Development of homogeneous luminescence assays for histone demethylase catalysis and binding.
Development of homogeneous luminescence assays for histone demethylase catalysis and binding.
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DOI：
10.1016/j.ab.2010.04.030
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发表时间：
2010-09-01
影响因子：
2.9
通讯作者：
Schofield C
中科院分区：
文献类型：
--
作者：
Kawamura A;Tumber A;Rose NR;King ON;Daniel M;Oppermann U;Heightman TD;Schofield C
组蛋白的共价修饰在染色质动力学和基因表达调控中起着重要作用。含有JmjC的组蛋白去甲基酶(HDMS)催化组蛋白尾巴上的甲基化赖氨酸残基去甲基化。在这里，我们报告了基于均相发光的分析方法的发展，以测量多肽的催化活性和与HDMS的结合亲和力。该分析采用放大发光邻近均相分析(Alpha)技术，灵敏、可靠，可用于HDMS的小分子抑制剂筛选。我们已经分析了JMJD2E的已知抑制剂，并证明了由Alpha确定的抑制剂效力与其他类型的分析之间的相关性。虽然这项研究的重点是JMJD2E亚型，但这里描述的催化周转和结合分析方法也可以用于其他HDMS的研究。该方法可用于HDM异构体选择性小分子抑制剂的开发。
Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JmjC-containing histone demethylases (HDMs) catalyse 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 and are sensitive, robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate 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.