Proteomic Identification of Protein Glutathionylation in Cardiomyocytes

Reactive oxygen species (ROS) are
important signaling molecules, but their overproduction is associated
with many cardiovascular diseases, including cardiomyopathy. ROS induce
various oxidative modifications, among which glutathionylation is
one of the significant protein oxidations that occur under oxidative
stress. Despite previous efforts, direct and site-specific identification
of glutathionylated proteins in cardiomyocytes has been limited. In
this report, we used a clickable glutathione approach in a HL-1 mouse
cardiomyocyte cell line under exposure to hydrogen peroxide, finding
1763 glutathionylated peptides with specific Cys modification sites,
which include many muscle-specific proteins. Bioinformatic and cluster
analyses found 125 glutathionylated proteins, whose mutations or dysfunctions
are associated with cardiomyopathy, many of which include sarcomeric
structural and contractile proteins, chaperone, and other signaling
or regulatory proteins. We further provide functional implication
of glutathionylation for several identified proteins, including CSRP3/MLP
and complex I, II, and III, by analyzing glutathionylated sites in
their structures. Our report establishes a chemoselective method for
direct identification of glutathionylated proteins and provides potential
target proteins whose glutathionylation may contribute to muscle diseases.