The mechanism of poly-NEDD8 chain formation in vitro
The mechanism of poly-NEDD8 chain formation in vitro
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DOI：
10.1016/j.bbrc.2009.02.090
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发表时间：
2009-04-10
影响因子：
3.1
通讯作者：
Chiba, Tomoki
中科院分区：
文献类型：
--
作者：
Ohki, Yu;Funatsu, Norikazu;Chiba, Tomoki
NEDD 8是一种小的泛素样蛋白，其在类似于泛素化的反应中修饰靶蛋白。在该反应中，需要三种酶并且这三种酶是足够的：NEDD 8激活El样酶（APP-BP 1/Uba 3）、NEDD 8。特异性E2酶（Ubc 12）和环指蛋白ROC 1（NEDD 8 E3连接酶）。与已知形成聚泛素链的泛素不同，对聚NEDD 8链的形成知之甚少。在这里，我们显示了使用完整的体外重建NEDD 8缀合系统在Cullin-1上形成聚NEDD 8链的机制。有趣的是，聚-NEDD 8链是由油催化Ubc 12的半胱氨酸残基组成的。此外，Ubc 12形成poly-NEDD 8链而没有ROC 1的活性。相反，ROC 1突变体，缺陷的泛素连接酶活性，显着增强聚NEDD 8链的形成。反过来，ROC 1对于多聚NEDD 8链从Ubc 12转移到Cul-1是必不可少的。这些结果表明ROC 1对聚NEDD 8链形成的重要调节作用，（C）2009 Elsevier Inc. All rights reserved.
NEDD8 is a small ubiquitin-like protein that modifies target proteins in a reaction similar to Ubiquitination. In this reaction, three enzymes are required and sufficient: NEDD8 activating El-like enzyme (APP-BP1/Uba3), NEDD8. specific E2 enzyme (Ubc 12) and RING-finger protein ROC1 (NEDD8 E3 ligase). Unlike ubiquitin, which is well known to form poly-ubiquitin chain, little is known about the formation of poly-NEDD8 chain. Here we show the mechanism of poly-NEDD8 chain formation on Cullin-1 using a complete in vitro reconstituted NEDD8 Conjugation system. Intriguingly, poly-NEDD8 chain was build up oil catalytic Cysteine residue of Ubc12. Furthermore, Ubc12 formed poly-NEDD8 chain without the activity of ROC1. Rather ROC1 mutant, defective for ubiquitin ligase activity, dramatically enhanced the poly-NEDD8 chain formation. In turn, ROC1 was essential for the transfer of poly-NEDD8 chain from Ubc 12 to Cul-1. These results Suggest the important regulatory role of ROC1 for poly-NEDD8 chain formation, (C) 2009 Elsevier Inc. All rights reserved.