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A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases

Xiao, Junpeng and Broz, Petr and Puri, Aaron W. and Deu, Edgar and Morell, Montse and Monack, Denise M. and Bogyo, Matthew. (2013) A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases. Journal of the American Chemical Society, Vol. 135, H. 24. pp. 9130-9138.

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Official URL: http://edoc.unibas.ch/dok/A6164931

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Abstract

Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Infection Biology (Broz)
UniBasel Contributors:Broz, Petr
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:American Chemical Society
ISSN:0002-7863
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:13 Sep 2013 07:59
Deposited On:13 Sep 2013 07:52

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