Large-Scale Identification of Lysine Crotonylation Reveals Its Potential Role in Oral Squamous Cell Carcinoma

PURPOSE: Lysine crotonylation, an emerging posttranslational modification, has been implicated in the regulation of diverse biological processes. However, its involvement in oral squamous cell carcinoma (OSCC) remains elusive. This study aims to reveal the global crotonylome in OSCC under hypoxic conditions and explore the potential regulatory mechanism of crotonylation in OSCC. METHODS: Liquid-chromatography fractionation, affinity enrichment of crotonylated peptides, and high-resolution mass spectrometry were employed to detect differential crotonylation in CAL27 cells cultured under hypoxia. The obtained data were further subjected to bioinformatics analysis to uncover the involved biological processes and pathways of the dysregulated crotonylated proteins. A site-mutated plasmid was utilized to investigate the effect of crotonylation on Heat Shock Protein 90 Alpha Family Class B Member 1 (HAP90AB1) function. RESULTS: A large-scale crotonylome analysis revealed 1563 crotonylated modification sites on 605 proteins in CAL27 cells under hypoxia. Bioinformatics analysis revealed a significant decrease in histone crotonylation levels, while up-regulated crotonylated proteins were mainly concentrated in non-histone proteins. Notably, glycolysis-related proteins exhibited prominent up-regulation among the identified crotonylated proteins, with HSP90AB1 displaying the most significant changes. Subsequent experimental findings confirmed that mutating lysine 265 of HSP90AB1 into a silent arginine impaired its function in promoting glycolysis. CONCLUSION: Our study provides insights into the crotonylation modification of proteins in OSCC under hypoxic conditions and elucidates the associated biological processes and pathways. Crotonylation of HSP90AB1 in hypoxic conditions may enhance the glycolysis regulation ability in OSCC, offering novel perspectives on the regulatory mechanism of crotonylation in hypoxic OSCC and potential therapeutic targets for OSCC treatment.