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Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis
E Swanton1, N Bishop, P Woodman
1School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
The Journal of Biological Chemistry
|December 23, 1999
Summary
Caspase-3 selectively cleaves human rabaptin-5 during apoptosis, generating domains that bind rabex-5. This conserved cleavage impacts endosomal membrane fusion.
Area of Science:
- Cell biology
- Molecular biology
- Apoptosis research
Background:
- Xenopus rabaptin-5 cleavage in apoptotic extracts reduces endosomal membrane fusion.
- Rabaptin-5 plays a role in endosomal trafficking and membrane fusion.
Purpose of the Study:
- To investigate the conservation of rabaptin-5 cleavage in human apoptotic cells.
- To identify the specific caspase responsible for rabaptin-5 cleavage.
- To determine the functional consequences of rabaptin-5 cleavage.
Main Methods:
- Analysis of human rabaptin-5 cleavage in apoptotic HeLa cell extracts.
- Use of caspase-3 inhibitors (Ac-DEVD-CHO) and immunodepletion.
- Cleavage assays with recombinant caspase-3, caspase-2, and caspase-7.
- Characterization of cleaved rabaptin-5 domains and their binding properties.
Main Results:
- Human rabaptin-5 is cleaved at two specific sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts.
- Caspase-3 is the primary enzyme responsible for rabaptin-5 cleavage; caspases-2 and -7 do not cleave rabaptin-5.
- Cleavage by caspase-3 generates two distinct domains, with the C-terminal domain retaining binding to rabex-5.
Conclusions:
- Caspase-dependent cleavage of rabaptin-5 is a conserved event in apoptosis.
- Caspase-3 selectively targets rabaptin-5, impacting its function in endosomal trafficking.
- The C-terminal domain of cleaved rabaptin-5 maintains interaction with rabex-5, suggesting a role in regulating Rab5-mediated endocytosis during apoptosis.