Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by H2O2 increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH. H2O2 reacts reversibly with CO2 to form a more reactive oxidant, peroxymonocarbonate (HCO4-), which is most likely responsible for the enhanced inactivation. However, to account for the extent of enhancement, we propose that GAPDH must facilitate formation and/or targeting of HCO4- to promote its own inactivation. Inactivation of intracellular GAPDH was also strongly enhanced by bicarbonate: treatment of Jurkat cells with 20 µM H2O2 in 25 mM bicarbonate buffer for 5 min caused almost complete GAPDH inactivation, but no loss of activity when bicarbonate was not present. H2O2-dependent GAPDH inhibition in bicarbonate buffer was observed even in the presence of reduced peroxiredoxin 2 and there was a significant increase in cellular glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. Our results identify an unrecognized role for bicarbonate in enabling H2O2 to influence inactivation of GAPDH and potentially reroute glucose metabolism from glycolysis to the pentose phosphate pathway and NAPDH production. They also demonstrate what could be wider interplay between CO2 and H2O2 in redox biology and the potential for variations in CO2 metabolism to influence oxidative responses and redox signaling.

Rat Liver Mitochondrial Dysfunction Induced by an Organic Arsenical Compound 4-(2-Nitrobenzaliminyl) Phenyl Arsenoxide.

Arsenic is successfully used in cancer chemotherapy and several cancer treatments on account of its apoptogenic effects. However, it is environmentally hazardous with potential for toxicity when distributed in the soil, water, and food, and long exposure to water contaminated with Arsenic may induce cancers. Some research studies have reported that liver is the storage site and an important target organ for Arsenic toxicity. In the present work, a new kind of organic arsenic compound, 4-(2-nitrobenzaliminyl) phenyl arsenoxide (NPA), was synthesized, and its potential involvement of mitochondria was explored. The results presented that the toxicology of NPA, at least in part, mediated mitochondrial function and may thoroughly destroy mitochondrial membrane physiological functions. NPA induced mitochondrial permeability transition pore (mtPTP) opening that induces mitochondrial biochemical abnormalities as evidenced by mitochondrial swelling, mitochondrial membrane potential breakdown, membrane fluidity alterations, and the strikingly remarkable protection of CsA. Meanwhile, both the decreased respiration rate of state 4 and the increased inner membrane H(+) permeabilization revealed that the inner membrane function regarding important energy production chain was destroyed. The toxicity of NPA is due to its interaction with mitochondrial membrane thiol protein. This conclusion is based on the protective effects of RR, DTT, and MBM(+).

Comparative proteomic analysis of thiol proteins in the liver after oxidative stress induced by diethylnitrosamine.

Conversion of protein -SH groups to disulfides is an early event during protein oxidation, which has prompted great interest in the study of thiol proteins. Chemical carcinogenesis is strongly associated with the formation of reactive oxygen species (ROS). The goal of this study was to detect thiol proteins that are sensitive to ROS generated during diethylnitrosamine (DEN) metabolism in the rat liver. DEN has been widely used to induce experimental hepatocellular carcinoma. We used modified redox-differential gel electrophoresis (redox-DIGE method) and mass spectrometry MALDI-TOF/TOF to identify differential oxidation protein profiles associated with carcinogen exposure. Our analysis revealed a time-dependent increase in the number of oxidized thiol proteins after carcinogen treatment; some of these proteins have antioxidant activity, including thioredoxin, peroxirredoxin 2, peroxiredoxin 6 and glutathione S-transferase alpha-3. According to functional classifications, the identified proteins in our study included chaperones, oxidoreductases, activity isomerases, hydrolases and other protein-binding partners. This study demonstrates that oxidative stress generated by DEN tends to increase gradually through DEN metabolism, causes time-dependent necrosis in the liver and has an oxidative effect on thiol proteins, thereby increasing the number of oxidized thiol proteins. Furthermore, these events occurred during the hepatocarcinogenesis initiation period.

Identification of a protein with antioxidant activity that is important for the protection against beer ageing.

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

Biological Production, Detection, and Fate of Hydrogen Peroxide.

SIGNIFICANCE: Hydrogen peroxide (H2O2) is generated in numerous biological processes. It transmits cellular signals, contributes to oxidative folding of exported proteins, and, in excess, can be damaging to cells and tissues. Although a strong oxidant, high activation energy barriers make it unreactive with most biological molecules. Its main reactions are with transition metal centers, selenoproteins and selected thiol proteins, with glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) being major targets. It reacts slowly with most thiol proteins, and how they become oxidized during redox signal transmission is not well understood. Recent Advances: Kinetic analysis indicates that Prxs and GPxs are overwhelmingly favored as targets for H2O2 in cells. Studies with localized probes indicate that H2O2 can be produced in cellular microdomains and be consumed by highly reactive targets before it can diffuse to other parts of the cell. Inactivation of these targets alone will not confine it to its site of production. Kinetic data indicate that oxidation of regulatory thiol proteins by H2O2 requires a facilitated mechanism such as directed transfer from source to target or a relay mediated through a highly reactive sensor. Critical Issues and Future Directions: Absolute rates of H2O2 production and steady-state concentrations in cells still need to be characterized. More information on cellular sites of production and action is required, and specific mechanisms of oxidation of regulatory proteins during redox signaling require further characterization. Antioxid. Redox Signal. 29, 541-551.

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals.

The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation.

The recent discovery of significant hydropersulfide (RSSH) levels in mammalian tissues, fluids and cells has led to numerous questions regarding their possible physiological function. Cysteine hydropersulfides have been found in free cysteine, small molecule peptides as well as in proteins. Based on their chemical properties and likely cellular conditions associated with their biosynthesis, it has been proposed that they can serve a protective function. That is, hydropersulfide formation on critical thiols may protect them from irreversible oxidative or electrophilic inactivation. As a prelude to understanding the possible roles and functions of hydropersulfides in biological systems, this study utilizes primarily chemical experiments to delineate the possible mechanistic chemistry associated with cellular protection. Thus, the ability of hydropersulfides to protect against irreversible electrophilic and oxidative modification was examined. The results herein indicate that hydropersulfides are very reactive towards oxidants and electrophiles and are modified readily. However, reduction of these oxidized/modified species is facile generating the corresponding thiol, consistent with the idea that hydropersulfides can serve a protective function for thiol proteins.

Are free radicals involved in thiol-based redox signaling?

Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.