Introduction Glutathione From Antioxidant to Redox Signal

Glutathione (GSH), the tripeptide Glu-Cys-Gly, is a low molecular weight thiol antioxidant present in high concentrations in most cells, with the exception of some bacteria and amoebae. Due to its thiol features, GSH may give rise to a variety of chemical reactions that are not typical of other nucleophiles, amino or hydroxyl groups (see Chapter 4). In fact, through nucleophilic addition or displacement, oxidation-reduction reactions, thiol/disulfide (SH/SS) exchange reactions (or thiol/ disulfide interchange reactions), GSH detoxifies electrophilic and oxidizing agents, preventing their attack on proteins and other macromolecules. The detoxification actions of GSH are often catalyzed by a variety of enzymes (such as glutathione transferase, glyoxalase, glutathione reductase, glutathione peroxidase, glutaredoxin), which form an efficient machinery capable of protecting cells even when GSH levels are low.

The protective role of GSH has long been demonstrated by experiments showing the exacerbating effects of GSH-depleting agents, the most used being diethylmale-ate (DEM) and buthionine sulfoximine (BSO) - in various animal models exposed to oxidative stress or chemical insults. Conversely - many studies have reported that cell-permeable GSH esters, or GSH synthesis precursors, such as N-acetyl-l-cysteine or l-2-oxothiazolidine-4-carboxylic acid, protect cells from damage caused by electrophilic or oxidizing agents.

Despite this, it should be mentioned, however, that few of the studies led to the registration and approval of thiol antioxidants in the therapy of the diseases for which there was preclinical evidence, recalling, to some extent, the failure of vitamin E supplementation in therapies against free radical diseases and cancer [1].

In addition to the protective function in toxicology, GSH has a well-known but still largely undefined role in regulating biochemical pathways through its reducing power. Physiological and toxicological roles of GSH actions are mediated by protein S-thiolation, that is the formation of protein-glutathione mixed disulfides (PrSSG) -

also named glutathiolation (and sometimes also referred to as glutathionation or glutathionylation) - and, according to more recent contributions, also with protein disulfide (PrSSPr) formation [2-4].

GSH controls a variety of key molecular mechanisms linked to cell signaling, immune response, cell proliferation, inflammation, apoptosis and cell death. In this context, redox regulation of gene expression is considered a fundamental mechanism in cell biology and many efforts have been directed at understanding redox sensors and redox-sensitive targets that in a great majority are represented by protein thiol groups (PrSH) [5]. Thus GSH, protein thiols and "low levels" of reactive oxygen species (ROS) (and reactive nitrogen species (RNS)) are all strictly interconnected in signaling where glutathiolation plays a major role. For example, it is known that H2O2 is implicated as an activator of the transcription factor NF-kB [6], that low concentration of H2O2 are mitogenic for vascular smooth muscle cells [2, 7-9], and that H2O2-mediated events are inevitably linked to glutathiolation.

Despite this, our knowledge of the biological roles of GSH remains limited and this is in part due to the many parts it plays in reactions of oxidation, thiol/disulfide (SH/SS) exchange and conjugation. For example, the widely recognized fundamental action of redox buffer exerted by the GSH/GSSG couple [10] is, to some extent, weakened by the fact that recent studies suggest that GSSG may have a minor role in protein glutathiolation (discussed below); in turn, thiolation by protein sulfenic acid intermediates is conditioned by possible GSH-conjugation reactions that in addition to oxidations reduce levels of GSH available to thiolate proteins.

Biological oxidations often involve the transformation of thiols to disulfides, and to less frequent forms of higher oxidation states (sulfinic or sulfonic groups, usually irreversible forms of oxidation). Disulfide formation implies accumulation of symmetrical (XSSX) or asymmetrical (XSSR), low or high molecular weight disulfides, whose return to basal conditions, often termed dethiolation (in the context of this chapter: deglutathiolation), is obtained at the expense of reducing agents (NADPH) and intervention of oxidoreductases (e.g. glutaredoxin, GSSG reductase, thioredoxin and thioredoxin reductase). At least in theory, dethiolation is rather complex because it can involve reshuffling of thiolated proteins by GSH and PrSH through complex SH/SS exchange reactions.

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