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Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.

Original publication

DOI

10.1016/j.ab.2010.04.030

Type

Journal article

Journal

Analytical biochemistry

Publication Date

09/2010

Volume

404

Pages

86 - 93

Addresses

Department of Chemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, UK.

Keywords

Humans, Peptides, Protein Isoforms, Enzyme Inhibitors, Luminescent Measurements, Protein Structure, Tertiary, Protein Binding, Biocatalysis, Jumonji Domain-Containing Histone Demethylases