Characterization of a novel cell death-inducing protein complex assembled in response to stress

Abstract

Pro-caspase-8 is the apical caspase in the extrinsic apoptosis which is initiated by the external stimulation of certain death receptors. Pro-caspase-8 is synthesised as an inactive zymogen, which must be cleaved to reach its full proteolytic activity. The canonical mechanism for pro-caspase-8 activation during extrinsic apoptosis involves its recruitment to Death Inducing Signalling Complex (DISC) where it can oligomerize and undergo autoactivation. Active caspase-8 is subsequently freed from DISC into cytosol where it initiates a caspase cascade leading to cell death. This thesis describes a novel pro-caspase-8 containing protein complex reported to form in cells devoid of the mitochondrial apoptosis pathway in response to prolonged endoplasmic reticulum (ER) stress. Throughout this thesis I refer to this protein complex as the stressosome. The stressosome consists of pro-caspase-8, the adaptor protein FADD and ATG5. Both pro-caspase-8 and FADD are well described mediators of cell death, however; they are usually associated with the external death receptor-mediated stress signalling rather than internal cytotoxic stimulation. ATG5 is an essential component of the autophagy machinery which is a highly pro-survival lysosome-dependent cellular degradation program regulating cellular homeostasis. The presence of ATG5 within the stressosome indicates that autophagy machinery may also participate in the execution of cell death through facilitation of pro-caspase-8 activation. This thesis investigates the stressosome formation and its regulation under different cellular stresses including ER stress (tunicamycin, brefeldin A), genotoxic stress (etoposide and γ-irradiation), heat shock and cytoskeletal disruption (taxol) in cell deficient in the mitochondrial apoptosis pathway through procaspase-9 knockdown. It also addresses the role of the stressosome in mediating cell death in procaspase-9 deficient cells by genetic manipulation of the stressosome components. Furthermore it investigates the stressosome composition by mass spectrometry analysis of the co-immunoprecipitated sample.