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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. [ABSTRACT FROM AUTHOR]

Sodium hypochlorite pentahydrate (NaClO·5H2O, 1) has recently been employed in organic synthesis as an oxidant for alcohols, sulfides, glycols, etc. In most of these reactions, however, reagent 1 functions just as a simple oxidant, and the variations of the reactions have not been well explored. In this study, we report another useful and fascinating reaction, in which reagent 1 functions as a green chlorinating reagent toward β,γ-unsaturated carboxylic acid (2). When substrate 2 was stirred at room temperature with 1 (2 eq) in acetonitrile for 1 h, α,β-unsaturated lactone (3) was obtained in moderate yields (up to 62%). The same reaction proceeded in various organic and aqueous solvents as well. When excess reagent 1 was employed, lactone 3 was further oxidized to the corresponding epoxide (4) for some cases. The conversion is initiated by electrophilic attack of HOCl to the C=C bond of 2 to generate a chloronium ion intermediate, which is cyclized to β-chlorolactone (5) and then 3 through the elimination of HCl. The usefulness of 1 as a chlorinating reagent was further demonstrated in the electrophilic substitution of activated aromatic compounds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

On the basis of the decolorization caused by the reaction of active oxygen species (AOSs) with methylene blue (MB), our group recently developed colorimetric indicators for hydroxyl radical (OH radical) by embedding MB in polymer thin films made of water-soluble pullulan or sodium alginate. In the present work, to elucidate the reason for the selective decolorization induced by the OH radical compared with other AOSs, such as ozone (O3) and hydrogen peroxide (H2O2), density-functional-theory calculations were performed at the B3LYP/6-31G(d) level for these AOSs and MB and its complexes with pullulan or sodium alginate model molecules. A frontier orbital analysis revealed that the π orbital of MB tends to delocalize on the whole molecule upon complexing with pullulan and sodium alginate, while the energy level is lower than the lowest unoccupied molecular orbital levels of O3 and H2O2 but higher than the singly occupied molecular orbital level of the OH radical. The results support the observation that only the OH radical, as the strongest oxidant, can react with MB in the polymer matrices. The selective decolorization of MB-dyed polymer films by the OH radical is due to not only the steric hindrance in the polymer matrix but also the perturbation of the π orbital of MB through the interaction with the polymer molecules. [ABSTRACT FROM AUTHOR]

It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76-94], des[64-80], and des [6-127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64-80] and des[6-127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5-45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1-4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1-4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76-94] was populated in the largest amount, but the oxidation to N was slower than that of des[64-80] and des[6-127]. At 35 °C, on the other hand, des[64-80] and des[6-127] were no longer stable, and only des[76-94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control. [ABSTRACT FROM AUTHOR]

In organic molecules a divalent sulfur atom sometimes adopts weak coordination to a proximate heteroatom (X). Such hypervalent nonbonded S···X interactions can control the molecular structure and chemical reactivity of organic molecules, as well as their assembly and packing in the solid state. In the last decade, similar hypervalent interactions have been demonstrated by statistical database analysis to be present in protein structures. In this review, weak interactions between a divalent sulfur atom and an oxygen or nitrogen atom in proteins are highlighted with several examples. S···O interactions in proteins showed obviously different structural features from those in organic molecules (i.e., πO → σS* versus nO → σS* directionality). The difference was ascribed to the HOMO of the amide group, which expands in the vertical direction (πO) rather than in the plane (nO). S···X interactions in four model proteins, phospholipase A2 (PLA2), ribonuclease A (RNase A), insulin, and lysozyme, have also been analyzed. The results suggested that S···X interactions would be important factors that control not only the three-dimensional structure of proteins but also their functions to some extent. Thus, S···X interactions will be useful tools for protein engineering and the ligand design. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+. [ABSTRACT FROM AUTHOR]

In this review from literature appearing over about the past 5 years, we focus on selected selenide reports and related chemistry; we aimed for a digestible, relevant, review intended to be usefully interconnected within the realm of fluorescence and selenium chemistry. Tellurium is mentioned where relevant. Topics include selenium in physics and surfaces, nanoscience, sensing and fluorescence, quantum dots and nanoparticles, Au and oxide nanoparticles quantum dot based, coatings and catalyst poisons, thin film, and aspects of solar energy conversion. Chemosensing is covered, whether small molecule or nanoparticle based, relating to metal ion analytes, H2S, as well as analyte sulfane (biothiols—including glutathione). We cover recent reports of probing and fluorescence when they deal with redox biology aspects. Selenium in therapeutics, medicinal chemistry and skeleton cores is covered. Selenium serves as a constituent for some small molecule sensors and probes. Typically, the selenium is part of the reactive, or active site of the probe; in other cases, it is featured as the analyte, either as a reduced or oxidized form of selenium. Free radicals and ROS are also mentioned; aggregation strategies are treated in some places. Also, the relationship between reduced selenium and oxidized selenium is developed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks. [ABSTRACT FROM AUTHOR]

The polypeptide backbone of proteins is held together by two main types of covalent bonds: the peptide bonds that link the amino acid residues and the disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds form as a protein folds in the cell and formation was assumed to be complete when the mature protein emerges. This is not the case for some secreted human blood proteins. The blood clotting protein, fibrinogen, and the protease inhibitor, α2-macroglobulin, exist in multiple disulfide-bonded or covalent states in the circulation. Thousands of different states are predicted assuming no dependencies on disulfide bond formation. In this study, probabilities for disulfide bond formation are employed to estimate numbers of covalent states of a model polypeptide with reference to α2-macroglobulin. When disulfide formation is interdependent in a protein, the number of covalent states is greatly reduced. Theoretical estimates of the number of states will aid the conceptual and experimental challenges of investigating multiple disulfide-bonded states of a protein. [ABSTRACT FROM AUTHOR]

Oxidative protein folding involves the formation of disulfide bonds and the regeneration of native structure (N) from the fully reduced and unfolded protein (R). Oxidative protein folding studies have provided a wealth of information on underlying physico-chemical reactions by which disulfide-bond-containing proteins acquire their catalytically active form. Initially, we review key events underlying oxidative protein folding using bovine pancreatic ribonuclease A (RNase A), bovine pancreatic trypsin inhibitor (BPTI) and hen-egg white lysozyme (HEWL) as model disulfide bond-containing folders and discuss consequential outcomes with regard to their folding trajectories. We re-examine the findings from the same studies to underscore the importance of forming native disulfide bonds and generating a "native-like" structure early on in the oxidative folding pathway. The impact of both these features on the regeneration landscape are highlighted by comparing ideal, albeit hypothetical, regeneration scenarios with those wherein a native-like structure is formed relatively "late" in the R→N trajectory. A special case where the desired characteristics of oxidative folding trajectories can, nevertheless, stall folding is also discussed. The importance of these data from oxidative protein folding studies is projected onto outcomes, including their impact on the regeneration rate, yield, misfolding, misfolded-flux trafficking from the endoplasmic reticulum (ER) to the cytoplasm, and the onset of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Heat-stable enterotoxin (STa) produced by enterotoxigenic E. coli causes acute diarrhea and also can be used as a specific probe for colorectal cancer cells. STa contains three intra-molecular disulfide bonds (C1–C4, C2–C5, and C3–C6 connectivity). The chemical synthesis of STa provided not only the native type of STa but also a topological isomer that had the native disulfide pairings. Interestingly, the activity of the topological isomer was approximately 1/10–1/2 that of the native STa. To further investigate the bioactive conformation of this molecule and the regulation of disulfide-coupled folding during its chemical syntheses, we examined the folding mechanism of STa that occurs during its chemical synthesis. The folding intermediate of STa with two disulfide bonds (C1–C4 and C3–C6) and two Cys(Acm) residues, the precursor peptide, was treated with iodine to produce a third disulfide bond under several conditions. The topological isomer was predominantly produced under all conditions tested, along with trace amounts of the native type of STa. In addition, NMR measurements indicated that the topological isomer has a left-handed spiral structure similar to that of the precursor peptide, while the native type of STa had a right-handed spiral structure. These results indicate that the order of the regioselective formation of disulfide bonds is important for the regulation of the final conformation of disulfide-rich peptides in chemical synthesis. [ABSTRACT FROM AUTHOR]

Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61-77, 73-91) and des[6-120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca 2+ . In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans -3,4-dihydroxyselenolane oxide (DHS ox ), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca 2+ . These intermediates could be assigned to (61-77, 73-91) and des[6-120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the β-sheet domain. Much faster folding to N was observed in the presence of Mn 2+ , whereas Na⁺, K⁺, Mg 2+ , and Zn 2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other., Competing Interests: The authors declare no conflict of interest.

We previously reported that water-soluble cyclic selenides can mimic the antioxidative function of glutathione peroxidase (GPx) in water through a simple catalytic cycle, in which the selenide (>Se) is oxidized by H₂O₂ to the selenoxide (>Se=O) and the selenoxide is reduced by a thiol back to the selenide. In methanol, however, the GPx-like activity could not be explained by this simple scenario. To look into the reasons for the unusual behaviors in methanol, monoamino-substituted cyclic selenides with a variable ring size were synthesized, and the intermediates of the catalytic cycle were characterized by means of 77 Se-NMR and LC-MS spectroscopies. In water, it was confirmed that the selenide and the selenoxide mainly contribute to the antioxidative function, though a slight contribution from the dihydroxy selenane (>Se(OH)₂) was also suggested. In methanol, on the other hand, other active species, such as hydroxyselenonium (>Se⁺-OH) and hydroxy perhydroxy selenane (>Se(OH)(OOH)), could be generated to build another catalytic cycle. This over-oxidation would be more feasible for amino-substituted cyclic selenides, probably because the ammonium (NH₃⁺) group would transfer a proton to the selenoxide moiety to produce a hydroxyselenonium species in the absence of an additional proton source. Thus, a shift of the major catalytic cycle in methanol would make the GPx-like antioxidative function of selenides perplexing.

A series of fatty acid conjugates of trans-3,4-dihydroxy-1-selenolane (DHS) were synthesized by reacting DHS with appropriate acid chlorides. The obtained monoesters were evaluated for their antioxidant capacities by the lipid peroxidation assay using a lecithin/cholesterol liposome as a model system. The observed antioxidant capacities against accumulation of the lipid hydroperoxide (LOOH) increased with increasing the alkyl chain length and became saturated for dodecanoic acid (C12) or higher fatty acid monoesters, for which the capacities were much greater than those of DHS, its tridecanoic acid (C13) diester, and PhSeSePh. On the other hand, the bacteriostatic activity of myristic acid (C14) monoester, evaluated through the colony formation assay using Bacillus subtilis, indicated that it has higher affinity to bacterial cell membranes than parent DHS. Since DHS-fatty acid conjugates would inhibit lipid peroxidation through glutathione peroxidase (GPx)-like 2e- mechanism, higher fatty acid monoesters of DHS can mimic the function of GPx4, which interacts with LOOH to reduce it to harmless alcohol (LOH). Importance of the balance between hydrophilicity and lipophilicity for the design of effective GPx4 mimics was suggested.