S-Thiolation mimicry: Quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography

Abstract

S-Glutathionylation is emerging as a novel regulatory and adoptive mechanism by which glutathione (GSH or GSSG) conjugation can modify functionally important reactive cysteines in redox-sensitive proteins. The dynamics of generation and reversal of this modification in cells is poorly understood. This study describes the ability and applicability of GSH- and GSSG-affinity matrices to quantitatively bind proteins which harbor reactive cysteines and undergo glutathionylation. We showed that purified proteins, known to be modified by S-thiolation, bind to these matrices, are selectively eluted by dithiothreitol and rapidly incorporate biotin-labeled GSH or GSSG in vitro. Chromatography of extracts from tumor cells that had been treated with oxidants (diamide, H 2O 2, tert-butyl hydroperoxide) on GSH–Sepharose showed the specific binding of many proteins, whose levels increased transiently (2- to 6-fold) soon after treatments. However, when these cells were post-incubated in drug/oxidant-free media, protein binding decreased gradually to control levels over 3–12 h, thereby demonstrating the central role of cysteine redox status in the binding. Immunoblotting of eluates from GSH–Sepharose showed the presence of known (actin, ubiquitin-activating enzyme E1, NF-κB, and proteasome) and putative (p53, glutathione- S-transferase P1) targets for glutathionation. After oxidant withdrawal, many of these proteins displayed unique kinetics in their loss of binding to GSH-matrix, reflecting their differential abilities to recover from cysteine redox changes in cellular milieu. Further, we correlated the kinetics of S-thiolation susceptibility of the proteasome and ubiquitin-E1 proteins with altered levels of protein ubiquitination in H 2O 2-treated cells. Our study reveals the hitherto underutilized ability of glutathione matrices for analyzing the kinetics of cysteine redox in cellular proteins and allows easy identification of S-thiolatable proteins.