人类组蛋白H3赖氨酸36甲基转移酶ASH1L和SETD2的动力学特征
Kinetic characterization of human histone H3 lysine 36 methyltransferases, ASH1L and SETD2.
作者信息
Eram Mohammad S, Kuznetsova Ekaterina, Li Fengling, Lima-Fernandes Evelyne, Kennedy Steven, Chau Irene, Arrowsmith Cheryl H, Schapira Matthieu, Vedadi Masoud
机构信息
Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 2M9, Canada.
出版信息
Biochim Biophys Acta. 2015 Sep;1850(9):1842-8. doi: 10.1016/j.bbagen.2015.05.013. Epub 2015 May 19.
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.
背景
组蛋白H3赖氨酸36（H3K36）的甲基化失调与包括癌症在内的多种疾病有关。ASH1L和SETD2是催化H3K36甲基化的两种酶。也有报道称ASH1L可催化H3K4甲基化。
方法
采用基于放射性的酶分析、使用特异性抗体的蛋白质免疫印迹法和分子建模来表征ASH1L和SETD2的底物特异性。
结果
在此我们报告了ASH1L和SETD2的分析方法开发、动力学表征及其体外底物特异性。两种酶都以重组核小体为底物表现出活性。然而，SETD2能甲基化组蛋白肽，而ASH1L不能，这表明SET结构域后碱性延伸与底物的相互作用对SETD2的活性可能并不关键。两种酶对含有H3K36A突变的核小体均无活性，表明它们对H3K36具有特异性。分析ASH1L和SETD2反应产物的甲基化状态也证实，两种酶都能使H3K36发生单甲基化和二甲基化，而对H3K4作为底物无活性，并且只有SETD2能够在体外使H3K36发生三甲基化。
结论
我们确定了ASH1L和SETD2活性的动力学参数，有助于筛选可用于进一步研究这两种蛋白质在健康和疾病中作用的抑制剂。ASH1L和SETD2都是H3K36特异性甲基转移酶，但只有SETD2能使该位点发生三甲基化。SET结构域后碱性延伸对ASH1L的活性至关重要，而对SETD2的活性并非关键。
普遍意义
我们提供了ASH1L和SETD2活性的完整动力学表征。
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