Identifying Protein Targets of KDAC4 and KDAC5

Abstract

Lysine acetylation is a common post‐translational modification that regulates many biological processes and that has been linked to numerous diseases. Protein lysine deacetylases (KDACs or HDACs) are enzymes that reverse this post‐translational modification by removing the acetyl group from the lysine residue in proteins. KDACs therefore play a critical role in the overall regulation of acetylation. Our objective is to understand the biological roles of KDAC4 and KDAC5, which are class IIa metal‐dependent KDACs, with an emphasis on identifying potential non‐histone substrates. Purified class IIa KDACs have greatly reduced catalytic activity in vitro compared to other KDACs, and only a few putative substrates have been identified for these enzymes in cell‐based experiments. To determine the possible targets of KDAC4 and KDAC5, we have recombinantly expressed and purified various enzyme constructs, including variants with site‐specific mutations that enhance activity to levels comparable with other metal‐dependent KDACs. Activity is measured using a combination of fluorescence‐based assays and mass spectrometry, using biologically relevant putative substrates. Activity assays using model peptides derived from known acetylated proteins with these gain‐of‐function variants reveal specific interaction preferences for the KDACs. Comparison of activity of the isolated catalytic domain with the activity of the full‐length proteins, which contain a second domain of unknown function, allows for determination of the impact of the second domain on interactions with possible target proteins. Together, our results provide insight into possible cellular targets of KDAC4 and KDAC5, as well as providing a mechanism to evaluate whether these enzymes are likely to be acting as deacetylases in vivo or interact with target proteins in a non‐catalytic manner. Understanding of the activity of these KDACs may lead to new therapeutic approaches for acetylation‐associated diseases.Support or Funding InformationNIH 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.