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Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
Published on: March 24, 2023
Executioner caspase-3 and caspase-7 are functionally distinct proteases
John G Walsh1, Sean P Cullen, Clare Sheridan
1Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Abstract:
Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.
Insights
Caspase-3 and caspase-7 are key apoptosis proteins. This study reveals they have distinct roles, with caspase-3 being more active and the primary executioner, challenging the idea of redundancy in cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Caspases are cysteine proteases crucial for apoptosis (programmed cell death).
- Caspase-3 and caspase-7 are considered major executioner caspases.
- Previous research suggested functional redundancy between caspase-3 and caspase-7 due to similar substrate activity.
Purpose of the Study:
- To investigate the functional differences between caspase-3 and caspase-7.
- To determine if these proteases have distinct roles in vivo despite similar in vitro activity.
- To provide a molecular basis for observed phenotypic differences in caspase-deficient mice.
Main Methods:
- Comparative analysis of caspase-3 and caspase-7 activity against natural substrates.
- In vivo studies using caspase-deficient mouse models.
- Substrate cleavage assays using proteins like Bid, XIAP, gelsolin, caspase-6, and p23.
Main Results:
- Caspase-3 and caspase-7 exhibit differential cleavage activity toward multiple natural substrates.
- Caspase-3 demonstrates broader substrate specificity (more promiscuous) compared to caspase-7.
- Caspase-3 is identified as the predominant executioner caspase during the demolition phase of apoptosis.
Conclusions:
- Caspase-3 and caspase-7 possess nonredundant roles in the cell death machinery.
- Differential substrate cleavage explains the distinct phenotypes observed in mice lacking either caspase-3 or caspase-7.
- The findings challenge the notion of functional redundancy and highlight specific roles for each executioner caspase.
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