Discovery of Chirally-dependent Protein O-2-Hydroxyglutarylation

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are common in multiple types of human cancer and cause accumulation of the oncometabolite D-2-hydroxyglutarate (D2HG), which promotes tumor progression by noncovalently inhibiting α-ketoglutarate–dependent enzymes. Here we discovered covalent O-2-hydroxyglutarylation by D2HG using chemical proteomics and further revealed distinct chiral preferences for D/L-2HG modifications. D2HG modifications are upregulated in IDH-mutant cells or upon D2HG treatment, while L2HG modifications increase under hypoxic conditions or following L2HG treatment. Notably, two kinases MRCKA and SLK are modified by D2HG and L2HG, respectively, and confirmed by synthetic peptide standards. Phosphoproteomics revealed reduced phosphorylation of MRCKA and SLK substrates, suggesting crosstalk between D/L-2HG modification and kinase activity. These findings highlight distinctive roles of D/L-2HG modifications in cancer progression and suggest avenues for therapeutic targeting of oncometabolite-induced PTMs.