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Title: Characterisation of the ~700kDa apoptosome complex from THP.1 cells
Author: Langlais, Claudia
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2002
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Apoptosis or programmed cell death is a process involving the regulated self-destruction of a cell. Execution of the apoptotic programme requires the hierarchical activation of a family of proteases, the caspases. Here, I describe the characterisation and purification of the apoptosome, a ~700kDa caspase activating complex. Gel filtration of dATP-activated THP.1 cell lysates showed that effector caspase activity is assessed with a large complex, the apoptosome, which contains Apaf-1 and active caspases-9, -3 and -7. Further analysis revealed two large complexes, a biologically inactive ~1.4MDa and a biologically active ~700kDa complex to be formed very rapidly. Experiments involving the use of the caspase inhibitor z-VAD.FMK demonstrated that complex formation is caspase-independent, however, release of processed caspase-9 from the complex and the recruitment of other procaspases to the complex, is caspase activity-dependent. In apoptotic cells, the ~700kDa apoptosome complex predominates and is therefore suggested to be the biologically relevant complex. Normal intracellular K+ concentrations inhibited formation of the ~700kDa apoptosome complex in lysates and in a reconstituted system. Increasing concentrations of cytochrome c partially reversed inhibition. A mechanism is proposed for the inhibitory effect of K+ during dATP-dependent caspase activation in cellular lysates. The ~700kDa apoptosome complex was purified using multi-step purification procedures. Several proteins were identified in the purified apoptosome fraction and these included Apaf-1 and capase-9 as well as XIAP, b-actin, CAP-1, CAZ1/CAZ2 and possibly Hsc70 are all involved with the cytoskeleton and are capable of binding actin, whereas rabaptin-5 and IKKg (NEMO) are known to be involved in endosomal transport and the NFkB pathway, respectively. The possible involvement of these proteins in apoptosome assembly and function is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available