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The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis
R Benetti1, G Del Sal, M Monte
1Laboratorio Nazionale CIB, Area Science Park, Padriciano 99, 34012 Trieste, Italy.
Abstract:
Gas2 is a caspase-3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis. Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity. To investigate possible pathways linking Gas2 to p53, a yeast two-hybrid screen swas performed, indicating m-calpain as a strong Gas2- interacting protein. Moreover, we demonstrate that Gas2 physically interacts with m-calpain in vivo and that recombinant Gas2 inhibits calpain-dependent processing of p53. Importantly, the Gas2 dominant-negative form (Gas2171-314) that binds calpain but is unable to inhibit its activity abrogates Gas2's ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53-dependent apoptosis. Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity.
Insights
Gas2 protein enhances apoptosis by stabilizing p53, a tumor suppressor. This occurs through Gas2
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Gas2 is a caspase-3 substrate involved in microfilament regulation and cell shape changes during apoptosis.
- The tumor suppressor p53 plays a critical role in cellular responses to DNA damage.
Purpose of the Study:
- To investigate the role of Gas2 in apoptosis and its connection to p53.
- To elucidate the molecular mechanisms by which Gas2 influences p53 stability and activity.
Main Methods:
- Yeast two-hybrid screening to identify Gas2-interacting proteins.
- In vivo co-immunoprecipitation to confirm Gas2-m-calpain interaction.
- In vitro assays using recombinant Gas2 to assess calpain activity.
- Apoptosis assays following various stress treatments (UV, etoposide, MMS).
- Analysis of p53 stability and transcriptional activity.
Main Results:
- Overexpression of Gas2 increases susceptibility to apoptosis induced by UV, etoposide, and methyl methanesulfonate.
- Gas2 enhances p53 stability and transcriptional activity.
- Gas2 physically interacts with m-calpain, and recombinant Gas2 inhibits calpain-dependent p53 processing.
- A dominant-negative form of Gas2 abrogates its ability to stabilize p53 and induce apoptosis.
- Gas2 regulates p53 levels independently of Mdm2, suggesting calpain inhibition as a mechanism.
Conclusions:
- Gas2 enhances p53 stability and transcriptional activity, thereby increasing apoptosis susceptibility.
- Gas2 interacts with and inhibits m-calpain, a key enzyme in p53 processing.
- Gas2 acts as a novel regulator of p53 stability and function, independent of the Mdm2 pathway.