Lysine Deacetylases Have Distinct Preferences for Multiply Acetylated Substrates

Abstract

Lysine deacetylases (KDACs) are enzymes that remove an acetyl group from a lysine, thereby regulating the post‐translational modification of lysine. KDACs are important in many different biological processes and are associated with various mechanisms of diseases in the human body. However, factors contributing to substrate specificity of the KDACs are poorly understood. Moreover, many potential substrate proteins contain adjacent or nearby lysine residues that can be independently acetylated and deacetylated. We hypothesized that KDACs will demonstrate preferences for a particular acetyllysine when reacting with multiply acetylated substrates. We additionally hypothesized that KDAC activity would exhibit a dependence on the acetylation status of nearby lysine residues. We identified several proteins reported as putative substrates of KDACs and which contain multiple nearby lysines, each of which has been reported as acetylated. Using peptides derived from these proteins, we measured activity of the putative substrates with each KDAC via a fluorescence‐based assay. The acetyllysine(s) targeted by each specific KDAC was determined by mass spectrometry. Molecular dynamics was used to model the interactions of the peptides with the KDACs. Our results demonstrate that nearby acetylation does influence the ability of individual KDACs to deacetylate a particular lysine residue, in a manner specific to each KDAC. We propose distinct contacts in the KDACs that may contribute to this selectivity. Future plans include evaluating the enzymatic activity of the KDACs with the full‐length putative substrates in cells to more firmly establish biological relevance.