Determinants of Lysine Deacetylase 8 Substrate Specificity

Abstract

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (HDACs or KDACs) are enzymes that reverse this post‐translational modification. Deacetylation is important for many cellular processes and aberrant KDAC activity has been linked to numerous diseases. While proper deacetylation is known to be critical for proper cellular function, details regarding substrates of particular KDACs and how substrate specificity is determined are lacking. To investigate how particular KDACs differentiate between potential substrates, we constructed a structural alignment of several KDAC catalytic domains. We observed that a loop important for substrate binding always contains an aspartic acid residue, but that the position of this residue differs substantially between KDAC8 and other KDACs. Furthermore, molecular dynamics simulations predict that for several peptide substrates which KDAC8 can deacetylate, ionic interactions likely occur between the aspartic acid in KDAC8 and positively charged residues adjacent to the acetylated lysine in substrates, but likely not with other KDACs. In vitro assays with purified, recombinant KDACs support these predictions. For KDAC8, but not other KDACs, a positively charged residue in the −1 position relative to the acetylated lysine significantly influences the enzyme activity with the peptide. In addition, KDAC8 variants lacking the aspartic acid exhibit greatly reduced activity with the same substrates, consistent with our model that this charged residue is important for KDAC8 substrate selection. However, the substrate discrimination within this set of peptides cannot be entirely explained by the aspartic acid residue, so we are investigating other neighboring residues in silico and in vitro for potential contributions. To complement these studies, we are also utilizing a cell‐based approach to identify novel biological substrates, which can be used to further test our hypothesis that there are critical ionic interactions allowing KDAC8 to bind its preferred substrates. In total, these experiments give us a better understanding of how particular KDACs discriminate between the thousands of potential substrates present in cells, as well as information about biological substrates for KDAC8. Support or Funding Information NIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.