Itaconate has been recently recognized as an anti-inflammatory metabolite involved in the pathogen–macrophage interface. Due to its weak electrophilicity, itaconate could modify cysteines of the protein KEAP1 and glutathione, which contribute to its anti-inflammatory effect. However, the substrates of itaconate modification in macrophages have not been systematically profiled, which largely impedes the understanding of its roles in immune responses. Here, we developed a specific thiol-reactive probe, 1-OH-Az, for quantitative chemoproteomic profiling of cysteine modifications by itaconate, and provided a global portrait of its proteome reactivity. We found that itaconate covalently modifies key glycolytic enzymes and impairs glycolytic flux mainly through inhibition of fructose-bisphosphate aldolase A (ALDOA). Moreover, itaconate attenuates the inflammatory response in stimulated macrophages by impairing the glycolysis. Our study provides a valuable resource of protein targets of itaconate in macrophages and establishes a negative-feedback link between glycolysis and itaconate, elucidating new functional insights for this anti-inflammatory metabolite.

衣康酸酯最近被认为是参与病原体-巨噬细胞界面的抗炎代谢产物。由于衣康酸弱的亲电性，它可以修饰蛋白KEAP1和谷胱甘肽的半胱氨酸，从而有助于其抗炎作用。但是，尚未对巨噬细胞中衣康酸酯修饰的底物进行系统分析，这在很大程度上阻碍了对其在免疫反应中作用的理解。在这里，我们开发了一种特定的硫醇反应性探针1-OH-Az，用于定量衣康酸对半胱氨酸修饰的化学计量分析，并提供了其蛋白质组反应性的整体描述。我们发现衣康酸酯主要通过抑制果糖-双磷酸醛缩酶A（ALDOA）共价修饰关键的糖酵解酶并损害糖酵解通量。而且，衣康酸酯通过损害糖酵解作用减弱了刺激的巨噬细胞的炎症反应。我们的研究为巨噬细胞中衣康酸酯的蛋白质靶标提供了宝贵的资源，并在糖酵解和衣康酸酯之间建立了负反馈联系，从而阐明了这种抗炎代谢产物的新功能见解。