Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor
Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor
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DOI：
10.1111/j.1742-4658.2012.08567.x
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发表时间：
2012-06-01
期刊：
影响因子：
5.4
通讯作者：
Gajhede, Michael
中科院分区：
文献类型：
--
作者：
Kristensen, Line H.;Nielsen, Anders L.;Gajhede, Michael
组蛋白赖氨酸残基的动态甲基化和去甲基化对于基因调控是重要的，并且由组蛋白甲基转移酶和组蛋白去甲基化酶（HDMs）促进。KDM 5 B/Jarid 1B/PLU 1是一种H3 K4 me 3/me 2特异性赖氨酸脱甲基酶，属于含JmjC结构域的组蛋白脱甲基酶（JHDMs）家族。一些研究将KDM 5 B与乳腺癌、前列腺癌和皮肤癌联系起来，突出了它作为药物靶点的潜力。然而，大多数抑制剂研究都集中在其他JHDM上，KDM 5 B的抑制剂仍有待探索。在这里，我们报告的表达，纯化和表征的催化核心的重组KDM 5 B（ccKDM 5 B，残基1-769）。我们表明，在昆虫细胞中重组表达的ccKDM 5 B在体外使H3 K4 me 3和H3 K4 me 2去甲基化。动力学表征表明，ccKDM 5 B的三甲基化底物H3（1-15）K4 me 3的表观米氏常数（Kmapp）值为0.5 μ m，与相关HDMs相比，表观底物亲和力显著增加。尽管存在PHD结构域，但催化活性不受H3 K9位置处的额外甲基化的影响，这表明对于ccKDM 5 B，H3 K4和H3 K9之间的体外染色质串扰不发生。ccKDM 5 B的抑制研究表明，2，4-吡啶二甲酸（2，4-PDCA）对ccKDM 5 B的体外和细胞抑制作用与HDM KDM 4C报告的效力相似。结构指导的序列比对结果表明，KDM 4A/C和KDM 5 B之间2，4-PDCA的结合模式是保守的。
Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (Kmapp) value of 0.5 mu m for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.