Understanding HDAC8-Catalyzed Deacetylation:From New Substrates to Diseases

[摘要] Lysine acetylation is a dynamic post-translational modification occurring ubiquitously in cells. The histone deacetylase (HDAC) family catalyzes the removal of an acetyl group from the ε-position of lysine residues in proteins. There are 11 metal-dependent deacetylases tasked with the proper regulation of thousands of proteins. Thus, it is important to define the specificity and reactivity of each isozyme.HDAC8 is a well characterized metal-dependent deacetylase that can be activated with Zn2+ or Fe2+ in vitro with a relatively unknown protein-substrate pool. To unveil new putative full- length protein substrates, we developed and optimized a chemical capture approach that can be used along with co-immunoprecipitation and proteomics to trap short-lived HDAC8-substrate interactions. Using this approach, we identified 11 potential HDAC8 substrates. These substrates were then validated using acetylated peptide mimics with an enzyme-coupled assay, determining rate constants that are at least two orders of magnitude faster than SMC3, one of the best in vivo validated HDAC8 protein substrates. Furthermore, we studied how Cornelia de Lange spectrum disorders are caused by HDAC8 missense mutations. Based on our results, we determined that these mutations can affect a variety of processes such as substrate binding, product release, and most interestingly, divalent metal binding; bringing to question the identity of HDAC8’s catalytic divalent metal ion. Finally, we have shown that the sequence specificity of HDAC8 towards peptide substrates is dependent on the identity of the catalytic divalent metal ion in addition to the immediate sequence and potentially other post-translational modifications. Overall, this work has provided insight into the substrate specificity and regulation of HDAC8 in the cell, in addition to providing a new, alternate approach for identifying potential HDAC-specific substrates that could be adapted for other deacetylase isozymes.

[发布机构] University of Michigan

[效力级别] protein substrates

[关键词] Histone deacetylase 8;protein substrates;Chemical Biology;Acetylation;Biological Chemistry;Science;Chemical Biology