Abstract
X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases 3, 7 and 9, and mitochondrial Smac (second mitochondria-derived activator of caspase) release during apoptosis inhibits the activity of XIAP. In this study we show 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 tumour necrosis factor-related apoptosis-inducing ligand/cycloheximide and staurosporine. In C-XIAP-C-expressing cells, cytochrome c release from mitochondria proceeded normally, whereas Smac release was significantly prolonged and incomplete. In addition, 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 mitochondrial Smac retention, as evidenced in experiments using XIAP mutants that cannot bind to Smac or effector caspases. Similarly, 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 permeabilization. Our findings suggest that although mitochondria may already contain pores sufficient for cytochrome c release, elevated amounts of XIAP can selectively impair and limit the release of Smac.
Highlights
X-linked inhibitor of apoptosis protein (XIAP) comprises three baculoviral IAP repeat domains (BIR1–3), as well as a zinc-finger RING domain, which exerts E3 ubiquitin ligase activity
Additional control experiments showed that the anti-apoptotic potential of C-XIAP-C was comparable to native XIAP (Figure 1g), and that C-XIAP-C could interact with its physiological binding partner and antagonist second mitochondriaderived activator of caspases (Smac) (Figure 1h), as well as with Smac-yellow fluorescent protein (YFP) fusion proteins (Supplementary Figure 1) that were used later in this study
We identified that the presence of XIAP can prolong and limit the release of Smac from the mitochondria during extrinsically and intrinsically induced apoptosis
Summary
XIAP comprises three baculoviral IAP repeat domains (BIR1–3), as well as a zinc-finger RING (really interesting new gene) domain, which exerts E3 ubiquitin ligase activity. The dimerization of caspase-9 monomers to yield the active caspase-9 conformation is inhibited by the BIR3 domain of XIAP through binding to the caspase-9 dimerization interface.[11] Executioner caspases 3 and 7 instead are inhibited by a region preceding the BIR2 domain. This region occupies the substrate-binding groove of executioner caspases 3 and 7, but requires further interactions of the effector caspases with the BIR2 domain to stabilize this inhibition.[12]. The main antagonist of XIAP is the second mitochondriaderived activator of caspases (Smac). Smac, similar to cyt-c, is released into the
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