Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism

Abstract

Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depends on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using a high-throughput Self-Assembled Monolayers for MALDI-TOF Mass Spectrometry (SAMDI) mass spectrometry screen. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the ratio of Fe(II)/Zn(II) HDAC8 activity varies widely (2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo which, in turn, may regulate its activity. However future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.

The enzyme activity of histone deacetylase 8 is modulated by a redox-switch

“…There is a large number of publications about the modulation of HDAC8 enzyme activity by inhibitors or incorporated metal ions [14] , [24] . In particular, the identification of N-acetylthiourea compounds as putative activators of HDAC8 raised considerable attention [25] .…”

“…HDAC8 activity is negatively regulated upon phosphorylation by cyclic AMP dependent protein kinase at position serine 39 [13] . Moreover, Fierke et al suggested that HDAC8 could be also regulated by metal switching in vivo [14] . Redox control of epigenetic processes has been proposed as a general principle to allow the cell to adapt to a changing environment.…”

A distal regulatory region of a class I human histone deacetylase

“…Protein expression, purification and mutagenesis. BL21(DE3) strain of Escherichia coli cells (New England Biolabs) expressing human HDAC8 were grown at 37°C in~99% D 2 O minimal M9 media containing 15 12 CD 3 )] (for labelling of valine and leucine residues) and 150 mg L −1 of methionine [ 13 C/ 1 H] (for labelling of methionine residues) 1 h prior to induction. Expression was induced at an OD 600nm between 0.5 and 1.0 by 1 mM ITPG.…”

“…While HDAC3 is inactive in isolation, it shows significant activity in complex with the SMRT DAD domain 11 . The isozyme HDAC8 is less active than other class I HDACs, however, HDAC8 displays significant activity on its own and also shows enhanced activity in the presence of metals, such as Fe(II) and Co(II) than Zn(II) 12 . HDAC8 was the first human HDAC for which high-resolution crystal structures were published 13,14 .…”

The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries

“…In addition, enzymatic activity patterns of HDACs in cell lysates could be determined (25). Moreover, the same technology enabled the discovery that HDAC8 substrate specificity is dependent on the nature of the metal ion within the active site (26).…”