Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

• This study shows that Trx can be glutathionylated under conditions of oxidative stress. Glutathionylation was confirmed by incubating rhTrx with GSSG at a concentration (5 mM) that can be observed in vivo under conditions of oxidative stress or in cellular compartments such as the endoplasmic reticulum where the GSH/GSSG ratio can be 1 (22). The mass spectrometric analysis showed that, at a GSSG concentration of 5 mM, only one GSH can be attached to rhTrx at Cys-72. This higher susceptibility of Cys-72 to oxidation is very likely caused by the fact that it is easily accessible in the active site surface of the three-dimensional structure of Trx (23). In fact, when we synthesized peptides reproducing partial Trx sequences and treated them with GSSG, we found that all Cys with the exception of one of those in the CGPC active site were equally susceptible to glutathionylation. Thus the susceptibility of Cys-72 appears to be observed only within the entire protein with its three-dimensional structure. Further supporting the concept that Cys-72 is the Cys residue of rhTrx most exposed to oxidizing agents, we noted that iodoacetamide treatment of rhTrx under nondenaturing conditions alkylated only Cys-72 (data not shown). Alkylation under nondenaturing conditions has been used to determine oxidant-sensitive Cys (also in terms of accessibility in the native structure; refs. 24 and 25). Thus, in the case of Trx, susceptibility to glutathionylation is mainly due to the three-dimensional structure rather than to the chemical-physical properties of the amino acids in the vicinity, as has been suggested for other proteins (26). A glutathione adduct on Cys-72 also might be favored by the primary structure, however, as this amino acid is next to a basic one, Lys-71, which might stabilize the adduct by interacting electrostatically with the -glutamyl group of GSH (27). Interestingly, Cys-72 is also involved in the dimerization of Trx (23, 28), which may occur under oxidative conditions or spontaneous oxidation, confirming that Cys-72 can be easily oxidized. The fact that the same Cys-72 of Trx can be involved in both the dimerization and in the glutathionylation of Trx suggests that glutathionylation prevents dimer formation. In fact, we found that in the absence of GSSG, the mass spectra of rhTrx revealed some Trx dimers, whereas in GSSG-treated rhTrx, no dimer was detectable (data not shown). Analysis by MALDI-TOF of rhTrx exposed to GSSG for longer periods (overnight) showed that two GSH residues can be attached to a single rhTrx molecule. This finding would imply that one of the two Cys that can undergo glutathionylation is Cys-72 and the other is either Cys-61 or Cys-68. In fact, a mixed disulfide between glutathione and one of the Cys of the CGPC active site (Cys-31, Cys-34) would be highly unstable (25). In agreement with this hypothesis, we observed that even when a synthetic peptide containing the CGPC active site was treated with GSSG, no adduct was ever formed with two GSH molecules. The fact that Trx can be glutathionylated raises the question of how this fact affects its activities. We investigated whether glutathionylation affected its enzymatic activity. Glutathionylated rhTrx lost all activity initially, but gradually, the activity was restored with time. This was probably due to the fact that Trx can reduce mixed disulfides (29), though not as efficiently as glutaredoxin, and its residual activity could reactivate itself according to the proposed scheme: The finding that Trx can be regulated by glutathionylation indicates the existence of crosstalk between the glutathione glutaredoxin system and the Trx system through which Trx activity can be influenced by the GSH/GSSG ratio, an indicator of the redox state of the cell. The observation that GSNO also can induce glutathionylation of Trx with the same efficiency as GSSG suggests that, through the formation of GSNO, NO can influence Trx activity. Although we demonstrate the occurrence of Trx glutathionylation in living cells exposed to an exogenous oxidant in the present work, other reports show that S-thiolathion of enzymes, namely glyceraldehyde-3-phosphate dehydrogenase, with loss of enzyme activity, can take place during physiologic oxidative stress such as that induced by monocyte activation (30). Therefore, it will be important to evaluate whether Trx is glutathionylated under pathological conditions resulting in oxidative stress. It should be noted that although glutathionylation reduces the enzymatic activity of Trx, it could have more subtle effects on other activities of this protein, altering its affinity for interacting proteins or even generating new activities. For instance, in a protein related to Trx, sharing its insulin disulfide reducing activity, glycosylation inhibiting factor macrophage migration inhibitory factor, S-thiolation caused conformational changes and generation of a growth factor like bioactivity (31). Trx has many biological activities, whose mechanisms are largely obscure: it acts extracellularly as a cytokine, chemokine, or growth factor (32–34) and intracellularly as a regulatory protein that influences several regulatory proteins such as NF- B (35) and ASK1 (36), and it is efficiently secreted through a still unknown mechanism. The relevance of the posttranslational modification of Trx reported here warrants further investigation.