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Updated: Aug 6, 2026
Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes
Alicia B Berger1, Martin D Witte, Jean-Bernard Denault
1Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, USA.
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.
Insights
Researchers developed new tools to monitor caspase activity during apoptosis. They discovered a novel intermediate form of caspase-7, crucial for understanding programmed cell death pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Caspases are critical cysteine proteases executing apoptotic cell death.
- Existing tools are insufficient for monitoring specific caspase regulation in distinct apoptotic pathways.
Purpose of the Study:
- To develop selective inhibitors and active site probes for monitoring executioner (caspase-3, -7) and initiator (caspase-8, -9) caspase activity.
- To dissect the kinetics of caspase activation in apoptosis using these novel reagents.
Main Methods:
- Development of highly selective caspase inhibitors.
- Design and application of active site probes for caspases.
- Kinetic analysis of caspase activation in cell-free extracts and intact cells.
Main Results:
- Successfully monitored executioner and initiator caspase activity.
- Identified a full-length caspase-7 intermediate activated early in apoptosis.
- Demonstrated that mature executioner caspases, not initiator caspases, mediate further processing of this intermediate.
- Observed distinct inhibitor sensitivity for the caspase-7 intermediate compared to processed forms.
Conclusions:
- The developed tools enable direct monitoring of caspase activity in apoptotic pathways.
- Caspase-7 activation involves a previously uncharacterized intermediate formed without initial zymogen cleavage.
- This intermediate's activation and processing kinetics offer new insights into apoptotic regulation.
