Protocols for the detection of s-glutathionylated and s-nitrosylated proteins in situ.
The oxidation of protein cysteine residues represents significant posttranslational modifications that impact a wide variety of signal transduction cascades and diverse biological processes. Oxidation of cysteines occurs through reactions with reactive oxygen as well as nitrogen species. These oxidative events can lead to irreversible modifications, such as the formation of sulfonic acids, or manifest as reversible modifications such as the conjugation of glutathione with the cysteine moiety, a process termed S-glutathionylation (also referred to as S-glutathiolation, or protein mixed disulfides). Similarly, S-nitrosothiols can also react with the thiol group in a process known as S-nitrosylation (or S-nitrosation). It is the latter two events that have recently come to the forefront of cellular biology through their ability to reversibly impact numerous cellular processes. Herein we describe two protocols for the detection of S-glutathionylated or S-nitrosylated proteins in situ. The protocol for the detection of S-glutathionylated proteins relies on the catalytic specificity of glutaredoxin-1 for the reduction of S-glutathionylated proteins. The protocol for the detection of S-nitrosylated proteins represents a modification of the previously described biotin switch protocol, which relies on ascorbate in the presence of chelators to decompose S-nitrosylated proteins. These techniques can be applied in situ to elucidate which compartments in tissues are affected in diseased states whose underlying pathologies are thought to represent a redox imbalance.
Aesif
Scott W
SW
Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA.
Janssen-Heininger
Yvonne M W
YM
Reynaert
Niki L
NL
D000818:Animals; D005978:Glutathione; D016591:Histocytological Preparation Techniques; D008168:Lung / Q000166:cytology; D026422:S-Nitrosoglutathione / Q000032:analysis* / Q000737:chemistry; D026403:S-Nitrosothiols / Q000032:analysis* / Q000737:chemistry
D026403:S-Nitrosothiols; D026422:S-Nitrosoglutathione; D005978:Glutathione