Thermal proteome profiling of itaconate interactome in macrophages

Itaconate (ITA) is an upregulated immunometabolite in macrophages during pathogen infection. It is known to influence oxidation stress, cellular metabolism, programmed cell death and many other biological processes to regulate the immune response <i>via</i> interaction with proteins. Previous studies capture covalently ITA-modified proteins by activity-based proteome profiling with bioorthogonal chemical probes; however, how itaconate interacts non-covalently with other proteins at the proteome level remains unexplored. Here we applied thermal proteome profiling (TPP) to globally identify a large number of ITA-interacting proteins in macrophage proteomes. Among these targets, we verified mitochondrial branched-chain aminotransferase (BCAT2) as a novel non-covalent binding target of itaconate <i>via</i> biochemical and structural experiments. The binding of itaconate could inhibit transamination activity of BCAT and regulate the metabolism of branched-chain amino acids (BCAAs) in lipopolysaccharide (LPS)-activated inflammatory macrophages. This study offers a valuable resource that helps decipher novel and comprehensive functions of ITA in macrophages during the immune response and other related biological processes.