Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.
Biochemistry. 2020 Oct 13;59(40):3796-3801. doi: 10.1021/acs.biochem.0c00701. Epub 2020 Oct 2.
Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.
蛋白质的泛素化和 SUMO 化对于各种生物反应至关重要。最近 SUMO 和泛素 (Ub) 之间的串扰被揭示，这迫切需要开发 Ub 标记 SUMO2 二聚体的合成平台，以破译其生物学功能。然而，在天然条件下方便地合成二聚体的平台仍未得到充分探索，仍然是主要的挑战。在这里，我们开发了一个平台，可以在天然条件下快速合成所有 8 种 Ub 标记 SUMO2 和 SUMO 化蛋白。扩展遗传密码 (EGC) 方法被用于在赖氨酸位置掺入 Se-烷基硒代半胱氨酸。氧化硒氧化物消除产生亲电中心，脱羟丙氨酸，它与 C 末端修饰的泛素的迈克尔加成，生成 Ub 标记 SUMO2。这些二聚体进一步用涉及 DNA 修复的 SUMO2 去泛素化酶 USP7 进行了检测，以了解对 Ub 标记 SUMO2 二聚体的特异性。我们的结果表明，USP7 的 C 末端结构域对于 USP7 的效率和对 Ub 标记 SUMO2 二聚体的选择性至关重要。
