Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology.
The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.
Wilhelm
S
S
Institut für medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Germany.
Häcker
G
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002860:Chromium Radioisotopes / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019286:Endopeptidase K / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D013997:Time Factors; D020298:U937 Cells
D000590:Amino Acid Chloromethyl Ketones; D002860:Chromium Radioisotopes; D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019286:Endopeptidase K; D020169:Caspases