Application of alpha-methyl selenocysteine as a tool for the study of selenoproteins.

Selenocysteine (Sec) is the 21st proteogenic amino acid and it is now widely accepted that Sec is involved in redox biochemistry when incorporated in proteins. However, many of the chemical mechanisms for Sec bioactivity remain unknown. Herein, we describe a derivative of Sec, alpha-methyl Sec ((αMe)Sec), that is a useful chemical tool to study selenoenzyme mechanisms. (αMe)Sec is identical to Sec except the Cα-H is replaced with a Cα-methyl group, which prevents this derivative from undergoing oxygen-mediated ß-syn elimination to dehydroalanine, which is a common problem with Sec-containing peptides and proteins. Thus, since (αMe)Sec-containing peptides and proteins cannot lose the side-chain selenium atom when oxidized, mechanistic studies can be performed that are not always possible with Sec. In this chapter, we provide detailed methods for the incorporation of (αMe)Sec into peptides using solid phase peptide synthesis and subsequent incorporation into mammalian thioredoxin reductase using protein semisynthesis. We then provide two examples of how (αMe)Sec has been used as a chemical tool to study selenoenzyme mechanism. Finally, we discuss future applications where we envision (αMe)Sec will be useful.

The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues.

The brevetoxins, neurotoxins produced by Karenia brevis, the Florida red tide dinoflagellate, effect fish and wildlife mortalities and adverse public health and economic impacts during recurrent blooms. Knowledge of the biochemical consequences of toxin production for K. brevis could provide insights into an endogenous role of the toxins, yet this aspect has not been thoroughly explored. In addition to neurotoxicity, the most abundant of the brevetoxins, PbTx-2, inhibits mammalian thioredoxin reductase (TrxR). The thioredoxin system, composed of the enzymes TrxR and thioredoxin (Trx), is present in all living organisms and is responsible in part for maintaining cellular redox homeostasis. Herein, we describe the cloning, expression, and semisynthesis of the selenoprotein TrxR from K. brevis (KbTrxR) and reductase activity toward a variety of substrates. Unlike mammalian TrxR, KbTrxR reduces oxidized glutathione (GSSG). We further demonstrate that PbTx-2 is an inhibitor of KbTrxR. Covalent adducts between KbTrxR and rat TrxR were detected by mass spectrometry. While both enzymes are adducted at or near the catalytic centers, the specific residues are distinct. Biochemical differences reported for high and low toxin producing strains of K. brevis are consistent with the inhibition of KbTrxR and suggest that PbTx-2 is an endogenous regulator of this critical enzyme.

Aggregation State of Synergistic Antimicrobial Peptides.

By integrating various simulation and experimental techniques, we discovered that antimicrobial peptides (AMPs) may achieve synergy at an optimal concentration and ratio, which can be caused by aggregation of the synergistic peptides. On multiple time and length scales, our studies obtain novel evidence of how peptide coaggregation in solution can affect the disruption of membranes by synergistic AMPs. Our findings provide crucial details about the complex molecular origins of AMP synergy, which will help guide the future development of synergistic AMPs as well as applications of anti-infective peptide cocktail therapies.

Can Selenoenzymes Resist Electrophilic Modification? Evidence from Thioredoxin Reductase and a Mutant Containing α-Methylselenocysteine.

Selenocysteine (Sec) is the 21st proteogenic amino acid in the genetic code. Incorporation of Sec into proteins is a complex and bioenergetically costly process that evokes the following question: "Why did nature choose selenium?" An answer that has emerged over the past decade is that Sec confers resistance to irreversible oxidative inactivation by reactive oxygen species. Here, we explore the question of whether this concept can be broadened to include resistance to reactive electrophilic species (RES) because oxygen and related compounds are merely a subset of RES. To test this hypothesis, we inactivated mammalian thioredoxin reductase (Sec-TrxR), a mutant containing α-methylselenocysteine [(αMe)Sec-TrxR], and a cysteine ortholog TrxR (Cys-TrxR) with various electrophiles, including acrolein, 4-hydroxynonenal, and curcumin. Our results show that the acrolein-inactivated Sec-TrxR and the (αMe)Sec-TrxR mutant could regain 25% and 30% activity, respectively, when incubated with 2 mM H2O2 and 5 mM imidazole. In contrast, Cys-TrxR did not regain activity under the same conditions. We posit that Sec enzymes can undergo a repair process via ß-syn selenoxide elimination that ejects the electrophile, leaving the enzyme in the oxidized selenosulfide state. (αMe)Sec-TrxR was created by incorporating the non-natural amino acid (αMe)Sec into TrxR by semisynthesis and allowed for rigorous testing of our hypothesis. This Sec derivative enables higher resistance to both oxidative and electrophilic inactivation because it lacks a backbone Cα-H, which prevents loss of selenium through the formation of dehydroalanine. This is the first time this unique amino acid has been incorporated into an enzyme and is an example of state-of-the-art protein engineering.

2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation.

There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

Facile removal of 4-methoxybenzyl protecting group from selenocysteine.

Historically, methods to remove the 4-methoxybenzyl (Mob)-protecting group from selenocysteine (Sec) in peptides have used harsh and toxic reagents. The use of 2,2'-dithiobis-5-nitropyridine (DTNP) is an improvement over these methods; however, many wash steps are required to remove the by-product contaminant 5-nitro-2-thiopyridine. Even with many washes, excess DTNP adheres to the peptide. The final product needs excess purification to remove these contaminants. It was recently discovered by our group that hindered hydrosilanes could be used to reduce Cys(Mob). We sought to apply a similar methodology to reduce Sec(Mob), which we expected to be even more labile. Here, we present a gentle and facile method for deprotection of Sec(Mob) using triethylsilane (TES), phenol, and a variety of other scavengers often used in deprotection cocktails. The different cocktails were all incubated at 40 °C for 4 hours. The combination of TFA/TES/thioanisole (96:2:2) appeared to be the most efficient of the cocktails tested, providing complete deprotection and yielded peptide that was mainly in the diselenide form. This cocktail also showed no evidence of side reactions or significant contaminants in the high-performance liquid chromatography (HPLC) and mass spectral (MS) analyses. We envision that our new method will allow for a simple and gentle "one-pot" deprotection of Sec(Mob) following solid-phase peptide synthesis and will minimize the need for extensive purification steps.

Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Synthesis of alpha-methyl selenocysteine and its utilization as a glutathione peroxidase mimic.

Selenocysteine (Sec) is the 21st amino acid in the genetic code where this amino acid is primarily involved in redox reactions in enzymes because of its high reactivity toward oxygen and related reactive oxygen species. Sec has found wide utility in synthetic peptides, especially as a replacement for cysteine. One limitation of using Sec in synthetic peptides is that it can undergo ß-syn elimination reactions after oxidation, rendering the peptide inactive due to loss of selenium. This limitation can be overcome by substituting Cα-H with a methyl group. The resulting Sec derivative is α-methylselenocysteine ((αMe)Sec). Here, we present a new strategy for the synthesis of (αMe)Sec by alkylation of an achiral methyl malonate through the use of a selenium-containing alkylating agent synthesized in the presence of dichloromethane. The seleno-malonate was then subjected to an enzymatic hydrolysis utilizing pig liver esterase followed by a Curtius rearrangement producing a protected derivative of (αMe)Sec that could be used in solid-phase peptide synthesis. We then synthesized two peptides: one containing Sec and the other containing (αMe)Sec, based on the sequence of glutathione peroxidase. This is the first reported incorporation of (αMe)Sec into a peptide as well as the first reported biochemical application of this unique amino acid. The (αMe)Sec-containing peptide had superior stability as it could not undergo ß-syn elimination and it also avoided cleavage of the peptide backbone, which we surprisingly found to be the case for the Sec-containing peptide when it was incubated for 96 hours in oxygenated buffer at pH 8.0.

Reduction of cysteine-S-protecting groups by triisopropylsilane.

Triisopropylsilane (TIS), a hindered hydrosilane, has long been utilized as a cation scavenger for the removal of amino acid protecting groups during peptide synthesis. However, its ability to actively remove S-protecting groups by serving as a reductant has largely been mischaracterized by the peptide community. Here, we provide strong evidence that TIS can act as a reducing agent to facilitate the removal of acetamidomethyl (Acm), 4-methoxybenzyl (Mob), and tert-butyl (But ) protecting groups from cysteine (Cys) residues in the presence of trifluoroacetic acid (TFA) at 37 °C. The lability of the Cys protecting groups in TFA/TIS (98/2) in this study are in the order: Cys(Mob) > Cys(Acm) > Cys(But ), with Cys(Mob) being especially labile. Unexpectedly, we found that TIS promoted disulfide formation in addition to aiding in the removal of the protecting group. Our results raise the possibility of using TIS in orthogonal deprotection strategies of Cys-protecting groups following peptide synthesis as TIS can be viewed as a potential deprotection agent instead of merely a scavenger in deprotection cocktails based on our results. We also tested other common scavengers under these reaction conditions and found that thioanisole and triethylsilane were similarly effective as TIS in enhancing deprotection and catalyzing disulfide formation. Our findings reported herein show that careful consideration should be given to the type of scavenger used when it is desirable to preserve the Cys-protecting group. Additional consideration should be given to the concentration of scavenger, temperature of the reaction, and reaction time.

Removal of the 5-nitro-2-pyridine-sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis.

We previously reported on a method for the facile removal of 4-methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2'-dithiobis-5-nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2-thio(5-nitropyridyl) (5-Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys-containing or Sec-containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5-Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.