Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones
Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones
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重组组蛋白的半胱氨酸氨乙基化辅助化学泛素化
DOI：
10.1021/jacs.8b13213
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发表时间：
2019-02-27
影响因子：
15
通讯作者：
Liu, Lei
中科院分区：
文献类型：
--
作者：
Chu, Guo-Chao;Pan, Man;Liu, Lei
组蛋白泛素化通过严格调控的动态可逆过程影响核小体的结构和功能。泛素化组蛋白及其类似物的高效制备对于组蛋白泛素化的生物化学和生物物理研究具有重要意义。在这里，我们报告了CAACU（半胱氨酸氨乙基化辅助化学泛素化）策略的有效合成泛素化组蛋白类似物。CAACU策略的关键步骤是通过半胱氨酸氨乙基化将N-烷基化的2-溴乙胺衍生物安装到重组组蛋白中，然后通过Seitz辅助的天然化学连接产生单和双泛素（Ub）和小泛素样修饰剂（SUMO）修饰的组蛋白类似物。该方法能够以约1.5-6 mg/L的表达量从重组蛋白快速生产修饰的组蛋白。产物中的含硫醚的异肽键是化学稳定的，并且与其天然对应物相比，在结构中仅具有一个原子取代。通过CAACU制备的泛素化组蛋白类似物可以很容易地重组成核小体，并被相关的相互作用蛋白选择性识别。含硫醚的异肽键也可以被去泛素化酶（DUBs）识别和水解。对含H2 BK（C）34 Ub的核小体进行冷冻电镜（cryo-EM）观察表明，所获得的CAACU组蛋白具有良好的结构研究质量。总的来说，这项工作证实了CAACU策略的实用性，用于简单有效地生产用于生物化学和生物物理研究的同质泛素化和SUMO化组蛋白。
Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BK(C)34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.