Dehydroglutathione to induce non-reducible glutathionylation for functional analysis

Protein cysteine is susceptible to diverse oxidations, including disulfide, S-sulfenylation, S-nitrosylation, and S-glutathionylation, that regulate many biological processes in physiology and diseases. Despite evidence supporting distinct biological outcomes of individual cysteine oxoforms, the approach for examining functional effects resulting from a specific cysteine oxoform, such as S-glutathionylation, remains limited. In this report, we devised a dehydroglutathione (dhG)-mediated strategy, named G-PROV, that introduces a non-reducible glutathionylation mimic to protein with the subsequent delivery of the modified protein to cells to examine “phenotype” attributed to “glutathionylation”. We applied our strategy to fatty acid binding protein 5 (FABP5), demonstrating that dhG induces selective modification at C127 of FABP5, resembling S-glutathionylation. dhG-modified glutathionylation in FABP5 increases its binding affinity to linoleic acid, enhances its translocation to the nucleus for activating PPARβ/δ, and promotes MCF7 cell migration in response to linoleic acid. Our data report a facile chemical tool to introduce a glutathionylation mimic to protein for functional analysis of protein glutathionylation.