Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase
Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase
复制标题
识别 LNX（一种包含 PDZ 结构域的 E3 泛素连接酶）底物的蛋白质组学策略
DOI：
10.1021/pr300674c
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发表时间：
2012-10-01
影响因子：
4.4
通讯作者：
Gao, Youhe
中科院分区：
文献类型：
--
作者：
Guo, Zhengguang;Song, Eli;Gao, Youhe
泛素连接酶（E3）通过识别靶底物赋予泛素化特异性。然而，大多数E3的底物尚未被广泛发现，需要新的方法来有效和全面地鉴定这些底物。大多数情况下，E3通过其蛋白质相互作用结构域特异性识别底物。我们开发了一种新的综合策略，以确定底物的E3蛋白质相互作用结构域的蛋白质组学规模。通过酵母双杂交系统筛选随机肽库，对蛋白质相互作用结构域的结合特性进行了表征。人工降解决定子，由一个优先的泛素化序列和特定的相互作用结构域结合基序组成，通过体外泛素化测定作为潜在的底物进行了测试。使用这种策略，不仅基板，但也非基板监管机构可以发现。详细的底物识别机制，这是有用的药物发现，也可以表征。我们使用了Numb蛋白X（LNX）家族的E3，一组PDZ结构域含有RING型E3泛素连接酶的配体，以证明这种策略的可行性。确定了LNX E3的许多潜在基板。其中8个在体外被泛素化，2个新的内源性底物，PDZ结合激酶（PBK）和断点簇区蛋白（BCR），在体内得到证实。我们进一步揭示了LNX 1介导的PI 3 K的泛素化和降解抑制细胞增殖并增强对阿霉素诱导的凋亡的敏感性。还对LNX E3的底物识别机制进行了表征;该过程涉及通过其特异性PDZ结构域与靶蛋白的C末端结合来识别底物。这种策略可以潜在地扩展到包含蛋白质相互作用结构域的各种E3，从而作为在蛋白质组学规模上全面鉴定其底物的有力工具。
Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.