Oxidation Of Thiol Groups Research Articles

Sodium transport and redox regulation in Saccharomyces cerevisiae under osmotic stress depending on oxygen availability

This study explores the molecular mechanisms behind the differential responses of Saccharomyces cerevisiae industrial strains (ATCC 9804 and ATCC 13007) to osmotic stress. We observed that, in contrast to ATCC 9804 strain, sodium flux in ATCC 13,007 is not N, N’-dicyclohexylcarbodiimide (DCCD)-sensitive under osmotic stress, suggesting a distinct ion homeostasis mechanism. Under aerobic conditions, osmotic stress increased reduced SH groups by 45% in ATCC 9804 and 34% in ATCC 13,007. In contrast, under microaerophilic conditions, both strains experienced a 50% reduction in thiol groups. Notably, ATCC 13,007 exhibited a 1.5-fold increase in catalase (CAT) activity under aerobic stress compared to standard conditions, while ATCC 9804 showed enhanced CAT activity due to SH group binding. Additionally, superoxide dismutase (SOD) activity was doubled during aerobic growth in both strains, with ATCC 13,007 showing a 1.5-fold higher SOD activity under osmotic stress. The results demonstrate that S. cerevisiae adapts to osmotic stress differently under aerobic and microaerophilic conditions, with aerobic conditions promoting Pma-Ena-Trk interplay, reduced thiol levels and increased catalase activity, while microaerophilic conditions demonstrate Pma-Nha-Trk interplay and shifts redox balance towards oxidized thiol groups and enhance superoxide dismutase activity. Understanding these mechanisms can aid in developing stress-resistant yeast strains for industrial applications.

Synthesis and Biological Activity of Homohypotaurine Obtained by the Enzyme-Based Conversion of Homocysteine Sulfinic Acid Using Recombinant Escherichia Coli Glutamate Decarboxylase.

l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, kcat (33.92 ± 1.07) and KM (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H2O2 in the presence of HHT.

Antiglycating Effects of Spirulina platensis Aqueous Extract on Glucose-Induced Glycation of Bovine Serum Albumin.

Glucose, the predominant carbohydrate in the human body, initiates nonenzymatic reactions in hyperglycemia, potentially leading to adverse biochemical interactions. This study investigates the interaction between glucose and Bovine Serum Albumin (BSA), along with the protective effects of Spirulina platensis PCC 7345 aqueous extract. Phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) in the extract were quantified using spectrophotometry. The extract's anti-glycation potential was assessed by analyzing its effects on albumin glycation, fluorescent advanced glycation end products (AGEs), thiol group oxidation, and β-amyloid structure generation. Additionally, its antidiabetic potential was evaluated by measuring α-amylase and α-glucosidase enzyme inhibition. Results indicate that the Spirulina extract significantly mitigated ketoamine levels, fluorescence, and protein-carbonyl production induced by glucose, demonstrating a 67.81 % suppression of AGE formation after 28 days. Moreover, it effectively inhibited amyloid formation in BSA cross-linkages. These findings suggest the potential of S. platensis as an anti-glycation and antidiabetic agent, supporting its consideration for dietary inclusion to manage diabetes and associated complications.

Tailoring ultrafiltration membranes with chemically modified human hair waste for improved permeability and antifouling performance

Human hair, a pervasive waste product, has the potential to pose environmental challenges due to its widespread accumulation. Therefore, exploring of alternatives that repurpose this waste into a valuable raw material aligns with the principles of the circular economy. This study introduces a novel protocol for crafting biohybrid ultrafiltration (UF) membranes with tailored charges, enhanced hydrophilicity, and notable attributes of high flux, rejection rate, and resistance to fouling. These membranes have been engineered by combining sulfonated poly(ethersulfone) (SPES) with chemically modified human hair (HH) filler, referred to as HH-SO3-NH2, utilizing the non-solvent induced phase separation (NIPS) approach. The preparation of HH-SO3-NH2 involved converting of oxidized thiol groups within human hair to sulfonic acid. Subsequently, 1 and 3 wt percent of HH-SO3-NH2 were integrated into the SPES matrix to formulate the biohybrid membranes. After incorporating HH-SO3-NH2, the biohybrid membranes changed in mechanical properties, porosity, and surface morphology, resulting in increased hydrophilicity. The pure water flux exhibited a systematic rise with higher HH-SO3-NH2 content-specifically, the hybrid membrane with 3 wt% HH-SO3-NH2 achieved a water flux of 239 L m−2 h−1 at 1 bar feed pressure, representing a 1.5-fold increase compared to the bare membrane's 175 L m−2 h−1 flux. The anti-fouling performance was assessed using humic acid (HA) and the hybrid membranes demonstrated removal efficiency of HA exceeding 99% without compromising rejection rates.

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Cross-linked polysiloxane-coated stable bond O-9-(2,6-diisopropylphenylcarbamoyl)quinine and quinidine chiral stationary phases as well as application in enantioselective cryo-HPLC.

In this work, brush-type chiral stationary phases (CSPs) with O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinidine (DIPPCQD-brush/-SH) and O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine (DIPPCQN-brush/-SH) were prepared as benchmarks for comparison with new corresponding polymeric CSPs with more stable bonding chemistry. These polymeric CSPs were prepared by coating a thin poly(3-mercaptopropyl)-methylsiloxane film together with the chiral selector onto vinyl-modified silica. In a second step, immobilization of the quinine/quinidine derivatives as well as cross-linking of the polysiloxane film to the vinyl-silica is achieved by a double thiol-ene click reaction. The polymeric CSPs exhibited similar enantioselectivity as the corresponding brush phases, but showed lower chromatographic efficiencies. Chiral acidic substances were separated into enantiomers (e.g., N-protected amino acids, herbicides like dichlorprop) in accordance with an enantioselective anion-exchange process. Oxidation of residual thiol groups of the polymer DIPPCQN-CSP introduced sulfonic acid co-ligands on the silica surface, which resulted in greatly reduced retention times. Acting as immobilized counterions, they allowed to reduce the concentration of counterions in the mobile phase, which is favorable for liquid chromatography (LC)-electrospray ionization-mass spectrometry application. Ibuprofen showed a single peak under ambient column temperature. However, application of cryogenic cooling of the column enabled to achieve baseline separation at -20°C column temperature. It can be explained by an enthalpically dominated separation, which leads to an increase in separation factors when the temperature is reduced. While it is quite uncommon to work at subzero degree column temperature, this work illustrates the potential to exploit such temperature regime for optimization of LC enantiomer separations.

Assembly of Tailored Porous Nanofibers from Mesoporous MCM‐41 Nanoparticles via Electrospinning

AbstractFor the first time the assembly of isolated and purified MCM‐41 nanoparticles into nanofibers was realized by electrospinning. Electrospinning was conducted with a dispersion of these nanoparticles and polyvinylpyrrolidone in ethanol. The nanofibers were characterized by electron microscopy, X‐ray diffraction, elemental analysis, gas physisorption and infrared spectroscopy in diffuse reflectance. The variation of the spinning parameters was investigated in detail towards the influence on the nanofiber diameter. The change of the applied voltage and the flow rate show strong impacts on the nanofiber diameter. Therefore, the nanofiber diameter can be tailored by choosing suitable electrospinning parameters. The fiber mats with thin nanofibers were then additionally functionalized with sulfonic acid groups in a proof of principal study. The nanofiber morphology was maintained both after functionalization with thiol group and subsequent oxidation to sulfonic acid groups.

Cellulose membrane coated Mo-doped TiO2 nanotube sheets for sustained oxidation of biomolecules

Titanium dioxide nanotubes (TNT) are widely researched materials for the photocatalytic generation of free radicals, which are useful in wastewater treatment. We aimed to prepare Mo-doped TNT sheets, covered with a cellulose membrane to avoid TNT surface inactivation by protein adsorption. We studied the susceptibility of serum albumin (SA) bound to different molar ratios of palmitic acid (PA) to denaturation and fibrillation by this system, which is meant to mimic oxidative stress conditions such as non-alcoholic fatty liver disease. The results demonstrated that cellulose membrane-covered TNT successfully oxidized the SA, identified by structural changes to the protein. Increasing the molar ratio of PA to protein-enhanced thiol group oxidation while protecting the protein against structural changes. Finally, we propose that in this photocatalyzed oxidation system, the protein is oxidized by a non-adsorptive mechanism mediated by H2O2. Therefore, we suggest that this system could be used as a sustained oxidation system to oxidize biomolecules as well as potentially in wastewater treatment.

Novel regulatory role of post-translational structural modification in modulating Src kinase activity in pulmonary arterial hypertension

Under conditions that lead to pulmonary arterial hypertension (PAH), the pro-proliferative oncoprotein Src kinase activity is elevated, contributing to pulmonary arterial smooth muscle cell remodeling, increasing vascular resistance, and leading to PAH. In addition, reactive oxygen species (ROS), including Hydrogen peroxide (H2O2), are elevated in the pulmonary vasculature under pro-PAH conditions. Therefore, we hypothesized that Src kinase is subject to redox modulation by elevated hydrogen peroxide (H2O2) under pro-PAH conditions. We intended to explore whether increased H2O2 can cause the oxidation of Src kinase thiol groups resulting in an inter-molecular disulfide bond forming Src kinase homodimer, increasing its catalytic activity. The results of this study detected evidence that Src kinase undergoes redox-induced homodimer formation as illustrated by western blot analysis under non-reducing conditions of rat MCT-induced PAH lung lysates. Furthermore, the redox-mediated Src kinase homodimer formation was associated with the increased catalytic activity of Src kinase, as seen in its intra-molecular auto-phosphorylation of its tyrosine 416 residue. This process was accompanied by activating one of Src kinase downstream substrates, STAT3, via the phosphorylation of its tyrosine 705 residue. Moreover, these mechanistic changes were associated with a PAH disease state confirmed with elevated right ventricle systolic pressure and right ventricle hypertrophy through right ventricle catheterization and the Fulton index, respectively. From the results above, we can conclude that in rat MCT-PAH lung lysates, Src kinase undergoes a redox-induced modulation that results in a structural modification associated with increased Src kinase activity, as observed in its autophosphorylation and the phosphorylation of its downstream substrate STAT3. In conclusion, under conditions that lead to PAH, Src kinase undergoes a post-translational structural modification. This modification occurs in redox-induced oxidation of Src kinase thiol groups. This process results in an inter-molecular disulfide bond formation yielding a homodimer associated with elevated Src kinase catalytic activity, further adding a mechanistic layer to Src kinase regulation in PAH. Kuwait University Research Sector This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The emerging key role of reactive sulfur species in abiotic stress tolerance in plants.

In plants, sulfur plays a critical role in the formation of important biomolecules such as cysteine, methionine, and tripeptide glutathione. Thiol groups, composed of sulfur, are essential to numerous metabolic processes. The easy and reversible oxidation and reduction of thiol groups have drawn attention to the redox regulation of cellular metabolism. Reactive sulfur species (RSS), including hydrogen sulfide (H2 S), persulfides and polysulfides, are synthetized in all living organisms, mainly from cysteine, and have been recognized in the last two decades as very important molecules in redox regulation. RSS are considered potent signaling molecules, being involved in the regulation of virtually all aspects of cell function. With regard to stress, reactive species and the antioxidant machinery maintain a delicate balance that gets disturbed under stress conditions, wherein reactive species biosynthesis, transportation, scavenging, and overall metabolism become decisive for plant survival. While reactive oxygen and nitrogen species have been much discussed over recent years, research into reactive sulfur species (RSS) biosynthesis, signaling, and relation to abiotic stresses is still nascent. RSS evolved long before reactive oxygen species, and because both are metabolized by catalase, it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may still do so today. In this review, we have tried to summarize the generation, signaling, and interaction of RSS in plant systems and to discuss in detail the roles under various abiotic stresses. This article is protected by copyright. All rights reserved.

Abstract 1386: Ag5 is highly potent, mitochondrially targeted agent with a differentiated mechanism of action that promises to be efficacious in solid cancers

Abstract Mitochondrial remodeling in cancer is important for tumorigenesis, metastasis and linked totreatment resistance and poor prognosis. Dysfunctional mitochondria are also driven by oncogenes (e.g. KRAS, MYC) resulting in an increased oxidation state triggering the antioxidant response in order to decrease ROS levels, and to trigger apoptosis when the oxidative stress cannot be overcome. So far, targeting cancer cell energy metabolism has been unsuccessful due to tumor evolution, low therapeutic index and redundancy of protective mechanisms. Taken together, the clinical sequelae of mitochondrial remodeling represent one of the largest areas of unmet need in cancer therapy. We are developing Ag5, a highly potent, mitochondrially targeted agent with a novel mechanism of action that promises to be efficacious in solid cancers. Ag5 selectively kills cancer cells by catalyzing the oxidation of thiol groups of members ofthese pathways, a rapid mechanism of action that can be reversed with ROS scavengers (e.g. NAC). As a consequence, Ag5 will preferentially kill cancer cells, but will spare normal cells due to their REDOX homeostasis. We characterized Ag5 efficacy with a range of in vitro viability assays and showed potency against a panel of established cancer cell lines with an IC50 in the low nM range, whereas Ag5 activity was significantly lower in non-malignant cells. Ag5 sensitivity was not only correlated with high ROS levels as measured by mitoSOX, but also with target protein oxidation (especially the mitochondrially expressed PRDX3), cardiolipin and cytochrome c release, thus highlighting the mitochondrial targeting of Ag5. Several well characterised gene mutations lead to alterations in mitochondrial antioxidant function which sensitise the tumors to Ag5, either by decreasing endogenous antioxidant synthesis or increasing reliance on antioxidants for cell survival. For example, ARID1A damaging mutations directly lead to decreased glutathione synthesis and are found in many solid cancers (e.g. NSCLC, CRC and clear cell ovarian cancers). Experimental data with pairs of ARID1A wild-type or KO cell lines showed strong sensitization to Ag5 activity when ARID1A is knocked out. Furthermore, Ag5 was shown to be effective in reducing tumor growth in KRAS G12C mutated NSCLC and other relevant cell lines in viability assays and an orthotopic in vivo model. Combination treatment with KRAS, SOS1 and SHP2 inhibitors demonstrated not only Ag5 synergy, but also superior efficacy when Ag5 was included over the combination of these inhibitors alone. In summary, Ag5 is a novel and innovative therapeutic candidate shown to be safe and effective in preclinical studies. Based on experimental data with clinically relevant mutations, we hypothesize that Ag5 is ideally investigated in enriched patient populations of lung cancerand other solid cancers. Citation Format: Martin Treder. Ag5 is highly potent, mitochondrially targeted agent with a differentiated mechanism of action that promises to be efficacious in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1386.

Rosemary (Rosmarinus officinalis L.) Glycolic Extract Protects Liver Mitochondria from Oxidative Damage and Prevents Acetaminophen-Induced Hepatotoxicity.

Rosmarinus officinalis L. (rosemary) is an aromatic culinary herb. Native to the Mediterranean region, it is currently cultivated worldwide. In addition to its use as a condiment in food preparation and in teas, rosemary has been widely employed in folk medicine and cosmetics. Several beneficial effects have been described for rosemary, including antimicrobial and antioxidant activities. Here, we investigated the mechanisms accounting for the antioxidant activity of the glycolic extract of R. officinalis (Ro) in isolated rat liver mitochondria (RLM) under oxidative stress conditions. We also investigated its protective effect against acetaminophen-induced hepatotoxicity in vivo. A crude extract was obtained by fractionated percolation, using propylene glycol as a solvent due to its polarity and cosmeceutical compatibility. The quantification of substances with recognized antioxidant action revealed the presence of phenols and flavonoids. Dereplication studies carried out through LC-MS/MS and GC-MS, supported by The Global Natural Product Social Molecular Networking (GNPS) platform, annotated several phenolic compounds, confirming the previous observation. In accordance, Ro decreased the production of reactive oxygen species (ROS) elicited by Fe2+ or t-BOOH and inhibited the lipid peroxidation of mitochondrial membranes in a concentration-dependent manner in RLM. Such an effect was also observed in liposomes as membrane models. Ro also prevented the oxidation of mitochondrial protein thiol groups and reduced glutathione (GSH). In model systems, Ro exhibited a potent scavenger activity toward 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radicals and superoxide anions. It also demonstrated an Fe2+ chelating activity. Moreover, Ro did not exhibit cytotoxicity or dissipate the mitochondrial membrane potential (∆Ψ) in rat liver fibroblasts (BRL3A cells). To evaluate whether such antioxidant protective activity observed in vitro could also be achieved in vivo, a well-established model of hepatotoxicity induced by acute exposure to acetaminophen (AAP) was used. This model depletes GSH and promotes oxidative-stress-mediated tissue damage. The treatment of rats with 0.05% Ro, administered intraperitoneally for four days, resulted in inhibition of AAP-induced lipid peroxidation of the liver and the prevention of hepatotoxicity, maintaining alanine and aspartate aminotransferase (ALT/AST) levels equal to those of the normal, non-treated rats. Together, these findings highlight the potent antioxidant activity of rosemary, which is able to protect mitochondria from oxidative damage in vitro, and effects such as the antioxidant and hepatoprotective effects observed in vivo.

Extract from Sea Buckthorn Seeds-A Phytochemical, Antioxidant, and Hemostasis Study; Effect of Thermal Processing on Its Chemical Content and Biological Activity In Vitro.

Sea buckthorn (Hippophae rhamnoides L.) is a small tree, valued for its medicinal properties throughout the ages. Sea buckthorn berries and leaves are a known source of phytochemicals and have been used in the treatment of inflammation, oedema, hypertension, ulcers, and wounds in folk medicine. Sea buckthorn seeds are natural dietary sources of various bioactive compounds as well, but the number of studies on their content and biological properties is still insufficient. For the first time, we examined the phytochemical content and biological activity of sea buckthorn seeds in vitro. We have studied the effect of two extracts-from regular (no thermal processing) and roasted (thermally processed) sea buckthorn seeds-on the levels of oxidative stress induced by H2O2/Fe2+ in plasma, coagulation times, and white thrombus formation (measured by Total Thrombus-formation Analysis System-T-TAS). We observed that sea buckthorn seeds contain diverse flavonoids, mostly glycosides of isorhamnetin, kaempferol, and quercetin, as well as smaller amounts of proanthocyanidins and catechin, triterpenoid saponins, and a number of unidentified polar and hydrophobic compounds. Both extracts inhibited lipid peroxidation and protein carbonylation, but only the extract from roasted seeds decreased oxidation of thiol groups in plasma treated with H2O2/Fe2+. They did not alter coagulation times, but the extract from roasted seeds at the highest concentration (50 µg/mL) prolonged the time needed for white thrombus formation. The results indicate that sea buckthorn seeds have antioxidant activity that is not impaired by thermal processing and possess anticoagulant potential, but more research is needed in order to ascertain which compounds are responsible for these effects, especially in in vivo models.

Efficient glucose-to-HMF conversion in deep eutectic solvents over sulfonated dendrimer modified activated carbon

In this study, a solid acid catalyst was designed by growing thiol-based dendrimer up to third generation on the surface of activated carbon (AC) by the thiol-ene click reaction and subsequent transformation to sulfonic acid groups. Characterization results displayed the successful grafting of sulfonic acid groups with high density on dendrimer functionalized AC through the oxidation of thiol groups. The efficiency of the obtained catalyst was explored in the acid-catalyzed transformation of glucose to 5-Hydroxymethylfurfural (HMF) using deep eutectic solvents (DESs). The most effective DES was choline chloride:urea (ChCl:urea), which provided an HMF yield of 96% after 1 h of reaction time at 100 °C from glucose. Some control experiments demonstrated that the alkalinity of nitrogen in the dendrimer structure combined with of urea has an important synergistic effect in enhancing the glucose isomerization to fructose, thus improving the reaction efficiency. Additionally, the presence of dendritic structure contributed to the catalytic activity by making the sulfonic acid group highly reactive. Both the catalyst and DES were used for five cycles without significant descent in catalytic activity.

Insights into H2O2-induced signaling in Jurkat cells from analysis of gene expression

Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2O2-dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signaling.

Synthesis and Use of the Bifunctional Sulfenic Acid Probe BCN-E-BCN for In Vitro and Cell-Based Assays of Protein Oxidation.

The reversible oxidation of cysteine thiol groups to sulfenic acid by reactive oxygen species (ROS) such as hydrogen peroxide can impact protein function, activity, and localization. As a consequence, ROS have profound effects on cell functions including proliferation, differentiation, and survival. Furthermore, there are clear associations between the effects of ROS on cells and the etiology of several diseases including cancer and neurodegeneration. In spite of the importance of cysteine sulfenylation as a validated post-translational modification, its labile nature impedes efficient and reproducible detection of proteins with cysteine sulfenic acid residues. To overcome this challenge, we developed a novel cell-permeable bifunctional reagent, consisting of two linked bicyclo[6.1.0]nonyne (BCN) moieties coupled with a short ethylenediamine-derived linker (BCN-E-BCN) that enables the detection of sulfenylated proteins in vitro and in intact cells. The two symmetrical BCN groups allow protein sulfenic acids to be selectively tagged with a BCN at one end while allowing for copper-free click chemistry with azide-tagged reagents of the opposite BCN. In this protocol, the synthesis of BCN-E-BCN and its use to detect cysteine sulfenic acids will be detailed. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Copper-mediated cyclopropanation of 1,5-cyclooctadiene Basic Protocol 2: Synthesis of endo- and exo-bicyclononyne Basic Protocol 3: Synthesis of endo-BCN-E-BCN Basic Protocol 4: BCN-E-BCN treatment of wild-type and cysteine-deficient mutant recombinant cofilin protein Basic Protocol 5: BCN-E-BCN labeling in live cells Basic Protocol 6: Western blotting and visualization of BCN-E-BCN-labeled samples.

Correcting the Experimental Enthalpies of Formation of Some Members of the Biologically Significant Sulfenic Acids Family.

Sulfenic acids are important intermediates in the oxidation of cysteine thiol groups in proteins by reactive oxygen species. The mechanism is influenced heavily by the presence of polar groups, other thiol groups, and solvent, all of which determines the need to compute precisely the energies involved in the process. Surprisingly, very scarce experimental information exists about a very basic property of sulfenic acids, the enthalpies of formation. In this Article, we use high level quantum chemical methods to derive the enthalpy of formation at 298.15 K of methane-, ethene-, ethyne-, and benzenesulfenic acids, the only ones for which some experimental information exists. The methods employed were tested against well-known experimental data of related species and extensive CCSD(T) calculations. Our best results consistently point out to a much lower enthalpy of formation of methanesulfenic acid, CH3SOH (ΔfH0(298.15K) = -35.1 ± 0.4 kcal mol-1), than the one reported in the NIST thermochemical data tables. The enthalpies of formation derived for ethynesulfenic acid, HC≡CSOH, +32.9 ± 1.0 kcal/mol, and benzenesulfenic acid, C6H5SOH, -2.6 ± 0.6 kcal mol-1, also differ markedly from the experimental values, while the enthalpy of formation of ethenesulfenic acid CH2CHSOH, not available experimentally, was calculated as -11.2 ± 0.7 kcal mol-1.

Redox regulation of enzymes involved in sulfate assimilation and in the synthesis of sulfur-containing amino acids and glutathione in plants.

Sulfur is essential in plants because of its presence in numerous molecules including the two amino acids, cysteine, and methionine. Cysteine serves also for the synthesis of glutathione and provides sulfur to many other molecules including protein cofactors or vitamins. Plants absorb sulfate from their environment and assimilate it via a reductive pathway which involves, respectively, a series of transporters and enzymes belonging to multigenic families. A tight control is needed to adjust each enzymatic step to the cellular requirements because the whole pathway consumes energy and produces toxic/reactive compounds, notably sulfite and sulfide. Glutathione is known to regulate the activity of some intermediate enzymes. In particular, it provides electrons to adenosine 5′-phosphosulfate reductases but also regulates the activity of glutamate-cysteine ligase by reducing a regulatory disulfide. Recent proteomic data suggest a more extended post-translational redox control of the sulfate assimilation pathway enzymes and of some associated reactions, including the synthesis of both sulfur-containing amino acids, cysteine and methionine, and of glutathione. We have summarized in this review the known oxidative modifications affecting cysteine residues of the enzymes involved. In particular, a prominent regulatory role of protein persulfidation seems apparent, perhaps because sulfide produced by this pathway may react with oxidized thiol groups. However, the effect of persulfidation has almost not yet been explored.

Redox Homeostasis is Disturbed by Redox Cycling between Reactive Cysteines of Thioredoxin 1 and 9,10-Phenanthrenequinone, an Atmospheric Electron Acceptor.

9,10-Phenanthrenequinone (9,10-PQ) is a toxicant in diesel exhaust particles and airborne particulate matter ≤2.5 μm in diameter. It is an efficient electron acceptor that readily reacts with dithiol compounds in vitro, resulting in the oxidation of thiol groups and concomitant generation of reactive oxygen species (ROS). However, it remains to be elucidated whether 9,10-PQ interacts with proximal protein dithiols. In the present study, we used thioredoxin 1 (Trx1) as a model of proteins with reactive proximal cysteines and examined whether it reacts with 9,10-PQ in cells and tissues, thereby affecting its catalytic activity and thiol status. Intratracheal injection of 9,10-PQ into mice resulted in protein oxidation and diminished Trx activity in the lungs. Using recombinant wild-type and C32S/C35S Trx1, we found that Cys32 and Cys35 selectively serve as electron donor sites for redox reactions with 9,10-PQ that lead to substantial inhibition of Trx activity. Addition of dithiothreitol restored the Trx activity inhibited by 9,10-PQ. Exposure of cultured cells to 9,10-PQ caused intracellular reactive oxygen species generation that led to protein oxidation, Trx1 dimerization, p38 phosphorylation, and apoptotic cell death. Overexpression of Trx1 blocked these 9,10-PQ-mediated events. These results suggest that the interaction of the reactive cysteines of Trx1 with 9,10-PQ causes oxidative stress, leading to disruption of redox homeostasis.

Selenium supplementation in the prevention of coronavirus infections* (*In Memory of Laszlo G. Egyud)

Selenium (Se) is a ubiquitous element akin to sulfur (S) existing in the Earth’s crust in various organic and inorganic forms. Selenium concentration varies greatly depending on the geographic area. Consequently, the content of selenium in food products is also variable. It is known that low Se is associated with an increased incidence of cancer and heart diseases. Therefore, it is advisable to supplement your diet with this element albeit in a proper form. Although blood increased concentrations of Se can be achieved with various pharmacological preparations only one chemical form (sodium selenite) can offer true protection. Sodium selenite, but not selenate, can oxidize thiol groups in the virus protein disulfide isomerase rendering it unable to penetrate the healthy cell membrane. In this way, selenite inhibits the entrance of viruses into the healthy cells and abolishes their infectivity. Therefore, this simple chemical compound can potentially be used in the recent battle against the coronavirus epidemic.