Cowling, Victoria Haigh; (2002) Regulation of caspase activation during programmed cell death. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Most triggers of programmed cell death or apoptosis promote cytochrome c release from the mitochondria into the cytosol which initiates the activation of a panel of pro-apoptotic caspases. Activation of caspase-8 in this pathway is not well defined but may be important because it activates Bid which promotes further cytochrome c release. A range of experimental approaches have suggested different mechanisms for caspase-8 activation in this pathway. In order to identify the proteases directly responsible for caspase-8 activation, caspase-8 cleaving activity was purified from cytochrome c activated cytosolic extracts. Caspase-6 was found to be the only soluble protease in cytochrome c activated cytosolic extracts that has significant caspase-8 cleaving activity. Furthermore, caspase-6 was sufficient to induce Bid-dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. The intrinsic activity of caspase-6 was found to be dependent on removal of the short prodomain. The requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis. The caspase activites which cleave the two cleavage sites of caspase-6 were purified from cytochrome c activated cell extracts. Caspase-3 and caspase- 7 were found to have different specificities for the caspase-6 cleavage sites, despite having the same preferred peptide substrates.

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