期刊
ANALYTICAL BIOCHEMISTRY
卷 350, 期 2, 页码 214-221出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.11.001
关键词
isopeptidases; protein degradation; N terminus; 3D polymerase
资金
- NIAID NIH HHS [R01 AI045818, R01 AI045818-08] Funding Source: Medline
- NIDDK NIH HHS [R43 DK071391, 1 R43 DK071391] Funding Source: Medline
- NINDS NIH HHS [R43 NS047948, 1R43 NS47948] Funding Source: Medline
The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as, plays an essential role in a number of biological schemes, and ubiquitin pathway small ubiquitin-like modifying protein (SUMO). enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases. (c) 2005 Elsevier Inc. All rights reserved.
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