Serious consideration of
reversible protein glutathionylation as a mechanism of regulation has a relatively brief history, probably less than 30 years. Nevertheless, this focused area of research has captured the imagination of a growing number of biologists, most intensively during the last 10 years. This emergence has been fostered largely by the convergence of understanding about reactive oxygen and reactive nitrogen species as second messengers in signal transduction, the importance of posttranslational modification of cysteine residues, and the special properties of the glutaredoxin (Grx) (thioltransferase) enzyme as a specific catalyst of deglutathionylation of protein–glutathione (GSH) mixed disulfides. An historical perspective on evolution of this focal area of biology can be gleaned from considering a number of key reviews that have appeared over the past 25 years. In reverse chronology, 25 years ago Ziegler presented the point of view that considered protein glutathionylation strictly in the context of thermodynamic redox equilibria coupled to the GSH/glutathione disulfide (GSSG) ratio, and he concluded that reversible glutathionylation as a regulatory phenomenon was highly unlikely (31
). According to his premise he was correct, because most cysteine residues have redox potentials that would require the intracellular GSH/GSSG ratio to change from about 100:1 to 1:1 in order for 50% of the protein-SH of interest to be converted to S-glutathionylated protein (protein-SSG) (12
). Thus, there must be mechanisms for activating the protein-SH or GSH thiol groups to facilitate protein-SSG formation under normal GSH/GSSG redox conditions where redox signaling occurs (see below). Certainly, under overt oxidative stress conditions where GSSG concentration is very elevated, GSSG may serve as the proximal mediator of protein-SSG formation. Thomas, a pioneer in developing tools for evaluating protein thiolation and protein-SSG formation in particular (26
), reviewed the role of enzymatic reversibility of protein disulfides in the context of oxidative stress. Besides homeostatic protection and repair as defenses against oxidative stress, Mieyal and coworkers (19
) posed the concept of regulation via
reversible S-glutathionylation, centering on the unique specificity of Grx (thioltransferase) for catalyzing reduction of glutathionyl mixed disulfides (13
The net reaction catalyzed by the Grxs is appropriately depicted as a thiol–disulfide exchange reaction involving sequential nucleophilic displacement reactions, rather than single-electron transfer reactions that would involve radical intermediates. Accordingly, the name “transhydrogenase,” which was applied originally to the enzyme activity from rat liver (23
), was replaced by the name “thioltransferase” (3
), because the latter more accurately describes the nature of the reaction that is catalyzed. Besides the original characterization of the mammalian thioltransferase enzyme (3
), analogous catalytic activity was discovered in bacteria and attributed to an enzyme that promoted the GSH-dependent turnover of ribonucleotide reductase in a mutant of Escherichia coli
that lacked thioredoxin. This E. coli
enzyme was named “glutaredoxin” (15
). Subsequent to these early studies, thioltransferase and glutaredoxin enzymes have been isolated from a variety of organisms, species, and tissues, and characterized as having homology of both amino acid sequence and three-dimensional structure. Consequently, it has been concluded that thioltransferase and glutaredoxin simply represent alternative names for the same family of enzymes. Although “thioltransferase” more accurately reflects the catalytic reaction, “glutaredoxin” has emerged internationally as the more commonly used name for this family of enzymes.
Documented by studies of various protein mixed disulfides and kinetic characterization (13
), the exquisite specificity of the human Grx enzyme for the glutathionyl moiety was further demonstrated by mass spectrometric analysis (29
). Thus, hGrx1 was reacted with cysteine-gluthathione mixed disulfide (Cys-SSG), which represents the prototype for all protein-Cys-SSG substrates. The Grx1 distinguished between the two sulfur atoms of Cys-SSG so that glutathionyl GRx mixed disulfide intermediate (Grx-SSG) was found to be the exclusive disulfide adduct. In contrast, the analogous form of human thioredoxin reacted with Cys-SSG to give Trx-SSCys and Trx-SSG in equal amounts. Focusing on the glutathionyl specificity of Grx, which was also described for E. coli
), Cotgreave and coworkers first demonstrated the proteomic approach of exploiting the deglutathionylation specificity of Grx to identify protein-SSG adducts (18
). A review by Cotgreave and Gerdes (6
) addressed the linkage between sulfhydryl modulation and cell proliferation, implicating the potential regulatory role of protein glutathionylation in cancer biology.
Klatt and Lamas presented a seminal review in 2000 (17
), which identified protein S-glutathionylation specifically as an emerging candidate mechanism by which redox signals mediated through reactive oxygen species (ROS) and reactive nitrogen species might be transduced, introducing the concept of cross interactions among protein thiol modifications initiated by nitrosative and/or oxidative stress. Since that time there has been a proliferation of original research articles and reviews on the topic of protein-S-glutathionylation as a regulatory and/or protective mechanism, providing a variety of points of view [e.g
)]. In particular, our previous review (24
) first introduced criteria for evaluating whether reports of S-glutathionylation of specific proteins are indicative of cellular regulatory events. presents these criteria in their current formulation.
Criteria that characterize S-glutathionylation as a regulatory mechanism under physiological conditions.
The frequency of reviews on the topic of protein S-glutathionylation has been increasing remarkably, coincident with the burgeoning interest in redox-activated signal transduction as a mechanism of cellular regulation that can be perturbed by the oxidative/nitrosative stress associated with many diseases. A survey of PubMed in 2011 revealed cumulatively more than 1200 articles have been published on Grx and/or thioltransferase; more than 500 articles have been published on glutathionylation or glutathiolation; and more than 120 articles have been published on Grx (or thioltransferase) and glutathionylation (or glutathiolation). Thus, awareness of the pivotal role of Grx in regulating reversible glutathionylation has expanded. However, future studies of cellular redox regulation via
S-glutathionylation are well advised to include examination of the role of Grx-mediated deglutathionylation in determining the steady-state protein-SSG status of specific proteins. Moreover, much more attention needs to be focused on potential enzymatic mechanisms of formation of protein-SSG, possibly involving glutathione-S-transferase or peroxidase enzymes (9