HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking

Abstract

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues, however, the specificity and substrate profile of each enzyme 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 photo-reactive, 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 greater catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery.

Critical review of non‐histone human substrates of metal‐dependent lysine deacetylases

“…Indeed, one report with KDAC8 indicated that the authors were able to validate only approximately 30% of the cross‐linked proteins as substrates by in vitro methods. Additionally, their approach did not identify a known substrate of KDAC8, SMC3 89 . Thus, this type of experiment can be useful for identifying potential substrate leads for further study, and perhaps strengthening evidence for suspected enzyme‐substrate pairs that have previously been identified by other means.…”

“…In this way, the catalytic efficiency of a particular KDAC for different acetylation sites can be compared 83,88 . Most in vitro studies screen several acetylated peptides, which correspond to partial sequences of acetylated proteins, as substrates 83,88‐91 . In other reports, individual peptides corresponding to a particular protein of interest or known substrate were shown to be deacetylated 74,92‐94 .…”

“…In addition to these experimental approaches, several groups have reported physical interactions between particular KDACs and other cellular proteins. These experiments range from co‐immunoprecipitation to using a KDAC variant that can be cross‐linked to proteins that bind near the active site in cells 89 . While these experiments do capture physical interactions, they do not, on their own, provide evidence of an enzyme‐substrate relationship.…”

“…It has been proposed that the acetylation/deacetylation of tubulin at this site serves to regulate the stability of microtubules and cilia 6,7,106 . KDAC8 has been reported to be capable of deacetylating tubulin at K40 as well, but the importance and prevalence of this event in vivo has not been well established or extensively replicated as it has with KDAC6 56,89 . The putative KDAC8‐α‐tubulin (K40) pair has been cited by multiple reports; however, the data in support of this are not strong.…”

“…For example, KDAC8 was shown to deacetylate a peptide derived from tubulin in vitro, containing an acetylated K394 residue, but, thus far, there is no evidence that this occurs in cells (Table 2). 89 …”