Requirements for proteolysis during apoptosis.
The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.
Vaux
D L
DL
The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Wilhelm
S
S
Häcker
G
G
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000473:pathology; D002474:Cell-Free System; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology*; D004720:Endonucleases / Q000378:metabolism*; D019286:Endopeptidase K / Q000494:pharmacology; D004789:Enzyme Activation; D051379:Mice; D008954:Models, Biological; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured
D003374:Coumarins; D009842:Oligopeptides; D011480:Protease Inhibitors; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; D004720:Endonucleases; D019286:Endopeptidase K; D003546:Cysteine Endopeptidases