|Ph.D Student||Engelman Rotem|
|Subject||Regulation of the Thioredoxin/Peroxiredoxin System by|
|Department||Department of Medicine||Supervisor||Professor Moran Benhar|
|Full Thesis text|
S-Nitrosothiols (SNOs), formed by nitric oxide (NO)-mediated S-nitrosylation, and hydrogen peroxide (H2O2), a prominent reactive oxygen species, are ubiquitous signaling molecules implicated in diverse physiological and pathological processes. Recent research has shown that the cellular action and metabolism of SNOs and H2O2 involves overlapping thiol-based mechanisms. Specifically, these reactive species were shown to regulate cellular responses by posttranslational modifications of cysteine thiols and to be metabolized by common thiol-based detoxification systems. These observations suggested the possibility of a biochemical crosstalk between H2O2 and NO. In this regard, peroxiredoxin (Prx) proteins and the thioredoxin (Trx) system have been shown to be important components in the cellular metabolism and signaling pathways related to both species and therefore were the focus of this research.
To examine the potential crosstalk between NO/SNO and H2O2 metabolism we first investigated how NO/SNO may affect the redox cycle of peroxiredoxin 1 (Prx1), a Trx-dependent thiol peroxidase that belongs to an antioxidant family of proteins highly important in the cellular response to H2O2. We found that Prx1 was readily nitrosylated by several NO/SNO donors, both in vitro and in cells. In addition, nitrosylation of Prx1 promoted structural and functional alterations. Specifically, nitrosylation of Cys52 and Cys83 promoted the disruption of the oligomeric structure of Prx1 and loss of peroxidase activity. Moreover, we found that relatively low concentrations of S-nitrosocysteine (CysNO) exerted a marked inhibitory effect on the regeneration of oxidized Prx1 by the Trx system. This effect appeared to be mainly due to direct modulation by CysNO of the activity of the selenoprotein Trx reductase (TrxR).
In the second part of this study we investigated the SNO-dependent regulation of TrxR, which is regarded as the “engine” of the Trx system. We examined the mechanism of SNO-mediated inhibition of TrxR, the nature of the modification and the targeted sites, and explored the possible biological significance of this mechanism. We showed that both recombinant and endogenous TrxR1 were subjected to inhibitory S-nitrosylation by several NO/SNO donors. In addition, we found CysNO to act as both substrate and inhibitor of TrxR1, as a function of its concentration. Mass spectrometry and biochemical analyses revealed nitrosylation at both active sites of the enzyme and highlighted its C-terminal active site selenocysteine residue to be key in conferring sensitivity to SNO-mediated inactivation. Our studies in vitro and in HeLa cancer cells showed that glutathione (GSH) protects TrxR1 from nitrosylation-dependent inactivation. We demonstrated that depletion of cellular GSH with L-buthionine-sulfoximine synergized with nitrosylating agents in promoting sustained nitrosylation and inactivation of TrxR1, events that led to a significant oxidation of Trx1 and extensive cell death.
Collectively, our findings expand the current understanding of how SNO affects a major thiol peroxidase in mammals and provide new insight into the role and regulation of the mammalian Trx system in relation to cellular nitroso-redox imbalance. This study further supports the rationale of dual treatment with NO donors and GSH inhibitors as an effective approach for promoting redox stress and death of cancer cells.