Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)
Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)
复制标题
通过工程串联混合泛素结合域 (ThUBD) 增强泛素化蛋白的纯化
DOI：
10.1074/mcp.o115.051839
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发表时间：
2016-04-01
影响因子：
7
通讯作者：
Xu, Ping
中科院分区：
文献类型：
--
作者：
Gao, Yuan;Li, Yanchang;Xu, Ping
泛素化是最常见的翻译后修饰之一，调节蛋白质的稳定性和功能。然而，由于泛素化蛋白质在细胞中的丰度较低，蛋白质组范围的图谱仍然具有挑战性。在这项研究中，我们系统地评估了泛素结合域(UBD)对不同类型泛素链的亲和力。通过选择高亲和力的UBD，并对不同长度和类型的UBD组合进行评价，我们构建了两个人工串联杂交UBD(ThUBD)，其中包括4个由DSK2p衍生的泛素相关(UBA)和泛素2衍生的UBA(ThUDQ2)组成的UBD，以及由DSK2p衍生的UBA和RABGEF1衍生的A20-ZnF(ThUDA20)组成的UBD。ThUBD与泛素化的蛋白质结合，亲和力明显高于自然产生的UBD。此外，它对所有七条赖氨酸连接链都表现出几乎无偏见的高亲和力。利用ThUBD结合质谱仪，我们从酵母和哺乳动物细胞中分别鉴定了1092和7487个可能的泛素化蛋白质，其中362和1125个蛋白质具有泛素修饰的位点。这些结果表明，ThUBD是一种精炼和有前途的方法，可以丰富泛素化蛋白质组，同时避免过度表达标记泛素变体和使用抗体识别泛素残基，从而为蛋白质泛素化研究社区提供了一个容易获得的工具。
Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.