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Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W
Nature Communications ( IF 14.7 ) Pub Date : 2021-07-29 , DOI: 10.1038/s41467-021-24669-6
Christopher W Davies 1 , Simon E Vidal 2 , Lilian Phu 3 , Jawahar Sudhamsu 4 , Trent B Hinkle 3 , Scott Chan Rosenberg 2 , Frances-Rose Schumacher 3 , Yi Jimmy Zeng 3 , Carsten Schwerdtfeger 5 , Andrew S Peterson 6 , Jennie R Lill 3 , Christopher M Rose 3 , Andrey S Shaw 7 , Ingrid E Wertz 2, 8 , Donald S Kirkpatrick 3, 9 , James T Koerber 1
Affiliation  

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.



中文翻译:

抗体工具包揭示了 UBE2W 的 N 端泛素化底物

泛素结合酶 UBE2W 催化蛋白质 N 端的非规范泛素化,尽管其底物谱仍不清楚。为了鉴定内源性 N 端泛素化底物,我们发现了四种单克隆抗体,它们选择性地识别带有 N 端双甘氨酸残基的胰蛋白酶肽,对应于 N 端泛素化位点。重要的是,这些抗体不能识别赖氨酸上与异肽连接的双甘氨酸(泛素)修饰。我们解析了一种与 Gly-Gly-Met 肽结合的此类抗体的结构,以揭示其选择性识别的分子基础。我们将这些抗体与质谱蛋白质组学结合使用,以在 UBE2W 的内源性底物上绘制 N 端泛素化位点。这些底物包括 UCHL1 和 UCHL5,其中 N 端泛素化明显改变了去泛素化酶 (DUB) 的活性。这项工作描述了一种抗体工具包,用于富集和全局分析内源性 N 端泛素化位点,同时揭示 UBE2W 的功能相关底物。

更新日期:2021-07-29
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