title: Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes.
abstract: Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, USA.
authors.forename: Alicia B
authors.initials: AB
authors.lastname: Berger
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.forename: Jean-Bernard
authors.initials: JB
authors.lastname: Denault
authors.forename: Amir Masoud
authors.initials: AM
authors.lastname: Sadaghiani
authors.forename: Kelly M B
authors.initials: KM
authors.lastname: Sexton
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002457:Cell Extracts; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry / Q000494:pharmacology*; D020543:Proteome; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D061945:Caspase Inhibitors; D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D015335:Molecular Probes; D020543:Proteome; D011994:Recombinant Proteins; D020169:Caspases