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Calpain activation in apoptosis
M K Squìer1, A C Miller, A M Malkinson
1Department of Immunology, University of Colorado Health Sciences Center, Denver 80262.
Abstract:
Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both "induction" and "release" models of apoptotic cell death.
Insights
Calpain, a calcium-dependent protease, plays a crucial role in programmed cell death (apoptosis). Inhibiting calpain effectively blocks apoptosis in various cell types, suggesting its essential function in this cell death process.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Programmed cell death (apoptosis) involves DNA fragmentation and cell disintegration.
- Common apoptotic themes include increased cytoplasmic calcium, cytoskeletal changes, and membrane lipid redistribution.
- Calpain, a calcium-dependent protease, is implicated in cytoskeletal and membrane dynamics.
Purpose of the Study:
- To investigate the role of calpain in apoptotic cell death.
- To examine calpain activation in thymocytes treated with dexamethasone.
Main Methods:
- Assays for calcium-dependent proteolysis in thymocyte extracts.
- Western blotting to detect calpain proenzyme cleavage.
- Inhibition of apoptosis using specific calpain inhibitors.
Main Results:
- Calpain activity increased significantly in thymocytes after dexamethasone treatment, peaking at 1 hour.
- Autolytic cleavage of the calpain I precursor was observed, indicating activation.
- Calpain inhibitors blocked apoptosis induced by dexamethasone, irradiation, or cycloheximide.
Conclusions:
- Calpain activation is a key event in dexamethasone-induced thymocyte apoptosis.
- Calpain plays a required role in both the induction and execution phases of apoptosis.
- Calpain inhibition represents a potential strategy to modulate apoptotic cell death.