S-glutathionylation: indicator of cell stress and regulator of the unfolded protein response

Abstract

The specific posttranslational modification of protein cysteine residues by the addition of the tripeptide glutathione is termed S-glutathionylation. This process is promoted by oxidative and nitrosative stress but also occurs in unstressed cells. Altered levels of S-glutathionylation in some proteins have been associated with numerous pathologies, many of which have been linked to redox stress in the endoplasmic reticulum (ER). Proper protein folding is dependent upon controlled redox conditions within the ER, and it seems that ER conditions can in turn affect rates of S-glutathionylation. This article seeks to bring together the ways through which these processes are interrelated and considers the implications of these interrelationships upon therapeutic approaches to disease.

Figure 1.. Reactivity of cysteinyl residues and the process of S-glutathionylation.

A. The cysteinyl residues of proteins (Cys; Cys–SH to stress the thiol form) of cells under oxidative/nitrosative stress can be oxidized to various acidic forms, including cysteine sulfenic acid (Cys–SOH), which is fairly labile and can be readily reduced back to the thiol (Cys–SH) or further oxidized to the more stable cysteine sulfinic acid form (Cys–SOOH). It is becoming increasingly clear that such oxidation may be essential to normal deactivation-and-reactivation cycles of proteins and enzymes. B. The more familiar reaction of protein cysteinyl residues, namely, in the formation of disulfide bonds (e.g., as a manifestation of protein secondary structure), is related to the process of protein S-glutathionylation, which is increasingly recognized as essential to cellular behavior in health and in disease states. In the presence of physiological concentrations of glutathione (GSH), specific cysteinyl residues, by virtue of their position and reactivity within protein microenvironments, can undergo such modification through reactions with oxidized glutathione disulfide (GSSG) or by glutathione-utilizing enzymes such as glutathione S-transferases (GST). This posttranslational modification becomes reversible under catalysis that involves small redox proteins such as sulfiredoxin and glutaredoxin. (Oxidation is indicated in blue; reduction is indicated in green.)

Figure 2.. The interplay of protein phosphorylation and protein S-glutathionylation pathways in cell signaling.

Under oxidative/nitrosative stress (indicated by ROS/ RNS), pi GST monomers associate with the target proteins c-Jun, JNK, and TRAF2, which are consequently S-glutathionylated (as indicated by –SSG). The immediate effect of S-glutathionylation could be TRAF2 activation, as well as the phosphorylative activation (indicated by –P) of c-Jun and JNK, culminating in the deployment of apoptotic or proliferative pathways. In addition, S-glutathionylation of pi GST results in its aggregation and concomitant inactivation. Dashed lines and arrows indicate hypothetical, rather than experimentally established, steps.

Figure 3.. The Unfolded Protein Response (UPR) and pro-apoptotic pathways.

The UPR is a complex signaling cascade that can be induced by stress and the accumulation of malfolded proteins in the ER. During homeostasis (upper schematic; blue), protein disulfide isomerase (PDI) promotes the proper folding of immature proteins (yellow strings). The ER-resident protein BiP associates with properly folded proteins and concomitantly inhibits three ER transmembrane proteins, namely, PERK, ATF6, and IRE1. Under conditions of oxidative and nitrosative stress (indicated by ROS/RNS and red background in lower ER schematic), PDI is modified (see text for discussion) and rendered inactive, and unfolded proteins (yellow strings) accumulate in the ER. BiP dissociates from improperly folded proteins and concomitantly surrenders negative regulatory interactions with PERK, ATF6, and IRE1. PERK thereby phosphorylates and inactivates (blunt arrow) eukaryotic translation initiation factor eIF2α, the phosphorylation of which is also associated with transcriptional activation of genes involved in the UPR. This transcriptional activation, which is also promoted by the activation of ATF6 and IRE1, can drive pro-apoptotic signaling, particularly through activation of CHOP (see text for details). IRE1 can also interact with TRAF2, which can function pro-apoptotically through association with ASK1 and JNK (see text; also see Figure 2). A third route to apoptosis is offered by caspase activity; intriguingly, regulation of caspase activity (particularly that of CASP3) may also be a function of GST.

Figure 4.. Regulation of PDI by its specific S-glutathionylation.

A. PDI is a member of the thioredoxin superfamily of small thiol-rich proteins. The protein sequence of PDI includes seven cysteine residues (highlighted in red). Basic amino acids that flank cysteine residues (green) may act to lower the pKa of the thiol group, making the relevant Cys more likely to be susceptible to S-glutathionylation. B. A cartoon structure of the domains in PDI shows a single cysteine in the N-terminal domain, four in the catalytic domains, and two in the b’ domain.