Szewczuk Lawrence M, Culhane Jeffrey C, Yang Maojun, Majumdar Ananya, Yu Hongtao, Cole Philip A
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 2007 Jun 12;46(23):6892-902. doi: 10.1021/bi700414b. Epub 2007 May 19.
Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.
赖氨酸特异性去甲基化酶1（LSD1）是一种转录抑制因子，也是一种黄素依赖性胺氧化酶，负责去除组蛋白H3赖氨酸4位上的甲基。在本研究中，我们表征了一种炔丙胺衍生化的组蛋白H3底物（1）对LSD1的抑制机制和范围。与氮丙啶基和环丙胺衍生化的组蛋白H3肽底物类似物不同，化合物1似乎能高效地共价修饰并不可逆地使LSD1失活。伴随这种失活的是光谱变化，其最大吸光度移至392nm。与1-LSD1复合物相关的光谱变化以及对降低pH值和硼氢化钠处理的反应性表明，其结构涉及黄素N5与抑制剂末端碳之间的黄素连接抑制剂共轭物。使用13C标记的抑制剂，对1-黄素共轭物的核磁共振分析与该结构归属一致。对1诱导的光谱位移进行动力学分析表明，在反应中形成的黄素加合物的动力学常数与LSD1失活过程的相似。综上所述，这些数据支持1使LSD1失活的机制，即胺氧化后迈克尔加成到炔丙基亚胺上。我们进一步研究了1的生物素化类似物（1-Btn）用作亲和下拉实验工具的潜力。使用1-Btn，从HeLa细胞核提取物中选择性下拉掺入的和内源性LSD1是可行的，这为基于活性的去甲基酶蛋白质组学奠定了基础。
