replaced by selenocysteines, was synthesized and the oxidative folding of a polypeptide containing three diselenide bonds was compared to that of the parent cysteine peptide. The crude high performance liquid chromatography (HPLC) profiles clearly showed that both the native EGF and its selenocysteine-analogue fold smoothly, yielding a single sharp peak, proving that even in the case of three disulfide-bonded polypeptides the disulfide-to-diselenide bond substitution is highly isomorphous, as confirmed by conformational circular dichroism measurements and particularly by the biological assays.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

Thioredoxin reductase selenoproteins from different organisms as potential drug targets for treatment of human diseases.

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

The role of glutathione peroxidase-1 in health and disease.

Glutathione peroxidase 1 (GPx1) is an important cellular antioxidant enzyme that is found in the cytoplasm and mitochondria of mammalian cells. Like most selenoenzymes, it has a single redox-sensitive selenocysteine amino acid that is important for the enzymatic reduction of hydrogen peroxide and soluble lipid hydroperoxides. Glutathione provides the source of reducing equivalents for its function. As an antioxidant enzyme, GPx1 modulates the balance between necessary and harmful levels of reactive oxygen species. In this review, we discuss how selenium availability and modifiers of selenocysteine incorporation alter GPx1 expression to promote disease states. We review the role of GPx1 in cardiovascular and metabolic health, provide examples of how GPx1 modulates stroke and provides neuroprotection, and consider how GPx1 may contribute to cancer risk. Overall, GPx1 is protective against the development and progression of many chronic diseases; however, there are some situations in which increased expression of GPx1 may promote cellular dysfunction and disease owing to its removal of essential reactive oxygen species.

On 'Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs' by R.E. Huber and R.S. Criddle.

Selenium was initially considered a toxic element found in plants growing in soils rich in this element. However, a few years later, selenocysteine was recognized as the 21st amino acid. Huber and Criddle's article has been crucial in discovering selenium-containing proteins and other related works on selenocysteine.

Expression of selenoproteins via genetic code expansion in mammalian cells.

Selenoproteins, which contain the 21st amino acid selenocysteine, play roles in maintaining cellular redox homeostasis. Many open questions remain in the field of selenoprotein biology, including the functions of a number of uncharacterized human selenoproteins, and the properties of selenocysteine compared to its analogous amino acid cysteine. The mechanism of selenocysteine incorporation involves an intricate machinery that deviates from the mechanism of incorporation for the canonical 20 amino acids. As a result, recombinant expression of selenoproteins has been historically challenging, and has hindered a deeper evaluation of selenoprotein biology. Genetic code expansion methods, which incorporate protected analogs of selenocysteine, allow the endogenous selenocysteine incorporation mechanism to be bypassed entirely to facilitate selenoprotein expression. Here we present a method for incorporating a photocaged selenocysteine amino acid (DMNB-Sec) into human selenoproteins directly in mammalian cells. This approach offers the opportunity to study human selenoproteins in their native cellular environment and should advance our understanding of selenoprotein biology.

Selenocysteine substitutions in thiyl radical enzymes.

Cysteine thiyl radicals are implicated as cofactors in a variety of enzymatic transformations, as well as transient byproducts of oxidative stress, yet their reactivity has undermined their detailed study. Selenocysteine exhibits a lower corresponding selenyl radical reduction potential, thus taming this radical reactivity without significant steric perturbation, potentially affording a glimpse into otherwise fleeting events in thiyl radical catalysis. In this chapter, we describe a suite of fusion protein constructs for general and efficient production of site-specifically incorporated selenoproteins by a recently developed nonsense suppression technology. As a proof of concept, we produced NikJ, a member of the radical S-adenosyl methionine enzyme family involved in the biosynthesis of peptidyl nucleoside antibiotics. We place emphasis throughout the plasmid assembly, protein expression, and selenium quantitation on accommodating the structural and functional diversity of thiyl radical enzymes. The protocol produces NikJ with near quantitative selenocysteine insertion, 50% nonsense read-through, and facile protein purification.

Chemoproteomic interrogation of selenocysteine by low-pH isoTOP-ABPP.

Selenoproteins comprise a small group of selenocysteine (Sec) containing proteins, often involved in redox homeostasis. While Sec is functionally similar to cysteine (Cys), with both acting as protein-centered nucleophiles, chemoproteomic strategies employing electrophilic probes have often failed to rigorously identify Sec residues, due to their relatively low abundance with respect to Cys across a proteome. To improve the enrichment and detection of selenoproteins, herein we describe a chemoproteomic strategy that relies on the unique properties of Sec as compared to Cys, such as reduced pKa and the unique isotopic distribution of selenium. Low pH electrophilic probe labeling of mouse proteomes reduces Cys reactivity, resulting in increased identification of most soluble selenoproteins. This quantitative chemoproteomic platform provides a method to reliably measure changes in selenoprotein abundance across growth conditions as well as quantify inhibition by selenoprotein specific inhibitors, such as Auranofin.

Assay of selenol species in biological samples by the fluorescent probe Sel-green.

Selenium (Se) is an essential trace element for diverse cellular functions. The biological significance of Se is predominantly dependent on its incorporation into the selenocysteine (Sec) for synthesis of selenoproteins (SePs), such as thioredoxin reductase family enzymes and glutathione peroxidase family enzymes. In general, the hyperactivity of the selenol group in Sec confers the Sec residue critical for functions of SePs. The Sec is much less abundant than its sulfur analog cysteine (Cys), and it remains a high challenge to detect Sec, especially in complex biological samples. We recently reported a selective fluorescent probe Sel-green for selenols and summarized the principles for design of selenol (and thiophenol) probes. Sel-green discriminates selenols from other biological species, especially thiols, under physiological conditions, and has been applied to detect both endogenous and exogenous selenol species in live cells. In this chapter, we describe a protocol and guideline for the selective detection of Sec by applying the Sel-green. This protocol is also suitable for detection of other selenol species. This practical and convenient assay would assist scientists to better understand the pivotal roles of Sec as well as SePs.

Thioredoxin Reductase Inhibition for Cancer Therapy.

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.

Chemoselective Copper-Mediated Modification of Selenocysteines in Peptides and Proteins.

Highly valuable bioconjugated molecules must be synthesized through efficient, chemoselective chemical modifications of peptides and proteins. Herein, we report the chemoselective modification of peptides and proteins via a reaction between selenocysteine residues and aryl/alkyl radicals. In situ radical generation from hydrazine substrates and copper ions proceeds rapidly in an aqueous buffer at near neutral pH (5-8), providing a variety of Se-modified linear and cyclic peptides and proteins conjugated to aryl and alkyl molecules, and to affinity label tag (biotin). This chemistry opens a new avenue for chemical protein modifications.

Reactivity of antitumor coinage metal-based N-heterocyclic carbene complexes with cysteine and selenocysteine protein sites.

The reaction of the antitumor M(I)-bis-N-heterocyclic carbene (M(I)-NHC) complexes, M = Cu, Ag, and Au, with their potential protein binding sites, i.e. cysteine and selenocysteine, was investigated by means of density functional theory approaches. Capped cysteine and selenocysteine were employed to better model the corresponding residues environment within peptide structures. By assuming the neutral or deprotonated form of the side chains of these amino acids and by considering the possible assistance of an external proton donor such as an adjacent acidic residue or the acidic component of the surrounding buffer environment, we devised five possible routes leading to the binding of the investigated M(I)-NHC scaffolds to these protein sites, reflecting their different location in the protein structure and exposure to the bulk. The targeting of either cysteine or selenocysteine in their neutral forms is a kinetically unfavored process, expected to be quite slow if observable at all at physiological temperature. On the other hand, the reaction with the deprotonated forms is much more favored, even though an external proton source is required to assist the protonation of the leaving carbene. Our calculations also show that all coinage metals are characterized by a similar reactivity toward the binding of cysteine and selenocysteine sites, although the Au(I) complex has significantly higher reaction and activation free energies compared to Cu(I) and Ag(I).