Requirement of both the second and third BIR domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by Smac

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.     

A dimeric Smac/diablo peptide directly relieves caspase-3 inhibition by XIAP. Dynamic and cooperative regulation of XIAP by Smac/Diablo

Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases from XIAP inhibition is not clear. It is generally believed that binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse inhibition of the effector caspases. Surprisingly, we show here that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined the dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP. We also show that XIAP homotrimerizes via its C-terminal Ring domain, making its inhibitory activity toward caspase-3 more susceptible to Smac.     

Smac3, a novel Smac/DIABLO splicing variant, attenuates the stability and apoptosis-inhibiting activity of X-linked inhibitor of apoptosis protein

X-linked inhibitor of apoptosis protein (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family, plays a crucial role in the regulation of apoptosis. XIAP is structurally characterized by three baculovirus IAP repeat (BIR) domains that mediate binding to and inhibition of caspases and a RING domain that confers ubiquitin ligase activity. The caspase inhibitory activity of XIAP can be eliminated by the second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) during apoptosis. Here we report the identification and characterization of a novel isoform of Smac/DIABLO named Smac3, which is generated by alternative splicing of exon 4. Smac3 contains an NH2-terminal mitochondrial targeting sequence required for mitochondrial targeting of Smac3 and an IAP-binding motif essential for Smac3 binding to XIAP. Smac3 is released from mitochondria into the cytosol in response to apoptotic stimuli, where it interacts with the second and third BIR domains of XIAP. Smac3 disrupts processed caspase-9 binding to XIAP, promotes caspase-3 activation, and potentiates apoptosis. Strikingly, Smac3, but not Smac/DIABLO, accelerates XIAP auto-ubiquitination and destruction. Smac3-stimulated XIAP ubiquitination is contingent upon the physical association of XIAP with Smac3 and an intact RING domain of XIAP. Smac3-accelerated XIAP destabilization is, at least in part, attributed to its ability to enhance XIAP ubiquitination. Our study demonstrates that Smac3 is functionally additive to, but independent of, Smac/DIABLO.     

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.     

HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.     

Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP.

XIAP is a mammalian inhibitor of apoptosis protein (IAP). To determine residues within the second baculoviral IAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for loss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe. Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3. Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the IAP antagonist DIABLO. Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo.     

Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.     

A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death

X chromosome-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspase-3, -7, and -9. Smac/DIABLO, an inhibitor of XIAP, is released from mitochondria upon receiving apoptotic stimuli and binds to the BIR2 and BIR3 domains of XIAP, thereby inhibiting its caspase-inhibitory activity. Here we report that a serine protease called HtrA2/Omi is released from mitochondria and inhibits the function of XIAP by direct binding in a similar way to Smac. Moreover, when overexpressed extramitochondrially, HtrA2 induces atypical cell death, which is neither accompanied by a significant increase in caspase activity nor inhibited by caspase inhibitors, including XIAP. A catalytically inactive mutant of HtrA2, however, does not induce cell death. In short, HtrA2 is a Smac-like inhibitor of IAP activity with a serine protease-dependent cell death-inducing activity.     

Regulation of XIAP and Smac/DIABLO in the rat hippocampus following transient forebrain ischemia

We investigated the expression of XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac/DIABLO, a newly identified mitochondrial apoptogenig molecule in the hippocampus following transient global ischemia. Transient global ischemia produced by two-vessel occlusion triggers the delayed neuronal death of CA1 neurons in the hippocampus. We demonstrate that CA1 neuronal loss induced by ischemia (10 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. XIAP (X chromosome-linked inhibitor of apoptosis protein) is a member of the inhibitor of apoptosis (IAP) gene family that, in addition to suppressing cell death by inhibition of caspases, is involved in an increasing number of signalling cascades. The present study shows alterations in the levels of XIAP and of Smac/DIABLO (second mitochondrial activator of caspase) after cerebral ischemia. The protein levels of XIAP and the number of XIAP-positive cells were regulated by cerebral ischemia in a strictly time and region dependent manner. The largest change in XIAP-IR was observed in the CA1 sub field, which is the most vulnerable area of hippocampus. The mitochondrial expression level of Smac/DIABLO increased during reperfusion. Smac/DIABLO expression was associated with alteration of the XIAP levels and the appearance of activated form of caspase-3 within the hippocampus during reperfusion in spatial and temporal manners.