The apoptosome is a caspase-activating complex consisting of Apaf-1, caspase-9 and cytochrome c, which is essential for the induction of stress-mediated apoptosis. This complex ranges in size from ~ 700 kDa to ~ 1.4 MDa, possibly due to the stable association of modulatory proteins. In the current study I employed two strategies to isolate and characterise the active ~ 700 kDa apoptosome in vitro. Firstly, I used GST-Casp91-130, which binds to the CARD domain of Apaf-1 in a dATP and cytochrome c-dependent manner, to affinity-purify an apoptosome containing only Apaf-1XL and cytochrome c. This result was confirmed by the second approach, which used an antibody to the caspase-9 to immunoprecipitate of the native apoptosome, which contained Apaf-1 and caspase-9 (p34/p35). However, in the absence of SMAC and Omi, the native apoptosome also contained caspase-3 and XIAP, both of which associated via the catalytic domains of caspase-9. When isolating the apoptosome from apoptotic cells using TAP-tagged caspase-9 variants, I discovered that the location of the TAP-tag can affect the ability of caspase-9 to interact with known binding partners and consequently can influence the induction of cell death. I have also studied the role of the apoptosome in the caspase-3 null cell line, MCF-7, and have demonstrated that an active ~ 700 kDa apoptosome complex is formed in both dATP-activated cell lysates and an apoptotic MCF-7 cells. Furthermore, the active apoptosome can directly process and activate caspase-7.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:697318 |
| Date | January 2005 |
| Creators | Twiddy, Davina Deborah |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/30776 |
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