Comparative Interactome Profiling of Itaconate and α-Ketoglutarate by the Peptide-Centric Local Stability Assay

Itaconate (ITA) is an immunoregulatory metabolite that is significantly upregulated in macrophages during bacterial infection. It can mediate immune and metabolic responses through both covalent modifications and noncovalent interactions with key proteins. While covalent itaconation has been systematically mapped using chemical probes, the global landscape of noncovalent targets of itaconate remains poorly explored. Here, we applied the peptide-centric local stability assay (PELSA) to globally profile the interactome of ITA in macrophage lysates. PELSA successfully identified known ITA targets and assigned ITA-responsive regions that correspond to authentic binding pockets. Comparative profiling with a structurally similar metabolite, α-ketoglutarate (AKG), further revealed the functional crosstalk between the two ligands. We biochemically validated the cytosolic isocitrate dehydrogenase 1 (IDH1) as a target of ITA, in which noncovalent interaction played a critical functional role. This study provides a valuable resource and underscores the importance of metabolic crosstalk between ITA and AKG.