Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails, and therefore have recently been renamed to acetyl-lysine deacetylases (KDACs). In particular, KDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as {square}-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of KDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of KDAC8 and other enzymes. The calibration process and comparison of the results between KDAC6 and KDAC8 highlighted structural differences that explain the already reported promiscuity of KDAC6. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/431878v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@86bf82org.highwire.dtl.DTLVardef@87007corg.highwire.dtl.DTLVardef@1310594org.highwire.dtl.DTLVardef@1656965_HPS_FORMAT_FIGEXP M_FIG C_FIG