Redox systems of the cell: Possible links and implications

Oxidation-reduction status (redox) is an important regulator of various metabolic functions of the cell. Perturbations in the redox status of cells by external or internal stimuli elicit distinct responses, resulting in alteration of cell function. Glutathione and thioredoxin are two major reducing systems of the eukaryotic cell that maintain redox balance, as well as interact with various transducer and effector molecules to bring about specific responses. However, these two systems differ greatly in their functions and responses to various types of stress. Oxidative stress profoundly impacts them both by direct or indirect oxidation of sulfhydryl groups. Glutathione is a small tripeptide with a single cysteine residue that undergoes oxidation-reduction (1). Thioredoxin (Trx) is an approximately 12-kD protein that contains five cysteine residues (two catalytic and three structural). These cysteines undergo oxidation-reduction reactions in response to oxidants or reductants in the environment (2).

To date, the glutathione and thioredoxin systems were considered parallel redox systems.

To date, the glutathione and thioredoxin systems were considered parallel redox systems, although their functions were distinct and divergent. However, an interesting study appearing in this issue of PNAS provides a potential basis for interaction of these two redox systems in response to oxidative stress. This study by Casagrande and colleagues (3) presents data that not only demonstrates a potential link between these two redox systems, but also delineates the mechanism by which glutathionylation of Trx can inactivate this multifunctional redox protein. This study opens an area for investigation of possible in vivo interactions between these two redox systems, and the functional significance of such interactions during oxidative stress. Thioredoxin is a low molecular weight protein with cytoplasmic, membrane, extracellular, and mitochondrial distribution (2). This protein was originally identified in Escherichia coli as a hydrogen donor for ribonucleotide reductase, the essential enzyme providing deoxyribonucleotide for DNA replication (2). The Trx system includes Trx and thioredoxin reductase (TR), uses NADPH as a source of reducing equivalents, and is an efficient protein disulfide reductase. Thioredoxin peroxidase (peroxiredoxin; Prx) is a 25-kD peroxidase, initially identified in yeast, that reduces H₂O₂ to water and molecular oxygen with the use of electrons provided by thioredoxin (4). There are at least one mitochondrial and two cytoplasmic forms of thioredoxin peroxidase (4). Thioredoxin, thioredoxin reductase, and thioredoxin peroxidase constitute the thioredoxin system, which is very similar to the glutathione system, but with a number of distinct and different functions. The active site of Trx, Trp-Cys-Gly-Pro-Cys, is highly conserved across species (2). Besides the active site cysteines, thioredoxin has three other structural cysteines. Thioredoxin previously was shown to dimerize under oxidative stress. When this dimerization occurs, Cys-72 forms the disulfide bridge (5). Casagrande and colleagues (3) have shown that Cys-72 also is the site where glutathionylation occurs, resulting in inactivation of the protein.

Thioredoxin expression increases during a variety of oxidative stress conditions. The expression of thioredoxin and of TR were increased in lungs of newborn infant baboons in response to elevated oxygen tension experienced as a result of premature birth (6). In addition, the expression of thioredoxin peroxidase also was increased in response to increased oxygen concentration in lungs of such premature newborn baboons (7). Thioredoxin protein expression increases in response to retinal ischemia-reperfusion (8), and in response to phorbol ester, interferon (9), retinoids (10), UV light exposure (9), and hydrogen peroxide (11). Thioredoxin can regenerate oxidatively damaged proteins in lens epithelial cells (12) and in porcine endothelial cells (13). Further, thioredoxin is resistant to H₂O₂-mediated oxidation as compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in porcine endothelial cells (14). Thioredoxin overexpression was found to prevent nitric oxide-induced diminution of NO synthase activity in lung endothelial cells (15). Thioredoxin is expressed in and secreted from both normal and neoplastic cells (16, 17), as well as cells transfected with plasmids containing the thioredoxin gene, in a leaderless pathway (17). Besides being an antioxidant itself (18–19), thioredoxin can be an important regulator of expression of other antioxidant genes such as manganese superoxide dismutase (MnSOD, ref. 20). Thus, thioredoxin has a variety of direct and indirect antioxidant functions, with potential for actions both within and outside the cell.

The ability of thioredoxin to increase expression of MnSOD and other defensive proteins appears to derive from its modulation of activation of redox-responsive transcription factors such as NF-κB (21, 22). Recent reports indicate that Trx can inhibit apoptosis signal-regulating kinase (23). The redox-active nature of thioredoxin is important in the proliferation of lymphoid cells as well as many types of malignant cells (24). Thioredoxin has been shown to enhance the DNA binding of hypoxia inducible factor (HIF-1) in hypoxic cell extracts. In addition, over-expression of thioredoxin potentiated hypoxia-induced induction of HIF-1-dependent reporter construct (25). Further, the expression of thioredoxin is increased in hypoxia (16). Hence, thioredoxin appears to be a key molecule in transducing hypoxia's ability to promote vascular (26–28) and tumor growth (29). Recently, it has been demonstrated that thioredoxin can mediate p53-dependent p21 activation (30), and thioredoxin translocates from the cytoplasm to the nucleus on stimulation by oxidative stress (31, 32). Thus, thioredoxin also may have a critical role in the normal protective growth arrest process that occurs in severe oxidative stress (33, 34). Even with these new findings, the role of thioredoxin in growth and development (35), signal transduction, and gene expression is only beginning to emerge.

S-thiolation, specifically S-glutathionylation, of critical proteins (GAPDH) resulting in their inactivation previously has been detected in vitro during the stimulated respiratory burst of human monocytes (36). This process was reversible, appeared dependent on hydrogen peroxide (37), and resulted in an increased cell uptake of glutathione (38). Although anticipated to be a mechanism that might shut off the respiratory burst, enhancement of oxidative stress during the burst caused a paradoxical priming, or further stimulation, of respiratory burst activity (39). Because decreased GAPDH activity would result, it is conceivable that increased glucose metabolism through the hexose monophosphate shunt and decreased glucose utilization through glycolysis could have a role in this effect, and, further, that such changes could impact inflammatory cell survival (40–43) and/or mode of death (44). Finally, it is worth noting that reversible protein S-glutathionylation has been observed in vivo during physiologic oxidative stress (45). Because thioredoxin is a principal protein thiol reductase, and it can become highly oxidized under such conditions (6), its recovery of function seems likely as such stress abates. The extent to which its function actually is impaired in vivo by S-glutathionylation, as indicated by the study of Casagrande et al. (3), warrants additional investigation.