Kinetic characterization of human histone H3 lysine 36 methyltransferases, ASH1L and SETD2
Kinetic characterization of human histone H3 lysine 36 methyltransferases, ASH1L and SETD2
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DOI：
10.1016/j.bbagen.2015.05.013
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发表时间：
2015-09-01
影响因子：
3
通讯作者：
Vedadi, Masoud
中科院分区：
文献类型：
--
作者：
Eram, Mohammad S.;Kuznetsova, Ekaterina;Vedadi, Masoud
背景：组蛋白H3(H3K36)赖氨酸36甲基化异常与包括癌症在内的多种疾病有关。Ash1和SETD2是催化H3K36甲基化的两种酶。H3K4甲基化在ASH1中也有报道。方法：采用基于放射性的酶分析、免疫印迹和特异性抗体免疫印迹以及分子模拟等方法研究AsH11和SETD2的底物特异性。结果：我们报道了ASH11和SETD2体外底物特异性的检测方法和动力学特征。以重组核小体为底物，两种酶均具有较强的活性。然而，SETD2而不是Ash1甲基化的组蛋白多肽也是如此，这表明基本的后置延伸与底物的相互作用可能不是SETD2活性的关键。这两种酶对含有H3K36A突变的核小体均不起作用，表明它们对H3K36具有特异性。通过对ASH1和SETD2反应产物甲基化状态的分析，也证实了H3K36和H3K4两种酶均不具有活性，只有SETD2能够在体外对H3K36进行三甲基化。结论：我们确定了ASH1和SETD2活性的动力学参数，为进一步研究这两种蛋白在健康和疾病中的作用提供了依据。ASH11和SETD2都是H3K36特异的甲基转移酶，但只有SETD2可以使该标记三甲基化。基本的后集扩展对Ash1活性是关键的，但对SETD2活性不是关键。一般意义：我们提供了Ash1和SETD2活性的完整动力学表征。(C)2015爱思唯尔B.V.保留所有权利。
Background: Dysregulation of methylation of lysine 36 on histone H3 (H3K36) have been implicated in a variety of diseases including cancers. ASH1L and SETD2 are two enzymes among others that catalyze H3K36 methylation. H3K4 methylation has also been reported for ASH1L.Methods: Radioactivity-based enzyme assays, Western and immunoblotting using specific antibodies and molecular modeling were used to characterize substrate specificity of ASH1L and SETD2.Results: Here we report on the assay development and kinetic characterization of ASH1L and SETD2 and their substrate specificities in vitro. Both enzymes were active with recombinant nucleosome as substrate. However, SETD2 but not ASH1L methylated histone peptides as well indicating that the interaction of the basic post-SET extension with substrate may not be critical for SETD2 activity. Both enzymes were not active with nucleosome containing a H3K36A mutation indicating their specificity for H3K36. Analyzing the methylation state of the products of ASH1L and SETD2 reactions also confirmed that both enzymes mono- and dimethylate H3K36 and are inactive with H3K4 as substrate, and that only SETD2 is able to trimethylate H3K36 in vitro.Conclusions: We determined the kinetic parameters for ASH1L and SETD2 activity enabling screening for inhibitors that can be used to further investigate the roles of these two proteins in health and disease. Both ASH1L and SETD2 are H3K36 specific methyltransferases but only SETD2 can trimethylate this mark. The basic post-SET extension is critical for ASH1L but not SETD2 activity.General significance: We provide full kinetic characterization of ASH1L and SETD2 activity. (C) 2015 Elsevier B.V. All rights reserved.