A novel mycothiol-dependent thiol–disulfide reductase in Corynebacterium glutamicum involving oxidative stress resistance

Abstractncgl2478 gene from Corynebacterium glutamicum encodes a thiol–disulfide oxidoreductase enzyme annotated as dithiol–disulfide isomerase DsbA. It preserves a Cys–Pro–Phe–Cys active-site motif, which is presumed to be an exclusive characteristic of the novel DsbA–mycoredoxin 1 (Mrx1) cluster. However, the real mode of action, the nature of the electron donor pathway and biological functions of NCgl2478 in C. glutamicum have remained enigmatic so far. Herein, we report that NCgl2478 plays an important role in stress resistance. Deletion of the ncgl2478 gene increases the size of growth inhibition zones. The ncgl2478 expression is induced in the stress-responsive extra-cytoplasmic function-sigma (ECF-σ) factor SigH-dependent manner by stress. It receives electrons preferentially from the mycothiol (MSH)/mycothione reductase (Mtr)/NADPH pathway. Further, NCgl2478 reduces S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol–disulfide and a dithiol–disulfide exchange mechanism, respectively. NCgl2478 lacks oxidase activity; kinetic properties of its demycothiolation are different from those of Mrx1. Site-directed mutagenesis confirms Cys24 is the resolving Cys residue, while Cys21 is the nucleophilic cysteine that is oxidized to a sulfenic acid and then forms an intramolecular disulfide bond with Cys24 or a mixed disulfide with MSH under oxidative stress. In conclusion, our study presents the first evidence that NCgl2478 protects against various stresses by acting as an MSH-dependent thiol–disulfide reductase, belonging to a novel DsbA–Mrx1 cluster.

An Orally Bioavailable (Mice) Prodrug of Glutathione

L-Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH), was found to be orally bioavailable in mice, and protected mice against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, a wide range of applicability for CySSG can be envisioned.

An Orally Bioavailable (Mice) Prodrug of Glutathione

Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH) --the “Master Antioxidant”, was found to be orally bioavailable in mice, and protected against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, this suggests a wide range of applicability for CySSG.

Identification of the Molecular Targets of Disulfide Bond Disrupting Agents

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, AGR3, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, these results reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, demonstrate a role for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and nominate ERp44 as a new molecular target for anticancer therapeutics.

Role of Glutaredoxin-1 and Glutathionylation in Cardiovascular Diseases

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body’s reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

Selenosugars Are the Major Selenometabolites in Liver of Turkeys Fed High Selenium

Objectives Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. Because the nature of this accumulated Se is not well characterized, we studied selenometabolomics in Se-deficient, Se-adequate, and high-Se liver. Methods Turkey poults were fed 0, 0.4, and 5 μg Se/g diet as Na2SeO3 in a Se-deficient (0.005 μg Se/g) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. Results In liver from turkeys fed a true Se-deficient diet and supplemented with inorganic Se (selenite), no selenomethionine (SeMet) was detected showing that the turkey cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient turkey liver, as expected in animals with negligible selenoprotein levels. Sec content in liver of turkeys fed high Se only doubled compared with Se-adequate liver, indicating that the 6-fold incryscease in liver Se in these birds was not due to increases in selenoproteins. What increased dramatically in high Se liver were the low molecular weight (MW) selenometabolites, glutathione-, cysteine- and methyl- conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). In addition, size-exclusion chromatography and followup analysis demonstrated that a substantial amount of Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide links, in addition to Sec-containing selenoproteins. In high-Se liver, these “selenosugar-decorated” proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to the low MW selenometabolites. Conclusions In Se-adequate liver, far more Se is present as the selenosugar moiety, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols, leading to the formation of methyl-SeGalNac. This suggested pathway, underlying adaptation to high Se status in animals, needs further investigation including study of the potential of selenosugar compounds as biomarkers of high Se status. Funding Sources USDA, Hatch project 1,004,389, and by the Wisconsin Alumni Foundation, Selenium Nutrition Research Fund 12,046,295.

Regulatory thiol oxidation in chloroplast metabolism, oxidative stress response and environmental signaling in plants

The antagonism between thiol oxidation and reduction enables efficient control of protein function and is used as central mechanism in cellular regulation. The best-studied mechanism is the dithiol-disulfide transition in the Calvin Benson Cycle in photosynthesis, including mixed disulfide formation by glutathionylation. The adjustment of the proper thiol redox state is a fundamental property of all cellular compartments. The glutathione redox potential of the cytosol, stroma, matrix and nucleoplasm usually ranges between −300 and −320 mV. Thiol reduction proceeds by short electron transfer cascades consisting of redox input elements and redox transmitters such as thioredoxins. Thiol oxidation ultimately is linked to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Enhanced ROS production under stress shifts the redox network to more positive redox potentials. ROS do not react randomly but primarily with few specific redox sensors in the cell. The most commonly encountered reaction within the redox regulatory network however is the disulfide swapping. The thiol oxidation dynamics also involves transnitrosylation. This review compiles present knowledge on this network and its central role in sensing environmental cues with focus on chloroplast metabolism.

Ultra-Sensitive Hydrogen Peroxide Sensor Based on Peroxiredoxin and Fluorescence Resonance Energy Transfer

In this paper, a fluorescence resonance energy transfer (FRET)-based sensor for ultra-sensitive detection of H2O2 was developed by utilizing the unique enzymatic properties of peroxiredoxin (Prx) to H2O2. Cyan and yellow fluorescent protein (CFP and YFP) were fused to Prx and mutant thioredoxin (mTrx), respectively. In the presence of H2O2, Prx was oxidized into covalent homodimer through disulfide bonds, which were further reduced by mTrx to form a stable mixed disulfide bond intermediate between CFP-Prx and mTrx-YFP, inducing FRET. A linear quantification range of 10–320 nM was obtained according to the applied protein concentrations and the detection limit (LOD) was determined to be as low as 4 nM. By the assistance of glucose oxidase to transform glucose into H2O2, the CFP-Prx/mTrx-YFP system (CPmTY) was further exploited for the detection of glucose in real sample with good performance, suggesting this CPmTY protein sensor is highly practical.

Influence of Oxidative Stress on Catalytic and Non-glycolytic Functions of Glyceraldehyde-3-phosphate Dehydrogenase

Background: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) is a unique enzyme that, besides its main function in glycolysis (catalysis of glyceraldehyde-3-phosphate oxidation), possesses a number of non-glycolytic activities. The present review summarizes information on the role of oxidative stress in the regulation of the enzymatic activity as well as non-glycolytic functions of GAPDH. Methods: Based on the analysis of literature data and the results obtained in our research group, mechanisms of the regulation of GAPDH functions through the oxidation of the sulfhydryl groups in the active site of the enzyme have been suggested. Results: Mechanism of GAPDH oxidation includes consecutive oxidation of the catalytic Cysteine (Cys150) into sulfenic, sulfinic, and sulfonic acid derivatives, resulting in the complete inactivation of the enzyme. The cysteine sulfenic acid reacts with reduced glutathione (GSH) to form a mixed disulfide (S-glutathionylated GAPDH) that further reacts with Cys154 yielding the disulfide bond in the active site of the enzyme. In contrast to the sulfinic and sulfonic acids, the mixed disulfide and the intramolecular disulfide bond are reversible oxidation products that can be reduced in the presence of GSH or thioredoxin. Conclusion: Oxidation of sulfhydryl groups in the active site of GAPDH is unavoidable due to the enhanced reactivity of Cys150. The irreversible oxidation of Cys150 is prevented by Sglutathionylation and disulfide bonding with Cys154. The oxidation/reduction of the sulfhydryl groups in the active site of GAPDH can be used for regulation of glycolysis and numerous side activities of this enzyme including the induction of apoptosis.

Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS

Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labeling. Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high-performance LC-MS without the need for metabolic labeling and derivatization.