Specificity of aza-peptide electrophile activity-based probes of caspases.
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.
Sexton
K B
KB
Department of Pathology, Stanford University School of Medicine, Stanford, CA 940305, USA.
Kato
D
D
Berger
A B
AB
Fonovic
M
M
Verhelst
S H L
SH
Bogyo
M
M
D001372:Aza Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D020169:Caspases / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004789:Enzyme Activation; D006801:Humans; D007202:Indicators and Reagents; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D013379:Substrate Specificity
D001372:Aza Compounds; D007202:Indicators and Reagents; D015335:Molecular Probes; D020169:Caspases; D003546:Cysteine Endopeptidases