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Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis
P G Villa1, W J Henzel, M Sensenbrenner
1INSERM U.382, Developmental Biology Institute of Marseille, Campus de Luminy, Marseille, France.
Abstract:
Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.
Insights
Programmed cell death involves specific proteases. This study identifies actin fragments as key substrates, suggesting calpain-like proteases play a crucial role in neuronal apoptosis and actin fragmentation.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Apoptosis (programmed cell death) is a regulated process involving proteases.
- Key substrates for these proteases in apoptosis remain largely unidentified.
- Previous work identified five unknown polypeptides increased in apoptotic neurons.
Purpose of the Study:
- To identify the previously observed polypeptides in apoptotic neurons.
- To elucidate the role of actin fragmentation in programmed cell death.
- To determine the specific proteases involved in neuronal apoptosis.
Main Methods:
- Microsequencing of polypeptides from apoptotic neurons.
- Analysis of actin fragmentation in various cell types undergoing apoptosis.
- Inhibition studies using calpain and caspase inhibitors.
Main Results:
- Two identified polypeptides were fragments of actin, including cleavage at the DNase I interaction site.
- Actin fragments were observed in multiple neuronal types and thymocytes during apoptosis.
- Calpain inhibitors blocked neuronal death and actin fragmentation, while caspase inhibitors did not prevent actin cleavage.
Conclusions:
- Actin fragmentation is a significant event in programmed cell death.
- Calpain-like proteases, not caspases, are key mediators of actin cleavage in neuronal apoptosis.
- Actin fragmentation is correlated with subsequent DNA fragmentation during apoptosis.