Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system

Ruth M. Kluck, Seamus J. Martin, Brian M. Hoffman, Jian S. Zhou, Douglas R. Green, Donald D. Newmeyer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

356 Scopus citations

Abstract

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redoxinactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.

Original languageEnglish (US)
Pages (from-to)4639-4649
Number of pages11
JournalEMBO Journal
Volume16
Issue number15
DOIs
StatePublished - Aug 1 1997

Keywords

  • Apoptosis
  • Caspases
  • Cytochrome c
  • Mitochondrial
  • Xenopus

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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