Abstract 4061: Depleting the cellular level of glutathione and subsequently S-glutathionylation of keap1 by electrophilic agents activates keap1-nrf2 pathway

S-glutathionylation is a reversible post-translational modification of protein cysteine with critical roles in oxidative stress and signal transduction. Keap1-Nrf2 pathway has emerged as a master regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that after depleting the cellular level of glutathione (GSH) by electrophilic agent (the GSH level decreased to 23 percent of control after the agent treatment for 4 h), Keap1 was modified by S-glutathionylation and Nrf2 expression increased both in nucleus and total cellular homogenates. The modification could be blocked by the addition of exogenous GSH, suggesting depleting the cellular level of GSH and alteration of intracellular GSH/GSSG ratio might be essential for S-glutathionylation of Keap1 and activating Keap1-Nrf2 pathway. Flexidock program was employed to elucidate the influence of Keap1 S-glutathionylation on Keap1-Nrf2 complex. After enforcing a disulfide bond between GSH and Cys434 residue in Keap1 and energy minimization, the two carboxylate oxygen atoms of GSH participate in hydrogen bond interaction with Arg483 and Arg380 residues in Keap1, which had been previously verified bound to DxETGE motif of Nrf2. The distance between GSH O1 and Arg483 NH1, GSH O2 and Arg380 NH1 were 2.587 A and 2.569 A, respectively, which could form potent hydrogen bonds and diminish the interaction between Keap1 and Nrf2, resulting in the accumulation of Nrf2 in the nucleus. We concluded from our findings that the activation of Keap1-Nrf2 pathway was triggered by Keap1 S-glutathionylation and conjugation with intracellular GSH might be a key event involved in the Nrf2 inducing activities of electrophilic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4061.