A spectrophotometric assay for histone deacetylase 8.
Inhibitors for the classical protein deacetylase enzymes have been actively pursued to develop the next generation of cancer therapy. Developing a novel convenient assay platform for the classical enzyme-catalyzed reactions could thus facilitate the drug discovery process. Based on our previous studies demonstrating the functional mimicry of N(epsilon)-thioacetyl-lysine for N(epsilon)-acetyl-lysine in the reaction catalyzed by the classical enzyme histone deacetylase 8 (HDAC8) on a peptide template derived from the C terminus of the human p53 tumor suppressor protein, we have developed a spectrophotometric HDAC8 assay via quantifying thioacetate produced from the enzymatic dethioacetylation with Ellman's reagent 5,5'-dithiobis(2-nitrobenzoate). We further demonstrated that this novel assay was selective for HDAC8 versus HDAC1 and 2 and for other classical protein deacetylase enzymes present in the HeLa nuclear extracts, thus making it potentially suitable not only for screening HDAC8-selective inhibitors but also for selectively assessing HDAC8 activity under (patho)physiological conditions.
Fatkins
David G
DG
Department of Chemistry, University of Akron, 190 E. Buchtel Commons, Akron, OH 44325, USA.
Zheng
Weiping
W
D000107:Acetylation; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D056572:Histone Deacetylase Inhibitors; D011994:Recombinant Proteins; D006655:Histone Deacetylases