Abstract 228: Glutathione S-transferase P-dependent Glutathiolation of Oxidized Aldose Reductase in the Ischemic Heart; a Novel Mechanism of Reversible Redox Signaling?
Aldose reductase (AR) is a stress-responsive enzyme reducing glucose and aldehydes derived from lipid peroxides generated during increased oxidative stress. We have recently reported that ischemia causes AR activation due to peroxynitrite-mediated oxidation of the active site cysteine residues (C298 and C303) to sulfenic acid. Herein we report that in the mouse heart, ischemic activation of AR was reversed upon reperfusion in a time-dependent manner and AR activity returned to the baseline after 30 min of reperfusion. Based on our previous observations that GSNO-mediated glutathiolation inhibits AR, we hypothesized that glutathiolation of the protein cysteines could cause deactivation of AR upon reperfusion. Our in vitro studies showed that treatment of human recombinant AR with peroxynitrite (0.1 mM) caused a 3-fold increase in AR activity (from 1.9 ± 0.09 to 5.8 ± 0.04 U/mg, n = 3; P < 0.1). Electrospray ionization mass spectrometric (ESI/MS) analysis revealed that peroxynitrite oxidized AR to sulfenic acid, ARSOH (mass increased by 16 Da). Addition of reduced glutathione (GSH, 2 mM) to ARSOH generated ARSSG (+ 305 Da) that was catalytically inactive. When recombinant glutathione S-transferase P (GSTP) was added into the reaction mixture, the rate of enzyme deactivation and ARSOH glutathiolation increased 2-fold, indicating that GSTP facilitates AR glutathiolation. To delineate the role of GSTP in AR deactivation upon reperfusion, enzyme activity was measured in the heart homogenates from GSTP- WT and -null mice subjected to 15 min of global ischemia followed by 30 min of reperfusion ex vivo. In contrast to WT hearts, in which AR was deactivated after 30 min of reperfusion, AR activation persisted in GSTP-null hearts. MALDI/TOF analysis of the heart homogenates revealed oxidation of AR at cysteines −187, −298 and −303 in GSTP-null hearts subjected to ischemia-reperfusion, whereas in GSTP WT hearts C303 remained oxidized and C187 and C298 were glutathiolated. Collectively, these findings indicate that GSTP plays crucial role in AR deactivation upon reperfusion by facilitating glutathiolation of its active site cysteine residues. The reversible redox modification reactions of AR may be paradigmatic of other thiol-based signaling mechanisms.