Detection of protein S-nitrosylation with the biotin-switch technique

Abstract

Protein S-nitrosylation, the posttranslational modification of cysteine thiols to form S-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for S-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during physiological and pathophysiological conditions. The focus of this review is the biotin-switch technique (BST), which has become a mainstay assay for detecting S-nitrosylated proteins in complex biological systems. Potential pitfalls and modern adaptations of the BST are discussed, as are future directions for this assay in the burgeoning field of protein S-nitrosylation.

Fig. 1

A general comparison of NO- and sulfur-based strategies for detecting protein S-nitrosylation. As an example, three lysates containing various amounts of protein S-nitrosylation are subjected to both NO- and sulfur-based assays. NO-based strategies include the Saville and diaminofluorescein (DAF) assays, which employ a chemical probe, and Hg-coupled photolysis-chemiluminescence (PCL), which detects NO gas liberated by SNO homolysis and can differentiate SNO from metal-NO. Importantly, this assay is highly sensitive (low nanomolar SNO concentrations can be detected) and has been well-validated with genetic models of disrupted NO/SNO metabolism [108, 109]. It therefore serves as a standard method for probing S-nitrosylation in vivo. With a complex biological sample (e.g. a lysate), these NO-based strategies can readily determine the absolute amount of SNO per sample, but cannot readily detect an individual protein-SNO. A sulfur-based strategy, such as the biotin switch technique (BST), employs covalent “tagging” at the sulfur atom of each SNO, thus facilitating relative quantitation and protein-SNO identification.

Fig. 2

Overview of the biotin switch technique. In the example shown, three lysates with various degrees of S-nitrosylation are subjected to the assay. The “blocking” step involves S-methylthiolation of each cysteine thiol with S-methylmethanethiosulfonate (MMTS). Next, ascorbate is employed to convert each SNO to a free thiol via a transnitrosation reaction to generate O-nitrosoascorbate. In the “labeling” step, each nascent free thiol (previously an SNO site) is biotinylated with biotin-HPDP. Biotinylated proteins are enriched by avidin affinity media, and analyzed by SDS-PAGE/immunoblotting. Total protein-SNOs or an individual protein-SNO can be detected with avidin-HRP or with an antibody against a protein of interest, respectively. As illustrated, the degree of pulldown correlates with protein S-nitrosylation. Prior to avidin pulldown, a small fraction of each sample is analyzed to determine protein “input.”

Fig. 3

A typical BST detects both endogenous and exogenous S-nitrosylation in cultured mammalian cells. A) Murine RAW264.7 macrophages were either untreated or cytokine-stimulated with lipopolysaccharide (500 ng/ml) and IFN-γ (100 U/ml) for 16 h, which drives NO production. Cellular extracts were subjected to the BST and probed for S-nitrosylated GAPDH (GAPDH-SNO), along with ascorbate and pre-photolysis controls. Notably, omission of ascorbate leads to nearly complete loss of biotinylation and pre-photolysis with a Hg vapor lamp greatly attenuates the same signal. B) Whole cellular protein-SNOs are detected by treating HEK293 cells with 200 µM S-nitrosocysteine (CysNO) for 10 min. Cellular extracts were subjected to the BST and 5% of each biotinylation reaction (~ 40 µg) was analyzed by immunoblotting with avidin-HRP and anti-GAPDH antibody (for “input”).

Fig. 4

The BST can be combined with pharmacological and genetic tools to study S-nitrosylation in primary cells and tissues in vivo. A) GRK2 is S-nitrosylated in HEK293 cells that stably overexpress the constitutive NOS isoforms (eNOS and nNOS). B) G protein coupled receptor (GPCR)-specific agonists (ATP, adenosine triphosphate; BK, bradykinin; ISO, isoproteronol; CTL, control), each of which leads to eNOS activation, increase GRK2 S-nitrosylation in human umbilical vein endothelial cells. C) GRK2 S-nitrosylation is diminished in lungs from eNOS^−/− mice relative to wild type (WT) mice. D) GRK2 S-nitrosylation is increased in lungs from mice lacking a major SNO-metabolizing enzyme, GSNO reductase (GSNOR^−/−). Figure adapted from reference [5].

Fig. 5

An illustration employing UV pre-photolysis and ascorbate controls within the BST for the analysis of a single lysate. One sample is split to two fractions, which are either untreated or exposed to a strong UV light source (“pre-photolysis”) prior to performing the BST. The pre-photolysis step leads to homolytic cleavage of the S-NO into NO and an unstable thiyl radical, which is either reduced to a thiol or oxidized to a higher S-oxide. In either case, the signal from an SNO will be attenuated by pre-photolysis, while free thiols or disulfides are unaffected. Inclusion or exclusion of ascorbate during the labeling step of the BST can also be employed to assay for protein-SNOs. This approach, which employs internal controls (e.g. pre-photolysis and ascorbate), is ideal when external controls such as NOS inhibition or activation are not feasible (e.g. human tissue samples) or ineffective (as in the case of reactions regulated by denitrosylation).