Photoaffinity labelling-based chemoproteomic strategy identifies PEBP1 as the target of ethyl gallate against macrophage activation

Abstract

Ulcerative colitis (UC) is an inflammatory disease of the colon with an unmet need for therapeutic targets. Ethyl gallate (EG) is a natural small molecule for UC treatment, but its cellular target is unknown. By labelling EG with a diazirine photocrosslinker and a click chemistry handle, we identified phosphatidyl-ethanolamine binding protein1 (PEBP1) as a direct cellular target of EG by forming hydrogen bonds with Asp70 and Tyr120. In particular, hydrogen/deuterium exchange mass spectrometry indicated that EG induced the sequence (residues 141–153) embedding to inhibit S153 phosphorylation of PEBP1. Additionally, the EG-mediated sequence (residues 108–122) exposure significantly enhanced PEBP1-Raf-1 interaction to block the downstream NF-κB inflammatory pathway in macrophages. Moreover, PEBP1 siRNA substantially reversed the EG-dependent down-regulation of the phosphorylation of IKKβ, IκBα and NF-κB, demonstrating that the NF-κB signal functioned as an essential anti-inflammation mechanism of PEBP1. Collectively, we revealed PEBP1 as a previously undescribed cellular target in macrophages for UC therapy and identified a new allosteric site for PEBP1 biology study using EG as a chemical probe.