Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.

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.

Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. Because these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH (nicotinamide adenine dinucleotide, reduced form) that is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by cobalt(II)-bound histone deacetylase 8 [Co(II)-HDAC8] compared with peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-, 2.8-, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.

Despite being extensively characterized structurally and biochemically, the functional role of histone deacetylase 8 (HDAC8) has remained largely obscure due in part to a lack of known cellular substrates. Herein, we describe an unbiased approach using chemical tools in conjunction with sophisticated proteomics methods to identify novel non-histone nuclear substrates of HDAC8, including the tumor suppressor ARID1A. These newly discovered substrates of HDAC8 are involved in diverse biological processes including mitosis, transcription, chromatin remodeling, and RNA splicing and may help guide therapeutic strategies that target the function of HDAC8.

The lysine deacetylase family of enzymes (HDACs) was first demonstrated to catalyze deacetylation of acetyllysine residues on histones. In subsequent years, HDACs have been shown to recognize a large pool of acetylated nonhistone proteins as substrates. Recently, thousands of acetylated proteins have been discovered, yet in most cases, the HDAC that catalyzes deacetylation in vivo has not been identified. This gap has created the need for better in vivo, in vitro, and in silico approaches for determining HDAC substrates. While HDAC8 is the best kinetically and structurally characterized HDAC, few efficient substrates have yet been substantiated in vivo. In this review, we delineate factors that may be important for determining HDAC8 substrate recognition and catalytic activity, including structure, complex formation, and post-translational modifications. This summary provides insight into the challenges of identifying in vivo substrates for HDAC8, and provides a good vantage point for understanding the variables important for predicting HDAC substrate recognition.

Francisella tularensis is the bacteria responsible for tularemia, or rabbit fever. Weaponized strains of F. tularensis were developed in the 1940's, and although all stockpiles of these strains were presumably destroyed, the bacterium still has the potential to be converted into a bioweapon. In order to treat weaponized strains, new targets and new treatments must be developed. To this end, we have characterized the acetate kinase from F. tularensis both structurally and kinetically. The acetate kinase gene was subcloned into a pProEX‐HTb expression vector, and was purified using a nickel‐affinity column. The structure of F. tularensis acetate kinase was determined to a resolution of 2.2 Å, and is similar to other known acetate and propionate kinases. The kinetic constants, kcat and KM, with acetate as the substrate were determined to be 363 s−1 and 14 mM, respectively. While the kinetic constants, kcat and KM, with propionate as the substrate were determined to be 66 s−1 and 75 mM, respectively. In addition, we utilized high‐throughput virtual screening to identify potential inhibitors of the acetate kinase, and we have demonstrated that the acetate kinase is amenable to inhibitor screening using a thermal stability assay. This research was funded by the Wabash College Byron K. Trippet Fund and the Haines Biochemistry Fund.