Proteins are critical mediators of redox signaling. Cysteines within its thiol groups sense the oscillations in oxidative state and undergo different reversible oxidative modifications. One of such modifications is protein S-glutathionylation (PSSG), a conjugation of glutathione (GSH) to protein thiols that not only protects them from oxidative damage, but also regulates a wide variety of cellular functions including signal transduction, cell survival and apoptosis. PSSG has been described to play an important role in cancer initiation, progression, response to anti-leukemic therapy and the emergence of drug resistance in leukemia cells. The process of PSSG is driven by fluctuations in the availability of GSH and oxidized glutathione (GSSG) and by enzymes catalyzing the removal and conjugation of GSH to thiol residues such as glutaredoxins and glutathione-S-transferases. Our recent data showed that response of leukemic cells to hypomethylation therapy is associated with disturbed GSH/GSSG ratio, lower expression of glutathione S-transferase P (GSTP1) and overexpression of glutaredoxin 1. Therefore, our aim was to find a pattern of thiol oxidation upon modulation of PSSG and next to identify the impact of perturbations in GSH metabolism on PSSG in healthy human embryonic stem cells (hESCs) and leukemic cell lines.

To do so, we induced oxidative stress in hESCs by treatment with hydrogen peroxide and monitored its impact on protein thiol modifications in the presence of GSSG and both GSSG and GSTP1 inhibitor. To identify and quantify free and oxidized protein cysteine sites we applied IodoTMT-based redox proteomic approach using LC-MS3 method. We identified 2485 cysteine containing proteins from which 773 had significantly changed thiol oxidation status. Inducing oxidative stress by H2O2 and increasing the level of oxidized glutathione by NOV-002 compound profoundly elevated the median protein oxidation from 4 to 6 %, while simultaneous inhibition of GSTP1 with Ezathiostat rescued the oxidation to control levels.

Next, we studied the impact of perturbations in GSH metabolism and inhibition of thioredoxin on protein PSSG using selective reduction and blocking of PSSG sites with subsequent TMT labeling in OCI-M2 leukemic cells. We found out that perturbation of GSH metabolism at the node of GSH synthesis (glutamate-cysteine ligase) led to significant alterations in protein S-glutathionylation and significantly inhibited leukemic cells growth.

Collectively, these data indicate that protein S-glutathionylation plays a critical role in antioxidant defense of the cell, and modulation of PSSG in leukemia can lead to proliferation arrest and induce cell death that could increase the efficacy of anti-leukemic therapy.