Protocols for the detection of s-glutathionylated and s-nitrosylated proteins in situ

Abstract

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.

Figure 17.1

Schematic representation of the Grx1-catalyzed cysteine derivatization protocol. Free thiols are blocked by alkylation with NEM (1), followed by Grx1-catalyzed reduction of S-glutathionylated proteins (2), and finally newly reduced cysteines are labeled using MPB and detected using streptavidin-conjugated fluorophores (3).

Figure 17.2

Representative image of untreated paraffin embedded murine lungs stained for S-glutathionylated proteins using the Grx1-catalyzed derivatization protocol and visualization via confocal laser scanning microscopy (B and D). Panels A and C represent nuclear counter stains for the tissues evaluated in (B) and (D), respectively. Panel D: Following the protocol outlined herein, reactivity is seen primarily within the epithelial cells of the conducting airways (inset) with some parenchymal reactivity. Panel B: As a negative control, GSH was omitted from the reaction mix, resulting in a loss of Grx-1-catalyzed labeling. Magnification = 200×. Insets: zoom = 4×.

Figure 17.3

Schematic representation of the protocol for detecting S-nitrosylated proteins in situ. Free thiols are blocked by alkylation with NEM (1), S-nitrosylated cysteines are reduced using ascorbate (2), and finally newly reduced cysteines are labeled using MPB and detected using streptavidin-conjugated fluorophores (3).