Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

The IAP family：endogenous caspase inhibitors with multiple biological activities

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains.These proteins have multiple biological activities that include binding and inhibiting caspases,regulating cell cycle progression,and modulating receptor-mediated signal transduction.Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells,and their degradation appears to be important for T cells to commit to death.In addition to three BIR domains,each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase(E3) activity to IAPs,and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus.Given the fact that IAPs can bind a variety of proteins,such as caspases and TRAFs,it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction,cell cycle,and apoptosis.     

Role of Melanoma Inhibitor of Apoptosis (ML-IAP) Protein, a Member of the Baculoviral IAP Repeat (BIR) Domain Family, in the Regulation of C-RAF Kinase and Cell Migration

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.     

Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.     

A highly conserved arginine is critical for the functional folding of inhibitor of apoptosis (IAP) BIR domains

The inhibitor of apoptosis (IAP) proteins are found in all animals and regulate apoptosis (programmed cell death) by binding and inhibiting caspase proteases. This inhibition is overcome by several apoptosis stimulators, including Drosophila Hid and mammalian Smac/DIABLO, which bind to 65-residue baculovirus IAP repeat (BIR) domains found in one to three copies in all IAPs. Virtually all BIRs contain three Cys and a His that bind zinc, a Gly in a tight turn, and an Arg. The functional and structural role of the Arg was investigated in isolated BIR domains from the baculovirus Orgyia pseudotsugata Op-IAP and the Drosophila DIAP1 proteins. Mutation of the Arg to either Ala or Lys abolished Hid and Smac binding to BIRs, despite the Hid/Smac binding site being located on the opposite side of the BIR domain from the Arg. The mutant BIR domains also exhibited weakened zinc binding, increased sensitivity to limited proteolysis, and altered circular dichroism spectra indicative of perturbed domain folding. Examination of known BIR structures indicates that the Arg side chain makes simultaneous bridging hydrogen bonds and a cation-pi interaction for which the Arg guanidino group is uniquely well suited. These interactions are likely critical for stabilizing the tertiary fold of BIR domains in all IAPs, explaining the conservation of this residue.     

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.     

Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs

Apoptosis is a physiological cell death process important for development, homeostasis and the immune defence of multicellular animals. The key effectors of apoptosis are caspases, cysteine proteases that cleave after aspartate residues. The inhibitor of apoptosis (IAP) family of proteins prevent cell death by binding to and inhibiting active caspases and are negatively regulated by IAP-binding proteins, such as the mammalian protein DIABLO/Smac. IAPs are characterized by the presence of one to three domains known as baculoviral IAP repeat (BIR) domains and many also have a RING-finger domain at their carboxyl terminus. More recently, a second group of BIR-domain-containing proteins (BIRPs) have been identified that includes the mammalian proteins Bruce and Survivin as well as BIR-containing proteins in yeasts and Caenorhabditis elegans. These Survivin-like BIRPs regulate cytokinesis and mitotic spindle formation. In this review, we describe the IAPs and other BIRPs, their evolutionary relationships and their subcellular and tissue localizations.     

The Activator of Apoptosis Smac-DIABLO Acts as a Tetramer in Solution

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.     

Anti- and pro-apoptotic activities of baculovirus and Drosophila IAPs in an insect cell line

The anti-apoptotic activities of two baculovirus IAPs, OpIAP and CpIAP, were directly compared with that of two Drosophila IAPs, DIAP1 and DIAP2, in the same insect cell line, SF-21 cells. Like OpIAP and CpIAP, DIAP1 inhibited actinomycin D-induced apoptosis and apoptosis induced by Doom. Removal of the RING finger of DIAP1 reduced but did not eliminate its anti-apoptotic activity. DIAP2 was unable to inhibit actinomycin-D induced apoptosis but was able to partially inhibit Doom-induced apoptosis. The baculoviral BIR and RING finger regions, when separated, were unable to block apoptosis induced by actinomycin D or Doom. Instead, the BIR regions of OpIAP and CpIAP as well as the RING finger regions of CpIAP and DIAP1 induced apoptosis. Thus, there were significant differences in the manner in which the different domains of the viral and cellular homologues of IAPs interacted with the components of the pathways regulating apoptosis in SF-21 cells.     

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding.     

Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.     

Solid phase synthesis of Smac/DIABLO-derived peptides using a ‘Safety-Catch’ resin: Identification of potent XIAP BIR3 antagonists

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide ‘Safety-Catch’ resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500 nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1 μM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.     

NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways

Inhibitor-of-apoptosis proteins (IAPs), including neuronal apoptosis inhibitory protein (NAIP), inhibit cell death. Other IAPs inhibit key caspase proteases which effect cell death, but the mechanism by which NAIP acts is unknown. Here we report that NAIP, through its third baculovirus inhibitory repeat domain (BIR3), binds the neuron-restricted calcium-binding protein, hippocalcin, in an interaction promoted by calcium. In neuronal cell lines NSC-34 and Neuro-2a, over-expression of the BIR domains of NAIP (NAIP-BIR1-3) counteracted the calcium-induced cell death induced by ionomycin and thapsigargin. This protective capacity was significantly enhanced when NAIP-BIR1-3 was co-expressed with hippocalcin. Over-expression of the BIR3 domain or hippocalcin alone did not substantially enhance cell survival, but co-expression greatly increased their protective effects. These data suggest synergy between NAIP and hippocalcin in facilitating neuronal survival against calcium-induced death stimuli mediated through the BIR3 domain. Analysis of caspase activity after thapsigargin treatment revealed that caspase-3 is activated in NSC-34, but not Neuro-2a, cells. Thus NAIP, in conjunction with hippocalcin, can protect neurons against calcium-induced cell death in caspase-3-activated and non-activated pathways.     

Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis

IAPs are a group of regulatory proteins that are structurally related. Their conserved homologues have been identified in various organisms. In human, eight IAP members have been recognized based on baculoviral IAP repeat (BIR) domains. IAPs are key regulators of apoptosis, cytokinesis and signal transduction. The antiapoptotic property of IAPs depends on their professional role for caspases. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. IAPs impede apoptotic process via membrane receptor-dependent (extrinsic) cascade and mitochondrial dependent (intrinsic) pathway. IAP-mediated apoptosis affects the progression of liver diseases. Therapeutic options of liver diseases may depend on the understanding toward mechanisms of the IAP-mediated apoptosis.     

Identification of a novel gene family,paralogs of inhibitor of apoptosis proteins present in plants,fungi, and animals

Only few orthologs of animal apoptosis regulators have been found in plants. Recently, the ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has been shown to affect plant programmed cell death. Here, we identified two novel proteins homologous to Arabidopsis thaliana IAP-like protein (AtILP) 1 and 2 by applying an improved motif searching method. Furthermore, homologs of AtILP1 were found to occur as a novel gene family in other organisms such as fungi and animals including Homo sapiens (HsILP1). Like baculovirus IAP repeats (BIRs) in IAPs, ILPs contain two highly conserved BIR-like domains (BLDs) with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Homology modeling revealed that the three-dimensional structure of BLD in HsILP1 is similar to that of BIR. Transient expression of HsILP1 resulted in inhibition of etoposide-induced apoptosis in HEK293 and HeLaS3 cells. These findings suggest that ILPs are conserved in a wide range of eukaryotes including plants, and that their functions are closely related to those of IAPs.     

ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.     

NF023 binding to XIAP‐BIR1: Searching drugs for regulation of the NF‐κB pathway

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac‐mimetics, the most promising IAP‐targeting compounds specifically recognize the IAP‐BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac‐mimetics interfere with the NF‐κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto‐ubiquitination process. It has been shown that the XIAP‐BIR1 (X‐BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF‐κB survival pathway. Preventing X‐BIR1 dimerization abolishes XIAP‐mediated NF‐κB activation, thus implicating a proximity‐induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X‐BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X‐BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1‐specific compounds acting synergistically with existing pro‐apoptotic drugs in cancer therapy. Proteins 2015; 83:612–620. © 2015 Wiley Periodicals, Inc.     

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.