The effect of the X-linked Inhibitor-of-Apoptosis Protein on the release of Smac from the mitochondria during Apoptosis

XIAP is a potent inhibitor of caspases-3, -7, and -9, and mitochondrial Smac release during apoptosis inhibits the activity of XIAP. The work in this thesis demonstrates that cytosolic XIAP also feeds back to mitochondria to impair Smac release. We constructed a fluorescent XIAP-fusion protein by labelling NH2- and COOH-termini with cerulean fluorescent protein (C-XIAP-C). Immunoprecipitation confirmed that C-XIAP-C retained the ability to interact with Smac and impaired extrinsically and intrinsically activated apoptosis in response to TRAILICHX and staurosporine. In C-XIAP-C expressing cells,cytochrome-c release from mitochondria proceeded normally whereas Smac release was significantly prolonged and incomplete. Also physiological expression of native XIAP prolonged or limited Smac release in HCT-116 colon cancer cells and primary mouse cortical neurons. The Smac-binding capacity of XIAP, but not caspase inhibition was central for mitochondria1 Smac retention, as evidenced in experiments employing XIAP mutants that cannot bind to Smac or effector caspases. Likewise, the release of a Smac mutant that cannot bind to XIAP was not impaired by C-XIAP-C expression. Full Smac release could however be provoked by rapid cytosolic C-XIAP-C depletion upon digitonin-induced plasma membrane permeabilisation. These findings suggest that even though mitochondria may already contain pores sufficient for cytochrome-c release, elevated amounts of XIAP can selectively impair and limit the release of Smac. Furthermore native Smac dimerises to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we demonstrate that upon mitochondria1 outer membrane permeabilisation physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended to release duration of dimeric Smac and resulted in the mitrochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. The findings presented therefore indicate that the dimerisation of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.