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

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.

Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine

“…Consistent with our study, the in silico predictions highlighted the preference for tyrosine and tryptophan in the +1 position; however, their model also suggested (but did not experimentally validate) a weaker preference for tryptophan in the +2 position that we did not observe (Figure ). Thus, these data only partially agree with our findings. The differences are likely due to the use of different experimental methods in each case.…”

“…We were primarily interested in KDAC8 (class I) and KDAC6 (class II). Both are relatively well-characterized KDACs with respect to crystal structures and previous screening work, and both have several putative substrates identified from in vitro and/or cell-based experiments. ,,,,− ,− We used KDAC1 for comparison as it is also a moderately well-characterized class I KDAC; however, it is primarily thought to reside in the nucleus and deacetylate histone proteins. ,, We used the FRK ac RW peptide as a starting point. This peptide was derived from an acetylated human protein (ADAP1; UniProtKB O75689), and we previously identified it as an in vitro substrate for KDAC1, KDAC6, and KDAC8. , Using a fluorescamine-based in vitro assay, we tested the activity of the KDACs with a set of peptides derived from FRK ac RW, where we replaced each of the residues following the acetyllysine (the +1 and +2 positions) with alanine and/or swapped their order to determine whether these residues influenced the activities of KDAC1, KDAC6, and KDAC8 with the peptide substrates.…”

“…Previous large-scale studies investigating substrate specificity at the +1 and +2 positions did not show strong agreement with one another. 15 , 16 , 43 One group performed a screen and found that KDAC8 showed a preference for phenylalanine in the +1 position; however, they did not find that KDAC8 had a preference for tyrosine at that position (tryptophan was not tested) and they did not see any notable preferences at the +2 position. 16 A more recent study used activity data from a small set of peptides to train a program that could predict peptide activity to reveal preferences for KDAC6.…”

Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6

“…In addition to protein substrates of HDAC6 CD2 identified through in vivo biochemical methods (Table S5), additional putative protein substrates have been identified by screening peptide fragments in vitro for activity and then correlating peptide sequences with the corresponding proteins for which crystal structures are available. , Analysis of protein structures identified through this approach yields additional examples of lysine residues contained in loops, α-helices, and one in a β-strand (Table S6).…”

Targeting HDAC6 to Overcome Autophagy-Promoted Anti-Cancer Drug Resistance

“…Histone deacetylase 6 (HDAC6) deacetylates non-histone proteins, including cortactin, peroxiredoxin, and α-tubulin [ 1 , 2 , 3 ]. For example, HDAC6 deacetylates α-tubulin and mediates the disassembly of primary cilia [ 4 ].…”