Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones

作者：Guo‐Chao Chu, Man Pan, Jiabin Li, Sanling Liu, Chong Zuo, Zebin Tong, Jingsi Bai, Qingyue Gong, Huasong Ai, Jian Fan, Xianbin Meng, Yichao Huang, Jing Shi, Haiteng Deng, Changlin Tian, Yi‐Ming Li, Lei Liu · 发表于：Journal of the American Chemical Society · 年份：2019 · DOI：10.1021/jacs.8b13213 · 被引用次数：106 · 研究领域：Ubiquitin and proteasome pathways、Protein Degradation and Inhibitors、Peptidase Inhibition and Analysis

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. Co...