Abstract
Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.
Highlights
X-linked inhibitor of apoptosis protein (XIAP) E3 ligase induces mitochondrial membrane permeabilization, resulting in intramitochondrial degradation of its inhibitor Smac
We propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical mitochondrial outer membrane permeabilization (MOMP) to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity
We further showed that in cells transiently overexpressing XIAP, mitochondrial uncoupling with carbonyl cyanide m-chlorophenylhydrazone (CCCP) is sufficient for induction of these events and triggers Bax translocation to and clustering at the outer mitochondrial membrane (OMM)
Summary
XIAP E3 ligase induces mitochondrial membrane permeabilization, resulting in intramitochondrial degradation of its inhibitor Smac. Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We show that mitochondrial XIAP action prominently occurs in response to drug-induced intrinsic and extrinsic apoptosis and phenotypically results in a reduced pre-MOMP delay, the lag time between the administration of an apoptotic stimulus and the onset of mitochondrial permeabilization. Combined mathematical modeling and experimental approaches revealed conditions whereby XIAP suppression of mitochondrial Smac significantly cooperates with downstream XIAP inhibition of caspases Based on these findings, we propose that following apoptosis induction XIAP-induced membrane permeabilization 1) is mechanistically and functionally distinct from canonical, pro-apoptotic MOMP, and 2) under increased XIAP levels functions as an anti-apoptotic tuning mechanism capable of reducing the mitochondrial apoptosis capacity via intramitochondrial Smac degradation
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