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.     

Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases.

Several human inhibitor of apoptosis (IAP) family proteins function by directly inhibiting specific caspases in a mechanism that does not require IAP cleavage. In this study, however, we demonstrate that endogenous XIAP is cleaved into two fragments during apoptosis induced by the tumor necrosis factor family member Fas (CD95). The two fragments produced comprise the baculoviral inhibitory repeat (BIR) 1 and 2 domains (BIR1-2) and the BIR3 and RING (BIR3-Ring) domains of XIAP. Overexpression of the BIR1-2 fragment inhibits Fas-induced apoptosis, albeit at significantly reduced efficiency compared with full-length XIAP. In contrast, overexpression of the BIR3-Ring fragment results in a slight enhancement of Fas-directed apoptosis. Thus, cleavage of XIAP may be one mechanism by which cell death programs circumvent the anti-apoptotic barrier posed by XIAP. Interestingly, ectopic expression of the BIR3-Ring fragment resulted in nearly complete protection from Bax-induced apoptosis. Use of purified recombinant proteins revealed that BIR3-Ring is a specific inhibitor of caspase-9 whereas BIR1-2 is specific for caspases 3 and 7. Therefore XIAP possesses two different caspase inhibitory activities which can be attributed to distinct domains within XIAP. These data may provide an explanation for why IAPs have evolved with multiple BIR domains.     

Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis

The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.     

An IAP-IAP complex inhibits apoptosis

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.     

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.     

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.     

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.     

Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family

Several of the inhibitor of apoptosis protein (IAP) family members regulate apoptosis in response to various cellular assaults. Some members are also involved in cell signalling, mitosis and targeting proteins to the ubiquitin-proteasome degradation machinery. The most intensively studied family member, X-linked IAP (XIAP), is a potent inhibitor of caspase activity; hence, it is generally assumed that direct caspase inhibition is an important conserved function of most members of the family. Biochemical and structural studies have precisely mapped the elements of XIAP required for caspase inhibition. Intriguingly, these elements are not conserved among IAPs. Here, we review current knowledge of the caspase-inhibitory potential of the human IAPs and show that XIAP is probably the only bona fide caspase inhibitor, suggesting that the other family members never gained the ability to directly inhibit caspase activity.     

The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.     

Survivin as a target for new anticancer interventions

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that has been implicated in both control of cell division and inhibition of apoptosis. Specifically, its anti-apoptotic function seems to be related to the ability to directly or indirectly inhibit caspases. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed as an attractive target for new anticancer interventions. Several preclinical studies have demonstrated that down-regulation of survivin expression/function, accomplished through the use of antisense oligonucleotides, dominant negative mutants, ribozymes, small interfering RNAs and cyclin-dependent kinase inhibitors, increased the apoptotic rate, reduced tumor-growth potential and sensitized tumor cells to chemotherapeutic drugs with different action mechanisms and gamma-irradiation in in vitro and in vivo models of different human tumor types.     

Backbone and side-chain 1H, 13C and 15N assignments of the ubiquitin-associated domain of human X-linked inhibitor of apoptosis protein

X-linked inhibitor of apoptosis protein (XIAP), a leading member of the family of inhibitor of apoptosis (IAP) proteins, is considered as the most potent and versatile inhibitor of caspases and apoptosis. It has been reported that XIAP is frequently overexpressed in cancer and its expression level is implicated in contributing to tumorigenesis, disease progression, chemoresistance and poor patient-survival. Therefore, XIAP is one of the leading targets in drug development for cancer therapy. Recently, based on bioinformatics study, a previously unrecognized but evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs was identified. The UBA domain is found to be essential for the oncogenic potential of IAP, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP to activate NF-κB. In the present study, we report the near complete resonance assignments of the UBA domain-containing region of human XIAP protein. Secondary structure prediction based on chemical shift index (CSI) analysis reveals that the protein is predominately α-helical, which is consistent with the structures of known UBA proteins.     

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.     

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.     

Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway

Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

Characterization of dequalinium as a XIAP antagonist that targets the BIR2 domain

Inhibitor of apoptosis proteins (IAPs) regulate the activity of caspases in apoptosis. The human X chromosome-encoded IAP (XIAP) is one of the more potent members of the IAP family and it has been described as a central regulator of apoptosis. Thus, molecules that inhibit XIAP could offer therapeutic opportunities to treat unwanted apoptosis inhibition. In the present study we have applied the selective optimization of side activities (SOSA) approach to the discovery of XIAP inhibitors. In this sense, we have identified dequalinium hydrochloride (Dq) as an inhibitor of the XIAP/caspase-3 interaction both in vitro and in cellular assays.