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Related Experiment Videos

Requirements for proteolysis during apoptosis

D L Vaux1, S Wilhelm, G Häcker

  • 1The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.

Molecular and Cellular Biology
|October 29, 1997
PubMed
Summary
This summary is machine-generated.

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Caspase activation during apoptosis triggers another enzyme, leading to DNA degradation and cell death. This newly identified enzyme, inhibited by N-ethylmaleimide, is crucial for apoptosis execution.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Apoptosis is a crucial process for development and tissue homeostasis.
  • Caspases are key effector proteases initiating apoptosis.
  • The precise downstream events following caspase activation remain under investigation.

Purpose of the Study:

  • To investigate the biochemical events following caspase activation in vitro.
  • To identify novel enzymes involved in the execution of apoptosis.
  • To elucidate the mechanism of DNA degradation during programmed cell death.

Main Methods:

  • In vitro activation of cytoplasmic extracts using proteinase K.
  • Assay of caspase-like activity using a fluorogenic substrate (DEVD-7-amino-4-methylcoumarin).

Related Experiment Videos

  • Inhibition studies using specific caspase and protease inhibitors (DEVD-aldehyde, iodoacetamide, N-ethylmaleimide).
  • Assessment of DNA degradation and morphological changes in isolated nuclei.

    • Main Results:

    • Activated cytoplasmic extracts cleaved the caspase substrate, indicating caspase-like activity.
    • This activity was inhibited by DEVD-aldehyde and iodoacetamide, but not N-ethylmaleimide.
    • Activated extracts induced internucleosomal DNA degradation and apoptotic morphology in isolated nuclei.
    • DNA cleavage and morphological changes were inhibited by N-ethylmaleimide, but not iodoacetamide.

    Conclusions:

    • Caspase activation during apoptosis leads to the activation of a distinct N-ethylmaleimide-sensitive cytoplasmic enzyme.
    • This newly identified enzyme is essential for the activation of endonucleases, DNA fragmentation, and nuclear morphological changes characteristic of apoptosis.