Exploration of protein lysine R2HGylation with synthetic probes

Abstract

R-2-Hydroxyglutarate (R2HG), elevated as an oncometabolite in isocitrate dehydrogenase 1/2 (IDH1/2)-mutated cancers, is found to competitively inhibit -ketoglutarate (KG)-dependent enzymes, thus affecting cancer cell fate determination and immune regulation. However, paradoxes remain in its pathological mechanisms, limiting the clinical application of IDH mutant inhibitors. Inspired by recently revealed lysine (K) acylations, we identified K-R2HGylation, the covalent attachment of R2HG on K209 of GSTP1 protein, as a novel post-translational modification (PTM) from IDH1-mutated cell lysates. Using synthetic R2HGylated peptides, we confirmed its C5-linked acylation structure, proved SIRT5 as its eraser, and found GAS41 as a potential reader under acidic conditions. The R2HGylated protein GSTP1 is a detoxification enzyme with important signalling and redox functions. To study the consequence of R2HGylation, we tried to generate K209-modified GSTP1 both in vitro and in vivo via expressed protein ligation (EPL) and unnatural amino acid (UAA) incorporation, where ligated proteins suggested a suppressive role of R2HGylation on the enzymatic activity of GSTP1. Failing in introducing R2HGylated lysine site-specifically into cells, GSTP1 with K209 mutated into E/F/R was stably transduced into cells to evaluate its roles in altered cell proliferation, differentiation, and lipopolysaccharide (LPS)-induced phosphorylation response upon R2HG pre-treatment. Meanwhile, we synthesized a series of cell permeable probes that metabolically label potential R2HGylation targets, which allowed visualization and protein profiling via conjugation to fluorophore and affinity tags by Cu(I)-catalysed azide-alkyne cyclization. MS-based quantitative proteomics highlighted most probable targets from R2HG-competed enrichment pool, and immunoprecipitation of overexpressed protein from IDH-mutated cells revealed another R2HGylation site. Assisted by computational algorithms, probe-linker fragments on enriched peptides were assigned, suggesting potential R2HGylation sites that require further validation. Encouraged by the importance of PTMs, we proposed that R2HG may contribute to cancer development by acylating functional targets. With our synthetic probes, we can find more R2HGylated proteins, profile modification sites, and possibly monitor R2HGylation levels in biological processes. By studying the function and regulation of modified proteins, we hope to understand the cell/enantiomer-specific effects of R2HG and find potential druggable targets or pathways for R2HG-elvated malignancies.