期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 444, 期 2, 页码 174-184出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2005.10.013
关键词
reactive cysteines; glutathionylation; GSH-sepharose; GSSG-sepharose; oxidative stress; post-translational modifications; ubiquitin-proteolysis
资金
- NCI NIH HHS [R01 CA97343] Funding Source: Medline
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, H2O2, 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 El, NF-kappa B, and proteasome) and putative (p53, glutathione-S-transferase PI) 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 H2O2-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. (c) 2005 Elsevier Inc. All rights reserved.
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