Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains
Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains
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DOI：
10.1021/pr201167n
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Layfield, Robert
中科院分区：
文献类型：
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作者：
Strachan, Joanna;Roach, Lucy;Layfield, Robert
泛素通过其翻译后对其他蛋白质的共价修饰，对多种影响进行了广泛的研究。然而，最近也有人提出了非锚定(非底物连接)多泛素链的作用。在这里，我们描述了使用泛素结合域来亲和纯化内源未锚定的多泛素链及其随后的质谱学表征。利用泛素受体ZNF216的A20Znf结构域，我们从骨骼肌中分离出一个蛋白质，通过纳米LC-MS和LC-MS/MS的组合显示，它代表了一个未修饰和未锚定的K48连接的泛素二聚体。使用USP5/异肽酶-T的Znf UBP(BUZ)结构域选择性纯化未锚定的多泛素链，可以分离出K48和K11连接的泛素二聚体，并揭示了包含多达15个泛素部分的较长链，其中包括K48连接。A20锌F纯化的泛素二聚体的自上而下的NanoLC-MS/MS产生的诊断离子与K48连接的存在一致，首次说明了这种方法在内源性多泛素修饰中探测连接性的潜力。除了对内源性非锚定多泛素链的分子组成提供初步的蛋白质组学见解外，这项工作还代表了体内多泛素链长度的第一个定义。
The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.