Molecular characterisation of the pro-apoptotic molecules APAF1 and Smac
thesisposted on 15.12.2014, 10:41 by Darren Leslie. Roberts
Apoptosis is a key process involved in cell number regulation and a range of pathological disorders. It is a highly structured process that removes the cell from the body without damage to surrounding cells. The mitochondria has been shown to play a key role in apoptosis induced by a wide range of stimuli and can trigger the apoptotic pathway by release of apoptogenic factors. One of these factors is cytochrome c, a protein that normally functions in the electron transport chain but upon release it binds to the first mammalian homologue of CED-4, Apoptotic Protease Activating Factor 1 (APAF1) and induces the formation of a large caspase-activating complex. Another protein released from the mitochondria is Smac, a pro-apoptotic protein that functions by antagonising Inhibitor of Apoptosis Protein function. The data presented in this thesis demonstrates the existence of multiple splice forms of both APAF1 and Smac and investigated their roles in the apoptotic pathway. APAF1 forms the large molecular weight complex known as the apoptosome in 293 cells although epitope tagged APAF1 formed an incorrectly formed this complex in the absence of additional stimuli. APAF1 was also found to be cleaved by Caspase-3 during apoptosis and that the resulting 30 kDa fragment is associated with the inactive complex. Data also indicates that caspase-9 recruitment to the apoptosome occurs via interactions between both the CARD and the CED-4 homologous regions of APAF1. The pro-apoptotic protein Smac exists as several forms due to alternative slicing and one of these forms, Smac , is pro-apoptotic even though it lacks the N-terminal Inhibitor of Apoptosis protein binding domain. Smac is localised to the cell cortex and does not redistribute upon induction of apoptosis. These data demonstrate that Smac is able to induce apoptosis independently of Inhibitor of Apoptosis Protein binding. A polyclonal antibody to Smac / Smac was also developed and characterised.