X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.     

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.     

Caspase-1 and caspase-8 cleave and inactivate cellular parkin

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

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.     

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.     

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.     

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.     

The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7

The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.     

IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms

Some members of the inhibitor of apoptosis (IAP) family suppress apoptosis by neutralizing caspases. The current model suggests that all caspase-regulatory IAPs function as direct enzyme inhibitors, blocking effector caspases by binding to their catalytically active pockets. Here we show that IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Whereas XIAP binds directly to the active-site pockets of effector caspases, we find that regulation of effector caspases by Drosophila IAP1 (DIAP1) requires an evolutionarily conserved IAP-binding motif (IBM) at the neo-amino terminus of the large caspase subunit. Remarkably, unlike XIAP, DIAP1-sequestered effector caspases remain catalytically active, suggesting that DIAP1 does not function as a bona fide enzyme inhibitor. Moreover, we demonstrate that the mammalian IAP c-IAP1 interacts with caspase-7 in an exclusively IBM-dependent, but active site pocket-independent, manner that is mechanistically similar to DIAP1. The importance of IBM-mediated regulation of effector-caspases in vivo is substantiated by the enhanced apoptotic potency of IBM-mutant versions of drICE, DCP-1 and caspase-7.     

Structural basis of caspase-7 inhibition by XIAP

The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. Here we present the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 A resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.     

Synergetic Effects of Caspase 3 and μ-Calpain in XIAP-Breakdown upon Focal Cerebral Ischemia

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to neurodegenerative disorders. The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of apoptosis. This protein binds to and inhibits both initiator caspases and effector caspases such as caspase-3. The aim of this study was to investigate the relationships between XIAP-breakdown, caspase activation in the development of delayed infarct upon ischemia. We demonstrated that endogenous XIAP is cleaved at least into two fragments during reperfusion following the ischemic insult. The two fragments produced seem to be related to caspase-3 and μ-calpain activities, which are massively enhanced in tissues challenged by ischemia. Therefore, degradation of XIAP by μ-calpain in our system may decrease the activation threshold of caspase-3 normally held in check by the IAPs and/or lead to auto-activation of other caspases. Special issue in honor of Naren Banik.     

Uncoupling of the signaling and caspase-inhibitory properties of X-linked inhibitor of apoptosis

In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-kappaB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-kappaB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-kappaB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled.     

NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP

The inhibitor of apoptosis proteins (IAPs) regulate the caspase family of cysteine proteases, which play an important role in the execution of programmed cell death. Human X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases-3, -7, and -9. Here we show that the Bir3 domain is the minimal region of XIAP that is needed for potent caspase-9 inhibition. The three-dimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five alpha-helices, a three-stranded beta-sheet, and a zinc atom chelated to three cysteines and one histidine. The structure of the Bir3 domain is similar to that of the Bir2 domain of XIAP but differs from the previously determined structure of the Bir3 domain of MIHB. Based on site-directed mutagenesis, we have identified the regions of the Bir3 domain of XIAP that are important for inhibiting caspase-9. Despite the structural similarities of the Bir2 and Bir3 domain of XIAP, a different set of residues were found to be critical for inhibiting the individual caspases. These results suggest that XIAP inhibits caspase-3 and caspase-9 in a different manner.     

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the lAP family members and is required for GIDE＇s E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE- induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over-or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.