Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates
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Updated: Oct 4, 2025
Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS
Published on: August 10, 2017
Julia K Varga1, Kelsey Diffley2, Katherine R Welker Leng2
1Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University of Jerusalem, Faculty of Medicine, POB 12272, 9112102, Jerusalem, Israel.
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.
Insights
Histone deacetylase 6 (HDAC6) regulates various cellular processes. This study identifies new HDAC6 peptide substrates and proposes roles in cancer via EGFR/Akt pathway regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Histone deacetylases (HDACs) have diverse biological functions beyond histone modification.
- HDAC6 specifically impacts apoptosis, cytoskeleton dynamics, and protein folding, targeting proteins like α-tubulin, Hsp90, and cortactin.
- Understanding HDAC6's substrate specificity is crucial for elucidating its multifaceted roles.
Purpose of the Study:
- To identify novel peptide substrates for HDAC6 using a combination of biochemical assays and computational prediction.
- To calibrate a structure-based substrate prediction protocol (Rosetta FlexPepBind) for HDAC6.
- To explore potential new functional and regulatory roles of HDAC6, particularly in cancer biology.
Main Methods:
- Biochemical enzymatic assays were used to measure HDAC6 activity on candidate unlabeled peptides.
- A structure-based substrate prediction protocol, Rosetta FlexPepBind, was calibrated using experimental data.
- A proteome-wide screen of reported acetylation sites was performed using the calibrated protocol and enzymatic assays.
Main Results:
- New peptide substrates for HDAC6 were identified.
- The calibrated protocol successfully predicted substrates and highlighted structural differences between HDAC6 and HDAC8, explaining HDAC6's promiscuity.
- Novel regulatory roles for HDAC6 in tumorigenesis and cancer cell survival, potentially through the EGFR/Akt pathway, were proposed.
Conclusions:
- HDAC6 exhibits promiscuous substrate specificity, influenced by its structure.
- The study provides new insights into HDAC6 function and identifies potential therapeutic targets in cancer.
- This work expands the understanding of HDAC6's involvement in cellular regulation and disease.
