Determining HDAC8 Substrate Specificity.

Abstract

Histone deacetylases (HDACs) are a group of 18 enzymes that catalyze the deacetylation of acetyl lysine residues in proteins. Acetyl lysine residues are present within thousands of proteins, and acetylation/deacetylation has been shown to affect protein properties integral to cellular homeostasis and disease states. Determining which protein is deacetylated by which HDAC isozyme is central to understanding biological regulation. To better identify HDAC substrates, I developed an assay to measure the acetate product formed by deacetylation catalyzed by metal-dependent HDACs for the evaluation of peptide substrates. Using this assay, I advanced a computational algorithm that predicts HDAC8 peptide substrates based on short range interactions. This algorithm accurately predicts the catalytic efficiency of 7-mer peptide substrates based on the sequence of the peptide. Using the deacetylation assay, I also demonstrated the reactivity of HDAC8 with peptide substrates derived from proteins with increased acetylation in vivo upon treatment with HDAC8 specific inhibitors. These experiments suggest that a subset of in vivo HDAC8 substrates can be predicted based on the six amino acids flanking the acetyl lysine. I have also utilized singly acetylated histone tetramers to establish that HDAC8 has enhanced activity in comparison to corresponding peptide substrates. Combined with the peptide work, these results suggest that molecular recognition by HDAC8 is determined by a combination of short and long-range interactions and acetyl lysine accessibility of in vivo substrates. Additionally, I have identified slow product dissociation as a novel regulatory method for HDAC activity. In the future we will expand these methods to identify substrates for other HDACs.