期刊
CELL DEATH AND DIFFERENTIATION
卷 14, 期 4, 页码 727-732出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/sj.cdd.4402074
关键词
activity based probes; caspases; aza-peptides; cysteine protease; active site labels; protease inhibitors
资金
- NATIONAL CENTER FOR RESEARCH RESOURCES [U54RR020843] Funding Source: NIH RePORTER
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [T32HG000044] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING [R01EB005011] Funding Source: NIH RePORTER
- NCRR NIH HHS [U54 RR020843-01] Funding Source: Medline
- NHGRI NIH HHS [5T32 HG00044] Funding Source: Medline
- NIBIB NIH HHS [R01 EB005011] Funding Source: Medline
Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.
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