Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States.
J Am Chem Soc. 2012 Mar 21;134(11):5138-48. doi: 10.1021/ja209574z. Epub 2012 Mar 12.
Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.
可逆的赖氨酸乙酰化和甲基化调节各种蛋白质的功能，包括组蛋白。在这里，我们合成了含有氮杂赖氨酸的肽，分别为乙酰化和非乙酰化形式，作为组蛋白去乙酰化酶（HDAC8、Sir2Tm 和 SIRT1）和组蛋白去甲基化酶 LSD1 的化学探针。我们已经表明，氮杂赖氨酸的乙酰化修饰是 sirtuins 的一个相当有效的底物，但对于经典的 HDAC8 来说，是一个较弱的底物。除了被 sirtuins 去乙酰化之外，乙酰化氮杂赖氨酸类似物还产生了一种新型的 ADP-核糖加合物，该加合物通过质谱、Western blot 分析和核磁共振波谱进行了表征。该肽-ADP-核糖加合物被提议对应于一个偏离的反应中间体，为 sirtuin 催化去乙酰化过程中形成的 O-烷基咪唑中间体的直接 2'-羟基攻击提供了独特的证据。含有未乙酰化氮杂赖氨酸的 H3 肽被证明是 LSD1 去甲基化酶的有效抑制剂，形成了与先前报道的相关结构特征一致的 FAD 加合物，为机制分析提供了新的化学探针。
