Efficacy of antioxidants in the yeast Saccharomyces cerevisiae correlates with their effects on protein thiols

We have found previously that only a limited number of antioxidants are able to protect yeast cells against endogenous and exogenous oxidative stress. In search of factors determining this selectivity of antioxidant action we compared the ability of a set of antioxidants to: (i) protect a thiol-dependent enzyme alcohol dehydrogenase (ADH) against inactivation by superoxide, peroxynitrite and hydrogen peroxide; (ii) prevent H(2)O(2)-induced activation of Yap1 p; and (iii) decrease extracellular redox potential of the medium. The results obtained provide demonstration with respect to yeast that the ability to lower redox potential and to maintain critical thiol groups in the reduced state is an important facet of the action of antioxidants.     

The cysteine proteome

The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metalation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to allow the discrimination of network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome.     

Measuring E(GSH) and H2O2 with roGFP2-based redox probes

Redox biochemistry plays an important role in a wide range of cellular events. However, investigation of cellular redox processes is complicated by the large number of cellular redox couples, which are often not in equilibrium with one another and can vary significantly between subcellular compartments and cell types. Further, it is becoming increasingly clear that different redox systems convey different biological information; thus it makes little sense to talk of an overall "cellular redox state". To gain a more differentiated understanding of cellular redox biology, quantitative, redox couple-specific, in vivo measurements are necessary. Unfortunately our ability to investigate specific redox couples or redox-reactive molecules with the necessary degree of spatiotemporal resolution is very limited. The development of genetically encoded redox biosensors offers a promising new way to investigate redox biology. Recently developed redox-sensitive green fluorescent proteins (roGFPs), genetically fused to redox-active proteins, allow rapid equilibration of the roGFP moiety with a specific redox couple. Two probes based on this principle are now available: Grx1-roGFP2 for the measurement of glutathione redox potential (E(GSH)) and roGFP2-Orp1 for measuring changes in H(2)O(2) concentration. Here we provide a detailed protocol for the use of these probes in both yeast and mammalian systems using either plate-reader- or microscopy-based measurements.     

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 μM) or DEM (5–25 μM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.     

Amino acids and their derivatives as radioprotective agents

Summary Numerous amino acids and their analogs are capable of protecting biological systems from the toxic effects of ionizing radiation. These radioprotective agents can be classified into two broad groups, depending upon the presence or absence of a free or potentially free sulfhydryl group. The sulfhydryl-containing compounds have been studied extensively and are thought to exert their radioprotective effects by several mechanisms, including free radical scavenging and hydrogen atom donation. Several non-sulfhydryl-containing amino acids are also being investigated for their radioprotective effects. These agents are less well known than the familiar sulfhydryl compounds, but possess very interesting protective qualities. In short, the study of amino acids and their derivatives as radioprotective agents continues to contribute to an understanding of processes involved in radiation toxicity and to offer new compounds with potential application to situations of human exposure.     

Genetic analysis of tissue glutathione concentrations and redox balance

Glutathione redox balance—defined as the ratio GSH/GSSG—is a critical regulator of cellular redox state, and declines in this ratio are closely associated with oxidative stress and disease. However, little is known about the impact of genetic variation on this trait. Previous mouse studies suggest that tissue GSH/GSSG is regulated by genetic background and is therefore heritable. In this study, we measured glutathione concentrations and GSH/GSSG in liver and kidney of 30 genetically diverse inbred mouse strains. Genetic background caused an approximately threefold difference in hepatic and renal GSH/GSSG between the most disparate strains. Haplotype association mapping determined the loci associated with hepatic and renal glutathione phenotypes. We narrowed the number of significant loci by focusing on those located within protein-coding genes, which we now consider to be candidate genes for glutathione homeostasis. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue-specific manner. This is the first quantitative trait locus study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of redox biochemistry and its regulation and inform future therapeutic applications.     

Free radical biology for medicine: learning from nonalcoholic fatty liver disease

Reactive oxygen species, when released under controlled conditions and limited amounts, contribute to cellular proliferation, senescence, and survival by acting as signaling intermediates. In past decades there has been an epidemic diffusion of nonalcoholic fatty liver disease (NAFLD) that represents the result of the impairment of lipid metabolism, redox imbalance, and insulin resistance in the liver. To date, most studies and reviews have been focused on the molecular mechanisms by which fatty liver progresses to steatohepatitis, but the processes leading toward the development of hepatic steatosis in NAFLD are not fully understood yet. Several nuclear receptors, such as peroxisome proliferator-activated receptors (PPARs) α/γ/δ, PPARγ coactivators 1α and 1β, sterol-regulatory element-binding proteins, AMP-activated protein kinase, liver-X-receptors, and farnesoid-X-receptor, play key roles in the regulation of lipid homeostasis during the pathogenesis of NAFLD. These nuclear receptors may act as redox sensors and may modulate various metabolic pathways in response to specific molecules that act as ligands. It is conceivable that a redox-dependent modulation of lipid metabolism, nuclear receptor-mediated, could cause the development of hepatic steatosis and insulin resistance. Thus, this network may represent a potential therapeutic target for the treatment and prevention of hepatic steatosis and its progression to steatohepatitis. This review summarizes the redox-dependent factors that contribute to metabolism alterations in fatty liver with a focus on the redox control of nuclear receptors in normal liver as well as in NAFLD.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.     

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

Thiols enhance NO formation from nitrate photolysis

Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to na?ve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.