A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors.
A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors.
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DOI：
10.1039/b921912a
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发表时间：
2010-05
影响因子：
--
通讯作者：
Simeonov A
中科院分区：
文献类型：
--
作者：
Quinn AM;Allali-Hassani A;Vedadi M;Simeonov A
由组蛋白甲基转移酶（HMT）催化的赖氨酸残基的甲基化是调节转录和染色质结构的核心组蛋白的许多修饰之一。G9a是哺乳动物常染色质中的主要HMT，最近的数据表明，需要它来使癌细胞中的恶性表型永久化，并且与转移有关，支持该HMT作为癌症和与表观遗传调控相关的其他疾病的治疗靶标。在目前用于测量甲基转移酶活性的方法中，许多方法涉及分离步骤或利用使实施和数据解释复杂化的偶联酶。在这里，我们描述了一种均相测定法，使用基于荧光的AlphaScreen免疫测定技术来测量G9a HMT活性。生物素化组蛋白肽的甲基化通过基于特异性抗体的检测，结合链霉亲和素包被的供体和二抗包被的受体珠来测量。该方法特别适用于检测通过所需组蛋白肽竞争机制起作用的抑制剂，并适用于检测其他HMT，在此用G9a同系物EHMT 1（也称为GLP）证明。
Methylation of lysine residues, catalyzed by histone methyltransferase (HMT) enzymes, is one of many modifications of core histone proteins that regulate transcription and chromatin structure. G9a is the predominant HMT in mammalian euchromatin and recent data suggest that it is required to perpetuate a malignant phenotype in cancer cells and is implicated in metastasis, supporting this HMT as a therapeutic target for cancer and other diseases associated with epigenetic regulation. Of the methods currently used to measure methyltransferase activity, many involve a separation step or utilize coupling enzymes complicating implementation and data interpretation. Here we describe a homogeneous assay to measure G9a HMT activity using the chemiluminescence-based AlphaScreen immunoassay technology. Methylation of biotinylated-histone peptide is measured through specific antibody-based detection, in conjunction with streptavidin-coated donor and secondary antibody-coated acceptor beads. The method is particularly well suited for detection of inhibitors acting by the desired histone peptide competitive mechanism and is applicable to testing other HMTs, demonstrated here with the G9a homolog EHMT1, also known as GLP.
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影响因子：
4.8
作者：
Patnaik, D;Chin, HG;Pradhan, S
通讯作者：
Pradhan, S
DOI：
10.1016/j.bbrc.2005.03.170
发表时间：
2005-05-27
影响因子：
3.1
作者：
Wang, CH;Leffler, S;Hrycyna, CA
通讯作者：
Hrycyna, CA
影响因子：
64.5
作者：
Trievel, RC;Beach, BM;Hurley, JH
通讯作者：
Hurley, JH
影响因子：
16
作者：
Kubicek, Stefan;O'Sullivan, Roderick J.;Jenuwein, Thomas
通讯作者：
Jenuwein, Thomas
影响因子：
10.5
作者：
Tachibana, M;Ueda, J;Shinkai, Y
通讯作者：
Shinkai, Y