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Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe
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In Vitro Assembly and Analysis of the Apoptosome Complex
Claudia Langlais1, Michelle A Hughes1, Kelvin Cain1
1MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
Cold Spring Harbor Protocols
|December 4, 2015
Summary
This study presents a cell-free system to investigate caspase activation and apoptosome assembly. Researchers can now monitor caspase-3 and -7 activity and confirm apoptosome formation using this novel in vitro model.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Caspase activation and apoptosome assembly are critical in apoptosis.
- Studying these processes in vitro requires robust and reproducible models.
- Existing methods may lack the resolution to study native complex formation and activity.
Purpose of the Study:
- To establish a cell-free in vitro model for studying caspase activation.
- To characterize the assembly mechanism of the native apoptosome complex.
- To provide a method for assessing caspase activity and apoptosome functionality.
Main Methods:
- Utilizing dATP-activated cell-free lysates for caspase activation.
- Employing fluorimetry with a tetrapeptide substrate (DEVD-AFC) for real-time caspase-3 and -7 activity detection.
- Using gel filtration to isolate the apoptosome complex and western blotting to confirm its assembly and activity.
Main Results:
- Demonstrated a method for real-time monitoring of caspase-3 and -7 (DEVDase) activity in cell-free extracts.
- Successfully isolated and confirmed the assembly of the native apoptosome complex (Apaf-1 and caspase-9).
- Validated apoptosome complex functionality by assessing its ability to process exogenous procaspase-3 and -7.
Conclusions:
- The described in vitro model enables detailed mechanistic studies of caspase activation and native apoptosome assembly.
- This protocol provides a valuable tool for researchers investigating the intrinsic apoptosis pathway.
- The method allows for the characterization of both complex formation and enzymatic activity in a controlled system.
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