DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells.
DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells.
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DOI：
10.1016/j.celrep.2013.10.008
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发表时间：
2013-11-14
期刊：
影响因子：
8.8
通讯作者：
Huang TT
中科院分区：
文献类型：
--
作者：
Békés M;Okamoto K;Crist SB;Jones MJ;Chapman JR;Brasher BB;Melandri FD;Ueberheide BM;Denchi EL;Huang TT
The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof of principle experiment, ectopic expression of the uncleavable ubiquitin stabilized mono-ubiquitinated PCNA in the absence of DNA damage, and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified previously unknown ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool to interrogate cell signaling pathways.
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DOI：
10.1074/jbc.m805871200
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期刊：
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影响因子：
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