S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography.
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(2)O(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 3-12h, 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-kappaB, 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(2)O(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.
Niture
Suryakant K
SK
Center for Cancer Biology, Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, 79106, USA.
Velu
Chinavenmeni S
CS
Bailey
Nathan I
NI
Srivenugopal
Kalkunte S
KS
D001665:Binding Sites; D032701:Biomimetics / Q000379:methods*; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008527:Medulloblastoma / Q000737:chemistry / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D009363:Neoplasm Proteins; D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine