DUB-Resistant Ubiquitin to Survey Ubiquitination Switches in Mammalian Cells

被引：36

作者：

Bekes, Miklos ^{[1
]}

Okamoto, Keiji ^{[2
]}

Crist, Sarah B. ^{[1
]}

Jones, Mathew J. ^{[1
]}

Chapman, Jessica R. ^{[3
]}

Brasher, Bradley B. ^{[4
]}

Melandri, Francesco D. ^{[4
]}

Ueberheide, Beatrix M. ^{[1
,3
]}

Denchi, Eros Lazzerini ^{[2
]}

Huang, Tony T. ^{[1
]}

机构：

[1] NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA

[2] Scripps Res Inst, Dept Mol & Expt Med, La Jolla, CA 92037 USA

[3] NYU, Sch Med, Prote Resource Ctr, New York, NY 10016 USA

[4] Boston Biochem, Cambridge, MA 02139 USA

来源：

CELL REPORTS | 2013年 / 5卷 / 03期

关键词：

DNA-DAMAGE RESPONSE; UNANCHORED POLYUBIQUITIN CHAINS; MONOUBIQUITINATED PCNA; POLYMERASE SWITCH; MECHANISM; COMPLEX; ACTIVATION; PROTEINS; REVEALS; SUMO;

D O I：

10.1016/j.celrep.2013.10.008

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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 monoubiquitinated 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 ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.

引用

页码：826 / 838

页数：13

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