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Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal
D A Martin1, R M Siegel, L Zheng
1Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.
Abstract:
Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, we find that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction was abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but was surprisingly unaffected by death inhibitor Bcl-2. Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely blocked apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm.
Insights
Oligomerization at the cell membrane triggers caspase-8 autoactivation and apoptosis. However, apoptosis may involve a death signal from the enzyme
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is essential for multicellular organisms.
- Caspase activation is a key step in many apoptosis pathways, including those initiated by death receptors like CD95/Fas/APO-1.
- The precise mechanisms regulating caspase activation remain incompletely understood.
Purpose of the Study:
- To investigate the requirements for activating caspase-8, a crucial protease in apoptosis.
- To determine if membrane localization and oligomerization are sufficient for caspase-8 autoactivation and subsequent apoptosis.
Main Methods:
- Utilized molecular chimeras, fusing caspase-8 with CD8 or Tac domains.
- Employed enzyme inhibitors (z-VAD-fmk, z-IETD-fmk, p35) and site-directed mutagenesis to probe caspase activity.
- Investigated the role of Bcl-2 and mutations affecting caspase release from the membrane.
Main Results:
- Membrane-associated oligomerization of caspase-8 potently induced autoactivation and apoptosis.
- Apoptosis was blocked by caspase inhibitors and active site mutations, but not by Bcl-2.
- Mutations preventing proteolytic release of caspase-8 from its membrane anchor abolished apoptosis.
Conclusions:
- Oligomerization at the cell membrane is sufficient to trigger caspase-8 autoactivation.
- Apoptosis induction may involve a death signal transmitted by the proteolytic release of active caspase-8 into the cytoplasm.