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Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP.
Communications Chemistry ( IF 5.9 ) Pub Date : 2019-09-19 , DOI: 10.1038/s42004-019-0211-7
Fabienne Burlina 1 , Abu-Baker M Abdel-Aal 2 , Richard Raz 2 , Irene Pinzuti 3 , George Papageorgiou 2 , Jiejin Li 2 , Robin Antrobus 4 , Stephen R Martin 2 , Simone Kunzelmann 2 , Benjamin Stieglitz 3 , John Offer 2
Affiliation  

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

中文翻译:

NEMO的辅助化学泛素化和HOIP的线性延伸。

NF-κB 必需调节剂 (NEMO) 的泛素化是细胞内免疫信号通路的一部分。单泛素化 NEMO 是探索 LUBAC(线性泛素链组装复合物)的 NEMO 线性泛素化机制所必需的,但无法通过生物技术获得。在这里,我们使用连接辅助剂对 NEMO 进行化学泛素化,这只需要两步合成,并且很容易安装到赖氨酸侧链上。化学连接直接发生在赖氨酸 ε 胺上,并在 pH 7 以下保持有效。我们表明,泛素化的 NEMO 与未修饰的 NEMO 对线性双泛素链具有相似的亲和力。化学合成的 NEMOCoZi-Ub 的近端泛素被认为是 HOIL-1 相互作用蛋白 (HOIP) 的 (RING-Between-RING) RBR 结构域线性延伸的底物。
更新日期:2019-09-19
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