期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：15
页码：4630-4635
DOI：10.1073/pnas.1422313112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe identification of specific ubiquitin ligase-substrate pairs is crucial for understanding the roles of protein ubiquitination in the regulation of diverse biological processes. Despite the development of various methodologies for substrate identification, it remains challenging to determine ubiquitin ligase substrates. Based on previously described tandem ubiquitin-binding entity(ies) (TUBE), we designed the trypsin-resistant (TR)-TUBE for expression in cells. The coexpression of TR-TUBE with an ubiquitin ligase stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and anti-Lys-{varepsilon}-Gly-Gly antibody, we successfully identified specific ubiquitin ligase-substrate pairs. Our methodology provides an effective means for the identification of ubiquitin ligase substrates and the detection of ubiquitin ligase activity. The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-{varepsilon}-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
关键词：ubiquitin-binding protein ; ubiquitin ligase ; ubiquitination
