HDAC8 is a member of the family of histone deacetylases (HDACs) that catalyze the deacetylation of acetyl lysine residues within histone and non-histone proteins. The recent identification of novel non-histone HDAC8 substrates such as SMC3, ERRα, and ARID1A indicates a complex functionality of this enzyme in cellular homeostasis. To discover additional HDAC8 substrates, we developed a comprehensive, structure-based approach based on Rosetta FlexPepBind, a protocol that evaluates peptide-binding ability to a receptor from structural models of this interaction. Here we adapt this protocol to identify HDAC8 substrates using peptide sequences extracted from proteins with known acetylated sites. The many new in vitro HDAC8 peptide substrates identified in this study suggest that numerous cellular proteins are HDAC8 substrates, thus expanding our view of the acetylome and its regulation by HDAC8.
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