Abstract

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. 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. We have observed that the activity of KDAC8 with charged substrates in vitro is sensitive to the ionic strength of the reaction buffer. This observation led us to hypothesize that charged residues near the acetylated lysine may be important for the interaction of KDAC8 with its substrates. To test this hypothesis, we have employed an in vitro assay which relies on the reaction of fluorescamine with lysine residues to produce a quantitative fluorescent signal upon deacetylation of a peptide substrate. Using this method, KDAC8 activity was measured with several substrates under conditions of varied ionic strength. Interestingly, for several positively charged peptide substrates, KDAC8 activity increases as ionic strength is decreased, but the opposite is often observed when negatively charged peptide substrates are present. Preliminary results with derivatives of a known peptide substrate suggest that a positively charged side chain adjacent to the acetylated lysine may make a specific contact with KDAC8, as the ionic strength effect varies with the position of the charge in the substrate sequence. Screening a larger set of peptides with charged residues at specific positions relative to the acetylated lysine for activity with KDAC8 provides insight into the contributions of charge to substrate specificity. Comparison of these data with molecular docking data of the same peptides with the active site of KDAC8 can predict specific interactions between charged residues in the KDAC8‐substrate pair. Together, these results will lead to a better understanding of KDAC8 substrate specificity. Extension of this approach to additional KDACs and substrate behavior in vivo will provide insight into substrate specificity of the KDAC family and the biological targets of each KDAC.Support or Funding InformationFunding was provided by NIH 5G12MD007595, R15GM129682, TL4GM118968, 5RL5GM118966, and the Louisiana Cancer Research Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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