Thioredoxin-Dependent Decomposition of Protein S-Nitrosothiols
The addition of nitric oxide to cysteine moieties of proteins results in the formation of S-nitrosothiols (SNO) that have emerged as important posttranslational signaling cues in a wide variety of eukaryotic processes. While formation of protein-SNO is largely nonenzymatic, the conserved family of Thioredoxin (TRX) enzymes are capable of selectively reducing protein-SNO. Consequently, TRX enzymes are thought to provide reversibility and specificity to protein-SNO signaling networks. Here, we describe an in vitro methodology based on enzymatic oxidoreductase and biotin-switch techniques, allowing for the detection of protein-SNO targets of TRX enzymes. We show that this methodology identifies both global and specific protein-SNO targets of TRX in plant cell extracts.
Key wordsBiotin switch technique S-nitrosylation Denitrosylation Thioredoxin Protein-SNO reductase Nitric oxide
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 678511), a Royal Society University Research Fellowship (UF090321), and a Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISSF).
- 3.Kovacs I, Lindermayr C (2013) Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation. Front Plant Sci 4:1–10Google Scholar
- 7.Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE (86). https://doi.org/10.1126/stke.2001.86.pl1