Cross-Species Profiling of Itaconate-Mediated Modifications by a Universal Probe

The immunometabolite itaconate can covalently modify proteins through cysteine itaconation and lysine itaconylation. Current bioorthogonal itaconate probes show biased specificity for profiling cysteine itaconation in macrophages versus bacteria and are not compatible with profiling lysine itaconylation at all. Here, we report isoC3A, a universal probe designed for the comprehensive chemoproteomic profiling of these two types of itaconate-mediated modifications in both eukaryotic and prokaryotic cells. In situ profiling of Salmonella-infected macrophages by isoC3A captures the dynamic changes of itaconate-mediated modifications in the proteomes of host cells and bacteria simultaneously. It also revealed that itaconate targets critical cysteine residues on the aconitate hydratase, enhances its dehydration activity to boost the cis-aconitate level, and drives a positive feedback loop to sustain itaconate biosynthesis. Together, these findings establish isoC3A as a universal probe for exploring the covalent targets of itaconate and suggest a metabolic amplification mechanism in innate immunity.