A targeted proteomic analysis of the ubiquitin-like modifier Nedd8 and associated proteins
A targeted proteomic analysis of the ubiquitin-like modifier Nedd8 and associated proteins
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DOI：
10.1021/pr700749v
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发表时间：
2008-03-01
影响因子：
4.4
通讯作者：
Huang, Lan
中科院分区：
文献类型：
--
作者：
Jones, Jeffrey;Wu, Kenneth;Huang, Lan
Nedd8是一种小的泛素样蛋白，可以在一个被称为泛素化的过程中与底物蛋白结合。尽管类化修饰在细胞增殖和发育中起着关键的调节作用，但对Nedd8底物的光谱及其相互作用网络的了解仍然很少。为了在蛋白质组水平上探索类化修饰途径，我们从稳定表达GST-Nedd8的HEK293细胞中亲和纯化了Nedd8修饰及相关蛋白，并采用LC-MS/MS进行后续蛋白鉴定。共鉴定出496个GST-Nedd8修饰及相关蛋白，包括所有参与类化和泛素-蛋白酶体降解途径的cullin家族8个成员（即cull -1、-2、-3、-4A、-4B、-5、-7和Parc）。此外，一组参与转录、DNA修复和复制、细胞周期调节和染色质组织以及重塑的蛋白质已被共纯化和鉴定。除了蛋白鉴定外，还通过MS/MS分析确定了cullins的类化修饰位点，这与之前的诱变研究结果一致。此外，质谱分析显示Nedd8 K11、K22、K48和K60在体内可形成链，而Nedd8 K22和K48在体外可发生类木化修饰。这些结果提供了体外和体内多泛素化的第一个分子证据，表明泛素和泛素样蛋白的链形成可能是这些修饰的普遍现象。尽管这些观察结果的生物学意义仍有待探索，但这项工作提供了关于Nedd8链组装及其相互作用网络的重要信息。由此获得的大量蛋白质组学信息可以为Nedd8的生物学作用提供线索，为深入分析Nedd8通路的调控奠定基础。
Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate-proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC-MS/MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here can provide clues on the biological role of Nedd8 and lay the foundation for an in-depth analysis of the regulation of the Nedd8 pathway.