In situ analysis of protein S-glutathionylation in lung tissue using glutaredoxin-1-catalyzed cysteine derivatization.
Protein S-glutathionylation (PSSG) is a posttranslational modification that involves the conjugation of the small antioxidant molecule glutathione to cysteine residues and is emerging as a critical mechanism of redox-based signaling. PSSG levels increase under conditions of oxidative stress and are controlled by glutaredoxins (Grx) that, under physiological conditions, preferentially deglutathionylate cysteines and restore sulfhydryls. Both the occurrence and distribution of PSSG in tissues is unknown because of the labile nature of this oxidative event and the lack of specific reagents. The goal of this study was to establish and validate a protocol that enables detection of PSSG in situ, using the property of Grx to deglutathionylate cysteines. Using Grx1-catalyzed cysteine derivatization, we evaluated PSSG content in mice subjected to various models of lung injury and fibrosis. In control mice, PSSG was detectable primarily in the airway epithelium and alveolar macrophages. Exposure of mice to NO(2) resulted in enhanced PSSG levels in parenchymal regions, while exposure to O(2) resulted in minor detectable changes. Finally, bleomycin exposure resulted in marked increases in PSSG reactivity both in the bronchial epithelium as well as in parenchymal regions. Taken together, these findings demonstrate that Grx1-based cysteine derivatization is a powerful technique to specifically detect patterns of PSSG expression in lungs, and will enable investigations into regional changes in PSSG content in a variety of diseases.
Aesif
Scott W
SW
Department of Pathology, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Anathy
Vikas
V
Havermans
Marije
M
Guala
Amy S
AS
Ckless
Karina
K
Taatjes
Douglas J
DJ
Janssen-Heininger
Yvonne M W
YM
D000818:Animals; D055162:Biocatalysis; D003545:Cysteine / Q000378:metabolism; D054477:Glutaredoxins / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D006651:Histocytochemistry / Q000379:methods*; D008168:Lung / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008171:Lung Diseases / Q000139:chemically induced / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D009585:Nitrogen Dioxide / Q000494:pharmacology; D010083:Oxidants, Photochemical / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D016612:Paraffin Embedding; D017293:Protein S / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D054477:Glutaredoxins; D010083:Oxidants, Photochemical; D017293:Protein S; D005978:Glutathione; D003545:Cysteine; D009585:Nitrogen Dioxide; D010100:Oxygen