Salicylaldehyde ester-mediated protein semi-synthesis enables studies on the tetra-acetylation of HMGB1

To answer how protein post-translational modifications (PTMs) affect protein function, conformation, sta-bility, localization and interaction with binders remains important in the biological study. However, the re-lated study has been dramatically hindered by the difficulty in obtaining homogenous proteins with site-specific PTMs of interest. Herein, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation). This methodology has enabled us to generate Lys (2/6/7/11) tetra-acetylated HMGB1 (high-mobility group box 1) protein, a 25 kDa proin-flammatory protein, in high purity. Further studies revealed that the tetra-acetylation may represent a regu-latory switch to control the HMGB1 signaling pathway by abolishing its interaction with lipopolysaccha-ride (LPS) and accelerating its degradation, consequently preventing cells from pyroptosis and lethality upon infectious injury.