Antioxidative and Antiglycative Stress Activities of Selenoglutathione Diselenide.

The damage caused by oxidative and glycative stress to cells accumulates on a daily basis and accelerates aging. Glutathione (GSH), a major antioxidant molecule in living organisms, plays a crucial role in detoxifying the stress-causing substances inherent in cells, such as H2O2 and methylglyoxal (MG), an important intermediate of advanced glycation end-products (AGEs). In this study, we focused on the enhanced antioxidant capacity of the selenium analog of GSH, i.e., selenoglutathione (GSeH), compared to GSH, and examined its effects on the detoxification of stress-causing substances and improvement in cell viability. In cell-free systems, GSeH (1 mM) generated in situ from GSeSeG in the presence of NADPH and glutathione reductase (GR) rapidly reduced more than 80% of 0.1 mM H2O2, indicating the significant glutathione peroxidase (GPx)-like antioxidant activity of GSeSeG. Similarly, around 50% of 0.5 mM MG was degraded by 0.5 mM GSeH within 30 min through a non-enzymatic mechanism. It was also found that GSeSeG (0.05-0.5 mM) showed glutathione S-transferase (GST)-like activity against 1-chloro-2,4-dinitrobenzene (CDNB), a model substance of oxidative stress-causing toxic materials in cells. Meanwhile, HeLa cells that had been pre-treated with GSeSeG exhibited increased viability against 1.2 mM H2O2 (at [GSeSeG] = 0.5-50 µM) and 4 mM MG (at [GSeSeG] = 3 µM), and the latter effect was maintained for two days. Thus, GSeSeG is a potential antioxidant and antiglycative stress agent for cells.

Seleno-relaxin analogues: effect of internal and external diselenide bonds on the foldability and a fibrosis-related factor of endometriotic stromal cells.

Human relaxin-2 (H2 relaxin) is a peptide hormone of about 6 kDa, first identified as a reproductive hormone involved in vasoregulation during pregnancy. It has recently attracted strong interest because of its diverse functions, including anti-inflammatory, anti-fibrotic, and vasodilatory, and has been suggested as a potential peptide-based drug candidate for a variety of diseases. Mature H2 relaxin is constituted by the A- and B-chains stabilized by two interchain disulfide (SS) bridges and one intrachain SS linkage. In this study, seleno-relaxins, SeRlx-α and SeRlx-ß, which are [C11UA,C11UB] and [C10UA,C15UA] variants of H2 relaxin, respectively, were synthesized via a one-pot oxidative chain assembly (folding) from the component A- and B-chains. The substitution of SS bonds in a protein with their analogue, diselenide (SeSe) bonds, has been shown to alter the physical, chemical, and physiological properties of the protein. The surface SeSe bond (U11A-U11B) enhanced the yield of chain assembly while the internal SeSe bond (U10A-U15A) improved the reaction rate of the folding, indicating that these bridges play a major role in controlling the thermodynamics and kinetics, respectively, of the folding mechanism. Furthermore, SeRlx-α and SeRlx-ß effectively reduced the expression of a tissue fibrosis-related factor in human endometriotic stromal cells. Thus, the findings of this study indicate that the S-to-Se substitution strategy not only enhances the foldability of relaxin, but also provides new guidance for the development of novel relaxin formulations for endometriosis treatment.

Diseleno-albumin, a native bio-inspired drug free therapeutic protein induces apoptosis in lung cancer cells through mitochondrial oxidation.

Macromolecular therapeutic is the emerging concept in the fields of drug delivery and drug discovery. The present study reports the design and development of a serum albumin based macromolecular chemotherapeutic by conjugating bovine serum albumin (BSA) with 3,3'-diselenodipropionic acid (DSePA), a pharmacologically active organo-diselenide (R-Se-Se-R). The reaction conditions were optimised to achieve the controlled conjugation of BSA with DSePA without causing any significant alteration in its physico-chemical properties or secondary structure and crosslinking. The chemical characterisation of the reaction product through various spectroscopic techniques viz., FT-IR, Raman, XPS, AAS and MALDI-TOF-MS, established the conjugation of about ∼5 DSePA molecules per BSA molecule. The DSePA conjugated BSA (Se-Se-BSA) showed considerable stability in aqueous and lyophilized forms. The cytotoxicity studies by involving cell lines of cancerous and non-cancerous origins indicated that Se-Se-BSA selectively inhibited the proliferation of cancerous cells. The cellular uptake studies by physically labelling Se-Se-BSA with curcumin and following its intracellular fluorescence confirmed that uptake efficiency of Se-Se-BSA was almost similar to that of native BSA. Finally, studies on the mechanism of action of Se-Se-BSA in the A549 (lung adenocarcinoma) cells revealed that it induced mitochondrial ROS generation followed by mitochondrial dysfunction, activation of caspases and apoptosis. Together, these results demonstrate a bio-inspired approach of exploring diselenide (-Se-Se-) grafted serum albumin as the potential drug free therapeutic for anticancer application.

Diselenide-bond replacement of the external disulfide bond of insulin increases its oligomerization leading to sustained activity.

Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S's) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 µg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.

Controlling the Redox Catalytic Activity of a Cyclic Selenide Fused to 18-Crown-6 by the Conformational Transition Induced by Coordination to an Alkali Metal Ion.

trans-3,4-Dihydroxyselenolane (DHS), a water-soluble cyclic selenide, exhibits selenoenzyme-like unique redox activities through reversible oxidation to the corresponding selenoxide. Previously, we demonstrated that DHS can be applied as an antioxidant against lipid peroxidation and a radioprotector by means of adequate modifications of the two hydroxy (OH) groups. Herein, we synthesized new DHS derivatives with a crown-ether ring fused to the OH groups (DHS-crown-n (n = 4 to 7), 1-4) and investigated their behaviors of complex formation with various alkali metal salts. According to the X-ray structure analysis, it was found that the two oxygen atoms of DHS change the directions from diaxial to diequatorial by complexation. The similar conformational transition was also observed in solution NMR experiments. The 1H NMR titration in CD3OD further confirmed that DHS-crown-6 (3) forms stable 1:1 complexes with KI, RbCl and CsCl, while it forms a 2:1 complex with KBPh4. The results suggested that the 1:1 complex (3·MX) exchanges the metal ion with metal-free 3 through the formation of the 2:1 complex. The redox catalytic activity of 3 was evaluated using a selenoenzyme model reaction between H2O2 and dithiothreitol. The activity was significantly reduced in the presence of KCl due to the complex formation. Thus, the redox catalytic activity of DHS could be controlled by the conformational transition induced by coordination to an alkali metal ion.

A protein identification method for proteomics using amino acid composition analysis with IoT-based remote control.

In the present study, we developed a protein identification method using low-cost and easy-to-operate amino acid composition analysis. The identification program automatically compares the quantitative result for each amino acid concentration obtained from the amino acid analysis to the amino acid composition data retrieved from the UniProt protein database. We found that the accuracy of protein identification using amino acid composition analysis was comparable to that of mass spectrometry analysis. The method was able to distinguish and identify differences in amino acid substitutions of several residues between proteins with high sequence homology. The identification accuracy of proteins was also improved by correcting the concentrations in the program for Cys, Trp, and Ile residues, which cannot be quantified by general sample preparation for amino acid analysis. Moreover, the amino acid analyzer was remotely controlled in accordance with the growing demand for remote work. The measured amino acid data were automatically uploaded to the IoT portal within a few minutes of each measurement, allowing researchers to download data and analyze them using the identification program anywhere and at any time by connecting to a network. The results indicated that the present method is useful for protein identification.

Molecular Interactions between Methylene Blue and Sodium Alginate Studied by Molecular Orbital Calculations.

A methylene blue (MB) indicator embedded in sodium alginate (SA) film was previously examined for detecting active oxygen species. In a previous study, spectrometry was used to identify and characterize the MB/SA complex. However, the decolorization mechanism was not fully assessed. In this study, our aim is to conduct computational calculations at the B3LYP/6-31G(d) level to clarify the exact types and positions of the interaction that cause the decolorization in MB. The results demonstrate that MB/SA interacts with carboxylates (-COO(superscript)-(superscript)) of SA and the N, C, and S atoms of MB, confirming previous experimental observations.

Synthesis of 4-Selenothreofuranose Derivatives via Pummerer-Type Reactions of trans-3,4-Dioxygenated Tetrahydroselenophenes Mediated by a Selenonium Intermediate.

Selenosugars are interesting targets of organic synthesis as they would possess potential biological activities. However, 4-selenotherofuranose derivatives, which have trans configuration for the two dihydroxy substituents at the 2,3-positions and a glycoside bond at the anomeric position, are not available in the current selenosugar library. In this study, racemic 4-selenothreofuranose derivatives were synthesized from trans-3,4-dioxygenated tetrahydroselenophenes in 77-99% yields with the α/ß selectivity about 7:3 via oxidation and subsequent seleno-Pummerer rearrangement. The acetoxy group introduced at the anomeric position was then substituted with various nucleophiles, including activated 6-chloropurine, which afforded 4'-selenothreonucleoside derivatives, in the presence of BF3·OEt2 or TMSOTf. The stereochemistry of the selenosugar products was established by 1H NMR spectroscopy as well as X-ray analysis. The similar α/ß selectivity observed in the latter glycosylation reaction to that in the former seleno-Pummerer rearrangement suggested the mediation of a common selenonium intermediate (-Se+=C<). It was also suggested that an unexpected interaction between the ester protecting group at the 3-position of the selenofuranose ring and the anomeric carbon atom decreases the α/ß selectivity.

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments.

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.

Glutathione peroxidase-like functions of 1,2-diselenane-4,5-diol and its amphiphilic derivatives: Switchable catalytic cycles depending on peroxide substrates.

Amphiphilic derivatives of (±)-trans-1,2-diselenane-4,5-diol (DSTox) decorated with long alkyl chains or aromatic substituents via ester linkages were applied as glutathione peroxidase (GPx)-like catalysts. The reduction of H2O2 with the diselenide catalysts was accelerated through a GPx-like catalytic cycle, in which the diselenide (Se-Se) bond was reduced to the diselenolate form ([Se-,Se-]) by coexisting dithiothreitol, and the generated highly active [Se-,Se-] subsequently reduced H2O2 to H2O retrieving the original Se-Se form. In the lipid peroxidation of lecithin/cholesterol liposomes induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), on the other hand, the Se-Se form directly reduced lipid peroxide (LOOH) to the corresponding alcohol (LOH), inhibiting the radical chain reaction, to exert the antioxidative effect. Thus, the two GPx-like catalytic cycles can be switched depending on the peroxide substrates. Furthermore, hydrophilic compounds with no or short alkyl groups (C3) showed high antioxidative activities for the catalytic reduction of H2O2, while lipophilic compounds with long alkyl chains (C6-C14) or aromatic substituents were more effective antioxidants against lipid peroxidation. In addition, these compounds showed low cytotoxicity in cultured HeLa cells and exhibited sufficient anti-lipid peroxidative activities, suggesting their potentials as selenium-based antioxidative drugs.

Relaxin-2 May Suppress Endometriosis by Reducing Fibrosis, Scar Formation, and Inflammation.

BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.

Application of a Distance-Dependent Sigmoidal Dielectric Constant to the REMC/SAAP3D Simulations of Chignolin, Trp-Cage, and the G10q Mutant.

The replica-exchange Monte Carlo method based on the single amino acid potential (SAAP) force field, i.e., REMC/SAAP3D, was recently developed by our group for the molecular simulation of short peptides. In this study, the method has been improved by applying a distance-dependent dielectric (DDD) constant and extended to the peptides containing D-amino acid (AA) residues. For chignolin (10 AAs), a sigmoidal DDD model reasonably allocated the native-like ß-hairpin structure with all-atom root mean square deviation (RMSD) = 2.0 Å as a global energy minimum. The optimal DDD condition was subsequently applied for Trp-cage (20 AAs) and its G10q mutant. The native-like α-rich folded structures with main-chain RMSD = 3.7 and 3.8 Å were obtained as global energy minima for Trp-cage and G10q, respectively. The results suggested that the REMC/SAAP3D method with the sigmoidal DDD model is useful for structural prediction for the short peptides comprised of up to 20 AAs. In addition, the relative contributions of SAAP to the total energy (%SAAP) were evaluated by energetic component analysis. The ratios of %SAAP were about 40 and 20% for chignolin and Trp-cage (or G10q), respectively. It was proposed that SAAP is more important for the secondary structure formation than for the assembly to a higher-order folded structure, in which the attractive van der Waals interaction may play a more important role.

Chemical synthesis of ferredoxin with 4 selenocysteine residues using a segment condensation method.

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe-2S] cluster. In this paper, we synthesized Se-Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se-Fd was successfully obtained.

Interaction kinetics of selenium-containing compounds with oxidants.

Selenium compounds have been identified as potential oxidant scavengers for biological applications due to the nucleophilicity of Se, and the ease of oxidation of the selenium centre. Previous studies have reported apparent second order rate constants for a number of oxidants (e.g. HOCl, ONOOH) with some selenium species, but these data are limited. Here we provide apparent second order rate constants for reaction of selenols (RSeH), selenides (RSeR') and diselenides (RSeSeR') with biologically-relevant oxidants (HOCl, H2O2, other peroxides) as well as overall consumption data for the excited state species singlet oxygen (1O2). Selenols show very high reactivity with HOCl and 1O2, with rate constants > 108 M-1 s-1, whilst selenides and diselenides typically react with rate constants one- (selenides) or two- (diselenides) orders of magnitude slower. Rate constants for reaction of diselenides with H2O2 and other hydroperoxides are much slower, with k for H2O2 being <1 M-1 s-1, and for amino acid and peptide hydroperoxides ~102 M-1 s-1. The rate constants determined for HOCl and 1O2 with these selenium species are greater than, or similar to, rate constants for amino acid side chains on proteins, including the corresponding sulfur-centered species (Cys and Met), suggesting that selenium containing compounds may be effective oxidant scavengers. Some of these reactions may be catalytic in nature due to ready recycling of the oxidized selenium species. These data may aid the development of highly efficacious, and catalytic, oxidant scavengers.

Modeling Thioredoxin Reductase-Like Activity with Cyclic Selenenyl Sulfides: Participation of an NH⋠⋠⋠Se Hydrogen Bond through Stabilization of the Mixed Se-S Intermediate.

At the redox-active center of thioredoxin reductase (TrxR), a selenenyl sulfide (Se-S) bond is formed between Cys497 and Sec498, which is activated into the thiolselenolate state ([SH,Se- ]) by reacting with a nearby dithiol motif ([SHCys59 ,SHCys64 ]) present in the other subunit. This process is achieved through two reversible steps: an attack of a cysteinyl thiol of Cys59 at the Se atom of the Se-S bond and a subsequent attack of a remaining thiol at the S atom of the generated mixed Se-S intermediate. However, it is not clear how the kinetically unfavorable second step progresses smoothly in the catalytic cycle. A model study that used synthetic selenenyl sulfides, which mimic the active site structure of human TrxR comprising Cys497, Sec498, and His472, suggested that His472 can play a key role by forming a hydrogen bond with the Se atom of the mixed Se-S intermediate to facilitate the second step. In addition, the selenenyl sulfides exhibited a defensive ability against H2 O2 -induced oxidative stress in cultured cells, which suggests the possibility for medicinal applications to control the redox balance in cells.

Oxidative folding pathways of bovine milk ß-lactoglobulin with odd cysteine residues.

Bovine ß-lactoglobulin (BLG) is a major whey protein with unique structural characteristics: it possesses a free Cys thiol (SH) and two disulfide (SS) bonds and consists of a ß-barrel core surrounded by one long and several short α helices. Although SS-intact conformational folding has been studied in depth, the oxidative folding pathways and accompanying SS formation/rearrangement are poorly understood. In this study, we used trans-3,4-dihydroxyselenolane oxide, a water-soluble selenoxide reagent which undergoes rapid and quantitative SS formation, to determine the oxidative folding pathways of BLG variant A (BLGA) at pH 8.0 and 25 °C. This was done by characterizing two key one-SS intermediates, a particular folding intermediate having a Cys66-Cys160 SS bond (I-1) and a particular folding intermediate having a Cys106-Cys119 SS bond (I-2), which have a native Cys66-Cys160 and Cys106-Cys119 SS bond, respectively. In the major folding pathway, the reduced protein (R) with abundant α helices was oxidized to I-1, which was then transformed to I-2 through SS rearrangement. The native protein (N) was formed by oxidation of I-2. The redundant Cys121 thiol facilitates SS rearrangement. N is also generated from an ensemble of folding intermediates having two SS bonds (2SS) intermediates with scrambled SS bonds through SS rearrangement, but this minor pathway is deteriorative due to aggregation or overoxidation of 2SS. During oxidative folding of BLGA, αâ†’ß conformational transition occurred as previously observed in SS-intact folding. These findings are informative not only for elucidating oxidative folding pathways of other members of the ß-lactoglobulin family, but also for understanding the roles of a redundant Cys thiol in the oxidative folding process of a protein with odd Cys residues.

Oxidative Protein Folding Using trans-3,4-Dihydroxyselenolane Oxide.

trans-3,4-Dihydroxyselenolane oxide (DHSox), a water-soluble cyclic selenoxide reagent, is useful for rapid and quantitative formation of disulphide (SS) bonds in a reduced state of SS-containing proteins because the selenoxide is a strong but selective oxidant for thiol substrates (RSH) in a wide range of pH. Due to this advantage over common disulphide reagents, such as oxidized dithiothreitol (DTTox) and glutathione (GSSG), DHSox enables clear characterization of oxidative folding pathways of proteins. DHSox is also useful for facile diagnosis of weakly folded structure, or reactivity (i.e., pKa) of the thiols, present in a reduced polypeptide chain and the partially oxidized folding intermediates, identification of the key SS intermediates that can be oxidized directly to the native state, and preparation of SS-scrambled misfolded protein species. In this chapter, these diverse utilities of DHSox in protein folding study are demonstrated.

Synthesis of selenocysteine-containing cyclic peptides via tandem N-to-S acyl migration and intramolecular selenocysteine-mediated native chemical ligation.

Selenocysteine-containing cyclic 8-mer peptides, which were designed to mimic the plausible catalytic tetrad of glutathione peroxidase, were successfully synthesized in one pot via tandem N-to-S acyl migration of N-alkylcysteine (NAC)-containing selenopeptides and intramolecular selenocysteine-mediated native chemical ligation (Sec-NCL) of the generated thioesters.

Protein Folding in the Presence of Water-Soluble Cyclic Diselenides with Novel Oxidoreductase and Isomerase Activities.

The protein disulfide isomerase (PDI) family, found in the endoplasmic reticulum (ER) of the eukaryotic cell, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions leads to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimer's, Parkinson's, diabetes mellitus, and atherosclerosis. Here we report that water-soluble cyclic diselenides mimic the multifunctional activity of the PDI family by facilitating oxidative folding, disulfide formation/reduction, and repair of the scrambled disulfide bonds in misfolded proteins.

Mechanism of radioprotection by dihydroxy-1-selenolane (DHS): Effect of fatty acid conjugation and role of glutathione peroxidase (GPx).

Dihydroxy-1-selenolane (DHS) previously reported to exhibit radioprotective activity was investigated to understand its mechanism of action in CHO cells of epithelial origin. DHS pre-treatment at 25 µM for 16 h significantly protected CHO cells from radiation (4-11 Gy)-induced delayed mitotic cell death. Further to examine, how increased cellular uptake can influence this mechanism, studies have been performed with DHS-C6, a lipophilic conjugate of DHS. Accordingly CHO cells pre-treated with DHS-C6, showed increased survival against radiation exposure. Notably treatment with both DHS and DHS-C6 significantly increased glutathione peroxidase (GPx) activity in cells by âˆ¼ 2.5 fold. Additionally, the compound DHS or DHS-C6 led to faster repair of DNA in irradiated cells and subsequently inhibited the G2/M arrest. Anticipating the role of GPx in radioprotection, our investigations revealed that addition of mercaptosuccinic acid, a pharmacological inhibitor of GPx reversed all the above effects of DHS or DHS-C6. Further inhibitors of check point kinase 1 (CHK1) and DNA-protein kinase (DNA-PK) although abrogated the radioprotective effect of DHS or DHS-C6 separately, did not show additive effect in combination with GPx inhibitor, suggesting their cross talk. In contrast to these results, both DHS and DHS-C6 treatment did not protect spleen lymphocytes from the radiation-induced apoptosis. Thus results confirmed that both DHS and DHS-C6 protected cells from radiation-induced mitotic death by augmenting DNA repair in a GPx dependant manner.