Department of Antibody Engineering, Genentech, Inc., South San Francisco, CA, USA.
Departments of Molecular Oncology and Early Discovery Biochemistry, Genentech, Inc., South San Francisco, CA, USA.
Nat Commun. 2021 Jul 29;12(1):4608. doi: 10.1038/s41467-021-24669-6.
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.
泛素连接酶 UBE2W 催化蛋白质 N 末端的非典型泛素化，但其底物谱仍不清楚。为了鉴定内源性 N 末端泛素化的底物，我们发现了四种单克隆抗体，它们选择性地识别具有 N 末端二甘氨酸残基的胰蛋白酶肽，对应于 N 末端泛素化的位点。重要的是，这些抗体不识别赖氨酸上的异肽键连接的二甘氨酸（泛素）修饰。我们解决了其中一种抗体与 Gly-Gly-Met 肽结合的结构，揭示了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，在 UBE2W 的内源性底物上绘制 N 末端泛素化位点。这些底物包括 UCHL1 和 UCHL5，其中 N 末端泛素化明显改变了去泛素化酶（DUB）活性。这项工作描述了一种用于富集和全局分析内源性 N 末端泛素化位点的抗体工具包，同时揭示了 UBE2W 的功能相关底物。
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