Unlocking an HDAC Toolbox: Methods toward Understanding Isozyme-specific Activity.

[摘要] Lysine acetylation is a dynamic post-translational modification occurring ubiquitously in cells.Histone deacetylases (HDACs) catalyze the enzymatic hydrolysis of acetyllysine.There are 18 HDACs, tasked with the negative regulation of acetylation of thousands of proteins.It is therefore critical to understand the reactivity and specificity of these enzymes.To probe the substrate specificity of HDAC isozymes, we developed and optimized a real-time enzyme-coupled assay that measures deacetylation via the formation of acetate and two mass spectrometric assays that measure the mass change associated with deacetylation.These assays were used to measure the reactivity of HDAC8 and HDAC11 with a variety of acetylated peptides and inhibitors, providing insight into the substrate selectivity.To characterize HDAC11, we recombinantly expressed a SUMO-HDAC11 fusion protein in E. coli in the presence of the molecular chaperone trigger factor.Interestingly, HDAC11 expressed in bacteria is inactive, in contrast to the catalytically active HDAC11 expressed and purified from eukaryotic cells.HDAC11 purified from insect cells catalyzes deacetylation of unlabeled peptide substrates and demonstrate unique selectivity that differs from HDAC8.To identify protein substrates, we introduced a chip-based proteomics method to screen the reactivity of HDAC isozymes with thousands of full-length human proteins.This work identified 44 and 25 potential HDAC8 and HDAC11 substrates, respectively.These substrates were then validated using peptide mimics with the enzyme-coupled and mass spectrometric assays mentioned above, determining rate constants spanning three orders of magnitude.Based on these results, isocitrate dehydrogenase 1 (IDH1) variants containing single, biologically relevant acetyllysine side chains were expressed and purified.HDAC8 catalyzes the deacetylation of these full-length protein substrates in vitro with high efficiency.Furthermore, the acetyllysine modifications decrease the catalytic activity of IDH1.Finally, the first HDAC prodrug, SAHA-TAP, is shown to covalently modify a conserved cysteine residue, Cys153 in HDAC8, leading to irreversible inactivation and simultaneous release of the competitive inhibitor SAHA.Overall, this work provides new methods for the characterization of HDAC reactivity, the identification of novel HDAC8 and HDAC11 substrates, and analysis of HDAC-inhibition.This work provides a foundation for understanding disease states that arise from aberrant acetylation and deacetylation.

[发布机构] University of Michigan

[效力级别] Histone Deacetylase 11

[关键词] Histone Deacetylase 8;Histone Deacetylase 11;HDAC;Biological Chemistry;Science;Chemical Biology