X-linked inhibitor of apoptosis protein : regulation and function

X-linked inhibitor of apoptosis protein (XIAP) is a direct endogenous inhibitor of caspases-3, -7, and -9 and also functions as a ubiquitin-signalling protein through its ubiquitin-associated and RING E3 ligase domains. Although XIAP has been extensively characterized biochemically, its physiological importance remains highly controversial. This dissertation examined XIAP regulation and function across three biological settings: trophic-factor-deprived embryonic sensory neurons, basal caspase regulation in developing and adult mouse tissues, and collective migration of metastatic breast cancer cells. First, I investigated XIAP regulation in embryonic dorsal root ganglion sensory neurons undergoing nerve growth factor deprivation-induced degeneration. XIAP protein declined early and reproducibly during deprivation, in parallel with caspase-3 activation and axonal degeneration. Although MRT67307 preserved XIAP and protected axons, IKKε deletion did not alter the deprivation response, and the more selective TBK1 inhibitor GSK8612 failed to preserve XIAP or prevent degeneration. TBK1 and IKKε could phosphorylate XIAP in HEK293T cells, but DRG data did not support these kinases as dominant physiological regulators of XIAP loss. Proteasome inhibition also failed to preserve XIAP despite ubiquitinated-protein accumulation, and caspase-3 deletion did not prevent XIAP depletion. Thus, XIAP loss during NGF deprivation occurs upstream of, or in parallel with, executioner caspase activation and was not explained by the tested kinase-, proteasome-, or caspase-dependent mechanisms. Second, I examined XIAP function in basal caspase-3 restraint across embryonic, postnatal, and adult tissues. My pilot data suggested that XIAP deletion was associated with increased cleaved caspase-3 accumulation in cultured E13.5 DRG explants and an apparent modest increase in embryonic cortex, whereas adult neuronal and non-neuronal tissues appeared to show little detectable XIAP-dependent caspase-3 restraint under basal conditions. Third, in MDA-MB-231 breast cancer cells, XIAP loss did not consistently alter collective wound closure, indicating that XIAP is not uniformly pro-migratory in this model. Together, these findings support XIAP as a context-dependent regulator of caspase restraint and cell migration rather than as a uniformly anti-apoptotic or pro/anti-migratory factor.