Review
Glutathione synthesis and its role in redox signaling

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Abstract

Glutathione (GSH) is the most abundant antioxidant and a major detoxification agent in cells. It is synthesized through two-enzyme reaction catalyzed by glutamate cysteine ligase and glutathione synthetase, and its level is well regulated in response to redox change. Accumulating evidence suggests that GSH may play important roles in cell signaling. This review will focus on the biosynthesis of GSH, the reaction of S-glutathionylation (the conjugation of GSH with thiol residue on proteins), GSNO, and their roles in redox signaling.

Highlights

Glutathione (GSH) is the most abundant antioxidant and a major detoxification agent in cells. ► Glutamate cysteine ligase, which catalyzes the first step in GSH synthetase is regulated by redox signaling. ► Accumulating evidence suggests that GSH plays important roles in cell signaling. ► Protein S-glutathionylation and S-nitrosylation play major roles in redox signaling.

Introduction

Several reactive species derived from oxygen and nitrogen are produced in cells from a variety sources such as NADPH oxidases (NOX) [1], leaks from the mitochondrial electron transport chain [2], redox cycling of quinones [3], and nitric oxide synthases (NOS) [4]. The principal reactive species produced enzymatically are superoxide (O2radical dot), hydrogen peroxide (H2O2), and nitric oxide (radical dotNO). These can be involved in cell injury but are also the principal actors in cell signaling, particularly the latter two [5], [6], [7]. Other species derived from these, hydroxyl radical (radical dotOH) and peroxynitrous acid (ONOOH), are extremely potent oxidants and are more likely involved in cell injury than signaling. Environmental exposure adds additional reactive species such as ozone and nitrogen dioxide [8]. Together these species are often referred to as reactive oxygen (ROS) and nitrogen species [9], terms we will use sparingly as they are not helpful in understanding mechanism. Indeed, an argument has been made for considering only H2O2 as the actual species involved among the ROS while radical dotNO and nitrosoglutathione (GSNO), a conjugate of radical dotNO and glutathione (GSH) that is produced by an as yet not definitively demonstrated oxidative mechanism, are responsible for ROS and RNS signaling. Finally, other reactive species, lipid hydroperoxides (ROOH) and α,β-unsaturated aldehydes, that are derived from lipid peroxidation can also participate in both injury and cell signaling [7].

As mentioned, O2radical dot, H2O2, and radical dotNO can contribute to pathology as well as signaling. Therefore, it is necessary for cells to maintain a narrow rate of production of these reactive species that is not directly harmful yet effective in mediating diverse physiologic functions through signaling.

Our focus here will be on the involvement of H2O2 and GSNO in signaling. The propensity of H2O2 to modify protein function is attributed to its ability to react with particular cysteine residues within proteins [10]. The possible oxidative modification of cysteine residues within proteins includes sulfenic acid (RSOH), intra- or intermolecular disulfide (RSSR), and S-glutathionylation (RSSG), sulfinic acid (RS(double bondO)OH) and sulfonic acid (RS(double bondO)2OH) acids, thiyl radicals (RS•), sulfenyl-amides, thiosulfinates, disulfide-S-monoxides; however, physiological signaling probably only involves the first three. Formation of the other species would influence signaling by producing species that are far more difficult to reduce back to a thiol than RSOH, RSSR and RSSG but are also less likely to form at low H2O2 concentration. Formation of RSOH, RSSG and RSSG can be transient and reversible, allowing these species to participate in biochemical functions, such as redox sensing and responding, catalysis, and signal transduction [11]. This redox-modification-based mode of signal transduction is called redox signaling. Only some select cysteine residues are involved in reactions with H2O2 or ROOH as even the more nucleophilic thiolate (single bondS) form is not strong enough to act in the absence of a nearby proton donor or metal to remove the OH that would be the leaving group if a thiolate alone reacted with ROOH or H2O2. This complex chemistry has been recently reviewed [5].

The concentration of GSH, γ-l-glutamyl-l-cysteinyl-glycine, which is the most abundant non-protein thiol in cells, is in the range of 1–10 mM in most mammalian cells [12]. GSH is also the most abundant antioxidant and a major detoxification agent in cells. Enzymes such as glutathione peroxidases (GPx) and one of the peroxiredoxins (Prdx VI) catalyze the reduction of H2O2 (or ROOH) by GSH into H2O (or the corresponding alcohol (ROH) and GSSG). GSSG is reduced back to GSH by glutathione reductase using NADPH to maintain a steady state GSH/GSSG ratio that is almost all GSH but varies with cell type and disease state [13]. GSH also conjugates with electrophiles and thus participates in the metabolism and detoxification of endogenous compounds and xenobiotic toxicants [14], [15], [16]. In addition, GSH is involved in many other metabolic reactions [9], [17], [18]. Therefore, it is not surprising that GSH plays roles in various cellular processes such as cell growth, proliferation, and apoptosis. Thus, it is clear that GSH is also involved in cell signaling through which it affects these important cell functions.

GSH participates in cell signaling through at least two mechanisms, protein S-glutathionylation and cysteine S-nitrosylation through thiol exchange with GSNO. The former is formed when GSH conjugates with reactive cysteine residues within proteins to form protein mixed disulfides (PSSG), and the latter is formed by reaction of a thiolate (single bondS) with GSNO. Emerging evidence suggest that both are controlled redox signaling mechanisms. How GSNO is formed has been the subject of intense investigation and several mechanisms have been proposed [6], [19]. Of course, GSH may also indirectly participate in the redox signaling by changing cellular redox homeostasis. The rest of this review will focus on the participation of protein S-glutathionylation and GSNO in signaling and on how cells regulate GSH homeostasis.

Section snippets

Regulation of glutathione content

In most cells, GSH is synthesized de novo through a two-step reaction. First γ-glutamylcysteine is formed from glutamate and cysteine catalyzed by glutamate cysteine ligase (GCL). Then glycine is added by glutathione synthetase to form GSH. GCL activity and cysteine availability are two rate-limiting factors in GSH synthesis. It is apparently so important for cells to maintain redox homeostasis and normal cellular function that both its concentration and GSH/GSSG ratio are tightly regulated.

Regulation of GCL expression

The regulation of both subunits of GCL, the catalytic subunit (GCLC) and the modifier subunit (GCLM), has been extensively studied in the past 20 years. A variety of signaling pathways, such as ERK1/2 [28], JNK1/2 [29], p38MAPK [30], PKC [31], and PI3K [32], [33], and transcription factors, such as c-Jun [29], NF-κB [27], JunD [34], and Nrf2 [25], etc., have been found to be involved in the regulation of GCLC and GCLM genes. The most significant and intriguing finding though, is that both genes

Protein S-glutathionylation

In a recent review [57], several reaction mechanisms of protein S-glutathionylation (PSSG) formation were proposed. We have added to and modified these in accord with the chemistry of cysteine oxidation in signaling proteins that was discussed in our recent review [5] (Fig. 1). Proton catalyzed nucleophilic substitution: To break the O-O bond in ROOH bond (where R is either a H or a lipid) and form a protein-cysteine sulfenic acid and ROH, there must be two conditions met; (a) the cysteine must

GSNO: reaction and regulation

radical dotNO is involved in a wide range of biochemical reactions and cell signaling pathways through protein modification. Although radical dotNO can diffuse freely, it is readily oxidized and this limits its function as a second messenger. Formation of S-nitrosothiols (single bondSNO) from radical dotNO and cysteine can protect radical dotNO from oxidative consumption and thereby extend radical dotNO bioavailability, both temporally and spatially. Protein S-nitrosylation can alter protein function and mediate signaling events and it has been considered as

Future direction

After decades of studies, it has been well established that redox-dependent reversible oxidative modification of proteins can occur under various pathophysiologic conditions, and is an important cell-signaling mode analogous to phosphorylation-based cell signaling mechanism. As the most abundant non-protein antioxidant in the cells, GSH is critical to maintain redox homeostasis and thus indirectly involved in redox signaling. Accumulating evidence suggest that GSH may directly participate in

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    This work was supported by ES005511 from the National Institutes of Health.

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