Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2013 Jul 2;8(7):e67653. doi: 10.1371/journal.pone.0067653. Print 2013.
Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k cat/K m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.
组蛋白 H3 尾部的翻译后修饰（PTMs），如甲基化、乙酰化和磷酸化，在表观遗传信号中发挥重要作用。在这里，我们使用模拟组蛋白 H3 的肽底物研究了这些 PTM 中的一些对体外甲基化赖氨酸 9 去甲基化率的影响。通过比较酶动力学特性，采用各种组合与其他 PTM 一起研究可能的交叉对话效应。我们比较了含有催化核心（cc）的截断组蛋白赖氨酸去甲基酶 KDM4A 和 KDM4C 的组蛋白尾部底物的动力学，并且对全长（FL）KDM4A 和 KDM4C 对一些组合进行了特征描述。我们发现，与 K4 和 K9 三甲基化结合的底物导致 FL-KDM4A 的催化活性显著增加。对于 KDM4A 和 KDM4C 的截断版本，可以观察到双三甲基化底物的催化活性增加了两倍。此外，全长去甲基酶与二甲基化和三甲基化底物之间的催化活性存在显著差异，这与之前报道的截断去甲基酶一致。磷酸化 T11 的组蛋白肽底物既不能被截断的也不能被全长的 KDM4A 和 KDM4C 去甲基化，这表明 T11 上的磷酸化阻止了同一肽上 H3K9me3 标记的去甲基化。K14 的乙酰化也被发现显著影响去甲基化率。因此，对于截断的 KDM4A，底物上 K14 的乙酰化导致酶催化效率（k cat/K m）增加，而对于截断的 KDM4C，它诱导降低，主要是由于 K m 的变化。这项研究表明，对同一肽上的其他 PTM 的去甲基化活性有显著影响，并且强调了在肽水平上研究这些相互作用的重要性，以更详细地了解表观遗传标记的动态。
