Journal
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
Volume 1760, Issue 3, Pages 380-387Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2006.01.006
Keywords
glutaredoxin; S-glutathionylation; cysteine derivatization; NADPH oxidase; oxidant; cell imaging
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Funding
- NHLBI NIH HHS [R01 HL60014, P01 HL67004, HL60812] Funding Source: Medline
- PHS HHS [P20 RL15557] Funding Source: Medline
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S-glutathionylation is rapidly emerging as an important post-translational modification, responsible for transducing oxidant signals. However, few approaches are available that allow visualization of glutathione mixed disulfides in intact cells. We describe here a glutaredoxin 1-dependent cysteine derivatization and labeling approach, in order to visualize S-glutathionylation patterns in situ. Using this new method, marked S-glutathionylation was observed in epithelial cells, which was predominant at membrane ruffles. As expected, the labeling intensity was further enhanced in response to bolus oxidant treatments, or in cells overexpressing Nox1 plus its coactivators. In addition, manipulation of endogenous levels of glutaredoxin-1 via RNAi, or overexpression resulted in altered sensitivity to H2O2 induced formation of glutathione mixed disulfides. Overall, the derivatization approach described here preferentially detects S-glutathionylation and provides an important means to visualize this post-translational modification in sub-cellular compartments and to investigate its relation to normal physiology as well as pathology. (C) 2006 Elsevier B.V. All rights reserved.
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