title: In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization.
abstract: 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 glutaredoxin1-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.
authors.affiliation: Department of Pathology, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Niki L
authors.initials: NL
authors.lastname: Reynaert
authors.forename: Karina
authors.initials: K
authors.lastname: Ckless
authors.forename: Amy S
authors.initials: AS
authors.lastname: Guala
authors.forename: Emiel F M
authors.initials: EF
authors.lastname: Wouters
authors.forename: Albert
authors.initials: A
authors.lastname: van der Vliet
authors.forename: Yvonne M W
authors.initials: YM
authors.lastname: Janssen-Heininger
mesh_terms: D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives*; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: C516006:Glrx protein, mouse; D054477:Glutaredoxins; D012710:Serum Albumin, Bovine; D010088:Oxidoreductases; D005978:Glutathione; D003545:Cysteine