HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking

The histone deacetylase family comprises
18 enzymes that catalyze
deacetylation of acetylated lysine residues; however, the specificity
and substrate profile of each isozyme remains largely unknown. Due
to transient enzyme–substrate interactions, conventional co-immunoprecipitation
methods frequently fail to identify enzyme-specific substrates. Additionally,
compensatory mechanisms often limit the ability of knockdown or chemical
inhibition studies to achieve significant fold changes observed by
acetylation proteomics methods. Furthermore, measured alterations
do not guarantee a direct link between enzyme and substrate. Here
we present a chemical crosslinking strategy that incorporates a photoreactive,
non-natural amino acid, p-benzoyl-l-phenylalanine,
into various positions of the structurally characterized isozyme histone
deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation,
and mass spectrometric analysis, we identified a subset of HDAC8 substrates
from human cell lysates, which were further validated for catalytic
turnover. Overall, this chemical crosslinking approach identified
novel HDAC8-specific substrates with high catalytic efficiency, thus
presenting a general strategy for unbiased deacetylase substrate discovery.