Abstract
Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 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 HDAC8 and other enzymes. 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. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.
Highlights
Histone deacetylases play important biological roles well beyond the deacetylation of histone tails
We trained the method on a set of selected peptides for which we measured catalytic activity, validated the model on a set of independently measured peptides and applied it to the human acetylome
In the following discussion we compare the selectivity determinants of HDAC6 and HDAC8, summarize potential roles for the newly predicted substrates, and point to challenges in the accurate and comprehensive characterization of a promiscuous enzyme such as HDAC6
Summary
Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. Schölz et al applied specific inhibitors against several HDACs in cell lines and quantified the change in acetylated lysine sites by SILAC-MS (dataset D-SILAC)[14]. In both studies, the experiments were run with at least five different HDACs, and both reached the conclusion that HDAC6 was, by far, the most promiscuous among the examined HDACs. Kutil et al assessed HDAC6 deacetylation of 13-mer peptides synthesized on an array, measuring activity with a mixture of anti-acetyl lysine antibodies (dataset D-13MER) and verified 20 substrate hits by high-performance liquid chromatography (HPLC; dataset D-HPLC)[3]. The predicted substrates were compared with hits from other studies, finding minimal overlap[3] (see Table S1 for a summary of all studies of HDAC6 substrates)
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