摘要
Ubiquitin(Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub(Ub_3^(11/48)(S-S)), the first example of an isopeptide mimic for the branched Ub chains,which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the El-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11 C, K48 C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub_3^(11/48)(S-S). Deubiquitination and pulldown assays indicated that the synthetic Ub_3^(11/48)(S-S) could be hydrolyzed by linkage-specific deubiquitinases(K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5 a.
Ubiquitin（Ub） chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub（Ub3^11/48（S-S））, the first example of an isopeptide mimic for the branched Ub chains,which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the El-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub（K11 C, K48 C）. The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub3^11/48（S-S）. Deubiquitination and pulldown assays indicated that the synthetic Ub3^11/48（S-S） could be hydrolyzed by linkage-specific deubiquitinases（K11-specific Cezanne and K48-specific OTUB1）, and recognized by proteasomal ubiquitin receptor S5 a.
基金
supported by the National Key R&D Program of China(2017YFA0505200)
the National Natural Science Foundation of China(91753205,21532004,21761142008)
the Program of Introducing Talents of Discipline to Universities of China(B16028)
关键词
二硫化物
半合成
模仿
连接
分叉
结构验证
生物化学
生物物理
ubiquitin chain isopeptide bond mimics, disulfide-directed strategy, branched Ub chains, crystal structure
