The Unfinished Puzzle of Glutathione Physiological Functions,an Old Molecule That Still Retains Many Enigmas

On the occasion of the centennial of glutathione (GSH) discovery, in 1988, Alston Meister wrote that the scientific history of this molecule began with a note from Joseph de Rey-Pailhade entitled “Sur un corps d'origine organique hydrogénant le soufre à froid,” which reported that hydrogen sulfide (H₂S) is produced when Brewer's yeast is crushed with elemental sulfur (S₈) (8). de Rey-Pailhade named philothion the compound responsible for this reaction, from the Greek words for love and sulfur, suggesting it contains labile hydrogen, to explain H₂S production. In 1907, de Rey-Pailhade gave credit to Heffter for suggesting that philothion labile hydrogen is from the sulfhydryl group of cysteines, which are responsible for the reducing properties of cells. In 1921, Hopkins purified a compound that had reducing properties, which were lost by oxidation and regained by reduction, and also produced H₂S in the presence of sulfur. Hopkins concluded that this compound is philothion, is made of glutamate and cysteine, and named it GSH. He finally acknowledged in 1929, based on the work of Hunter and Eagles, that GSH is a tripeptide made of glutamic acid, cysteine, and glycine. These seminal observations established the striking properties of GSH as a major cellular reducing power that undergoes reversible oxidation–reduction, also recognizing the importance of the cysteine residue in this attribute.

What is today our knowledge and understanding of the cellular functions of GSH? Common scientific knowledge holds that GSH's major cellular functions are linked to its antioxidant and redox buffering properties, which is not wrong, but not entirely correct. A fundamental issue should be now firmly established in the assessment of thiol-based cellular reactions that sets the kinetics over the thermodynamic properties of a redox system. As discussed by Deponte in this Forum (5), nonenzymatic redox buffering is based on the Gibbs free energy of redox reactions at equilibrium, whereas by use of enzymes, cellular systems work under steady-state conditions that deviate from thermodynamic equilibria.

Therefore, use of equilibrium-based redox potentials and “redox buffer” concepts to describe and rank redox reactions in vivo is inappropriate. Rather, GSH functions should be established based on comparison of available genetic and quantitative kinetic data (5). If not a redox buffer, GSH remains a major antioxidant, not by directly reducing reactive oxygen species, but by catalyzing the reduction of the selenothiol-based GSH peroxidases enzymes, at least in mammals, wherein these enzymes are found.

Genetic approaches in the yeast and mouse systems have revealed new unsuspected twists in the biology of GSH that make this molecule essential for eukaryotic life. The mouse knockout of γ-glutamyl cysteine synthetase gene, which encodes the rate-limiting enzyme in GSH synthesis, is indeed embryonic lethal, and both yeast and mammalian cells lacking it cannot grow without exogenous GSH supplementation. What makes GSH essential for life? Whether this requirement is linked to GSH “redox buffer” properties has been ruled out, based on the fact that only trace amounts of it are needed for viability in both yeast and mammals. Furthermore, the thioredoxin pathway should substitute for GSH, at least partially, if it were only for ensuring general cellular thiol–redox control. The yeast essential requirement for GSH is similarly not linked to its antioxidant properties, since strict anaerobiosis is unable to rescue the growth of GSH-depleted cells. Instead, GSH essential nature in yeast has been traced to its requirement for the cytosolic assembly of iron–sulfur clusters (Fe-S), which are the prosthetic groups of many essential proteins. Although neither shown, nor ruled out, GSH requirement for Fe-S assembly should apply to mammals too, but is presumably offset by another essential function of the redox tripeptide in assisting the phospholipid hydroperoxidase GPx4, the activity of which is essential for preventing ferroptosis, a newly described nonapoptotic cell death pathway mediated by lipid peroxides, and requiring traces of iron (9).

The physiology of GSH in the eukaryotic cell is made complex, not only by its vital functions already seen that offset other nonvital but nevertheless important functions but also by its very different roles in the different compartments of the eukaryotic cell. Research on GSH has started to address the compartment-specific basis of GSH metabolism, an approach that has been empowered by the introduction of GSH redox probes that can be expressed ubiquitously.

In this Forum, in addition to Deponte who has sought to revisit old concepts of GSH compartment-specific functions by a comparison of available genetic and quantitative kinetic data (5), two of the remaining five articles focus on specific cellular compartments. The review by Calabrese et al. (3) addresses GSH trafficking and functions in the mitochondrial matrix and intermembrane space, in which redox metabolism is intense but complex, and the mechanisms ensuring the homeostatic maintenance of the mitochondrial GSH redox poise (Fig. 1). Delaunay-Moisan et al. (4) address the still highly controversial, but passionate, debate of the role of GSH during oxidative protein folding in the endoplasmic reticulum (ER) and its interplay with the ER major redox enzymes, the ER oxidase Ero1 and protein disulfide isomerases. Delaunay-Moisan et al. conclude with the need of elucidating the still elusive mechanisms of GSH trafficking in and out of the ER to establish its function in this organelle, and on its possible shared function with the thioredoxin pathway as ER reducing powers (4). The review by Berndt and Lillig (2) addresses the still very mysterious function of GSH in iron metabolism. The review by Bachhawat and Kaur (1) considers recent data on GSH degradation that push away the old models of the GSH cycle. Lastly, the review by Hatem et al. (7) focuses on the outstanding links between GSH metabolism, carcinogenesis, and anticancer drug resistance.

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FIG. 1.

The diverse functions of GSH in the different compartments of the eukaryotic cell. GSH is synthesized in the cytosol, to reach very high mM levels, from which it traffics to each of the eukaryotic cell compartments (red arrows). A transport mechanism is only identified for GSH transport in the IMS. In the cytosol, GSH serves as reducing power for Gpx enzymes that scavenge hydroperoxides and is required for iron–sulfur (Fe-S) assembly/iron delivery, two functions underlying GSH essential requirement for viability. GSH degradation by the Dug pathway and Chac enzymes takes place in the cytosol, and degradation by γ-glutamyl-transpeptidase takes place in the vacuole in yeast and at the cell membrane in mammals. In mitochondria, GSH acts redundantly with the thioredoxin pathway to reduce oxidized thiols and to scavenge peroxide, and might also participate in Fe-S assembly. In the ER, GSH has a demonstrated role in counteracting the Ero1-PDI thiol oxidizing pathway, but whether it is required for oxidative protein folding and whether this function is shared with the thioredoxin pathway remains an open question. Owing to its crucial function in hydroperoxide scavenging and Fe-S assembly, and its major detoxification function in conjunction with GSH transferase enzymes and drug efflux pumps, GSH constitutes a major therapeutic target of cancer. Irrespective of the organelle, GSH mode of action appears dictated by kinetics rather than by thermodynamic parameters, which should rule out the concept of nonenzymatic redox buffering. ER, endoplasmic reticulum; GSH, glutathione; IMS, intermembrane space of mitochondria.

Of the many ideas and concepts developed in these articles, we should reemphasize the notion that redox metabolism is essentially controlled by kinetics rather than thermodynamic constraints, which in the case of GSH mandate abandoning the still too frequently used concepts of nonenzymatic redox buffering (4). If so, why is cellular GSH present in such high, millimolar amounts, which is probably the reason that has steered the concept of “redox buffer.” There is no definite answer to this outstanding question, but one should still have in mind the old concept developed at the dawn of GSH history that this molecule, in the same way as reduced nicotinamide adenine dinucleotide phosphate (NADPH), is a crucial cellular reducing currency, and as such should be available in large amounts. Such an idea has recently been elegantly illustrated by a fully viable mouse model harboring a double hepatic knockout of both GSH reductase and cytosolic thioredoxin reductase, in which the vital thiol reducing power usage totally switches from NADPH to GSH (6).

We hope readers will find this Forum thought stimulating and instructive.