Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.
Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.
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建立一个无小麦细胞的合成蛋白阵列，其中包含250个人和小鼠E3泛素连接酶，以鉴定E3连接酶与底物蛋白之间的新型相互作用。
DOI：
10.1371/journal.pone.0156718
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Sawasaki T
中科院分区：
文献类型：
--
作者：
Takahashi H;Uematsu A;Yamanaka S;Imamura M;Nakajima T;Doi K;Yasuoka S;Takahashi C;Takeda H;Sawasaki T
泛素化是真核生物众多生物学过程中的一种重要的翻译后修饰。泛素化的主要作用被认为是触发蛋白质降解和调节信号转导。在蛋白质泛素化过程中，底物特异性主要由E3泛素连接酶（E3）决定。尽管人类基因组中有600多个基因编码E3，但由于E3的多样性和细胞中泛素化蛋白的不稳定性，许多靶蛋白的E3仍然未知。我们在此展示了一种新的生物化学分析，用于鉴定靶向特定蛋白的E3。利用小麦无细胞蛋白质合成系统，合成了包含227个人和23个小鼠重组E3的蛋白质阵列。为了建立使用AlphaScreen技术的高通量结合试验，我们选择MDM2和p53作为E3与其靶蛋白的模型组合。AlphaScreen测定特异性地检测粗翻译混合物中p53和MDM2的结合。然后，使用E3蛋白阵列进行综合结合测定。11个E3显示出高结合活性，包括4个先前报道的E3（例如，MDM2、MDM4和WWP1）靶向p53。该结果证明了试验的可靠性。另一种相互作用物RNF6和DZIP3（尚未有与p53结合的报道）被发现在体外泛素化p53。进一步分析显示RNF6以E3活性依赖的方式降低H1299细胞中p53的含量。这些结果表明RNF6在细胞中泛素化和降解p53的可能性。本文建立的新型体外筛选系统是用于发现靶蛋白的新型E3的有力工具。
Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3—which there have been no report to bind p53—were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.