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 impairs Smac release from the mitochondria during apoptosis

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.     

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.     

Design,synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure–activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.     

Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP

Here we demonstrate that JNK3 can phosphorylate Smac. Smac phosphorylation attenuates its ability to activate apoptosome activity in HeLa S-100 cell lysates. Addition of the X-linked inhibitor of apoptosis protein (XIAP) to the S-100 markedly suppresses apoptosome activity, and this suppressive effect of XIAP is neutralized by adding unphosphorylated Smac, but not phosphorylated Smac. Furtherover, JNK3-mediated phosphorylation of Smac markedly attenuates the interaction between Smac and XIAP, as measured by BIACORE assays and non-denaturing gel shift assays. When JNK3 activity is down-regulated in etoposide-induced HeLa cells by transiently overexpressing a dominant negative version of JNK3 (DN-JNK3), the caspase-3 activity as well as PARP cleavages are markedly enhanced. And the interaction of Smac with XIAP also increases by down-regulating JNK3 activity under the same conditions. These results suggest that JNK3 activity can attenuate the progression of apoptosis through a novel mechanism of action, the down-regulation of interaction between Smac and XIAP.     

Dimerization of Smac is crucial for its mitochondrial retention by XIAP subsequent to mitochondrial outer membrane permeabilization

Following the apoptotic permeabilization of the outer mitochondrial membrane, the inter-membrane space protein second mitochondria-derived activator of caspases (Smac) is released into the cytosol. Smac efficiently promotes apoptosis by antagonizing x-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases-9, -3, and -7, via a short NH₂-terminal inhibitor of apoptosis protein (IAP) binding motif (AVPI). Native Smac dimerizes to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we here demonstrate that upon mitochondrial outer membrane permeabilization physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended the release duration of dimeric Smac and resulted in the mitochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. Our findings therefore indicate that the dimerization of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

杏仁核注射红藻氨酸诱导大鼠边缘叶癫痫发作时海马中Smac和XIAP蛋白的表达

应用红藻氨酸(kainic acid,KA)诱导的大鼠边缘叶癫痫发作模型,检测第二个线粒体源的半胱天冬蛋白酶激活物,直接与凋亡抑制蛋白结合的低等电点蛋白(second mitochondrial activator of caspases／direct inhibitor of apoptosis protein-binding protein of low isoelectric point[PI],Smac／DIABLO)和X染色体连锁的凋亡抑制蛋白(X-chromosome-linked inhibitor of apoptosis protein,XIAP)在癫痫大鼠海马神经元表达。单侧杏仁核内注射KA诱导癫痫发作,1h后用安定终止发作,然后分别用TUNEL染色和cresyl violet染色观察海马神经元存活和凋亡的变化,用免疫荧光和Western blot检测海马Smac／DIABLO、XIAP和半胱天冬蛋白酶-9(caspase-9)的表达。结果表明,发作终止2h时KA注射同侧海马CA3区细胞浆内Smac／DIABLO蛋白表达增加,4h时caspase-9出现裂解片断,8h时出现TUNEL阳性细胞,24h时达高峰。脑室内注射caspase-9抑制剂z-LEHD-fluoromethyl ketone(z-LEHD-fmk)可减少TUNEL阳性细胞,增加存活神经元。发作后KA注射同侧海马CA3区神经元caspase-9免疫反应性增强,Smac／DIABLO和XIAP弥散于整个神经元内。对侧海马未检测到TUNEL阳性细胞及Smac／DIABLO和XIAP蛋白的上述变化。以上结果提示,癫痫发作可诱导Smac／DIABLO蛋白从线粒体向细胞浆的移位、XIAP亚细胞分布改变和caspase-9的激活,Smac／DIABLO、XIAP和caspase-9可能参与了癫痫神经元损伤的病理生理机制,caspase-9可能是潜在的治疗靶点。     

Interaction of a cyclic, bivalent smac mimetic with the x-linked inhibitor of apoptosis protein

We have designed and synthesized a cyclic, bivalent Smac mimetic (compound 3) and characterized its interaction with the X-linked inhibitor of apoptosis protein (XIAP). Compound 3 binds to XIAP containing both BIR2 and BIR3 domains with a biphasic dose-response curve representing two binding sites with IC 50 values of 0.5 and 406 nM, respectively. Compound 3 binds to XIAPs containing the BIR3-only and BIR2-only domain with K i values of 4 nM and 4.4 microM, respectively. Gel filtration experiments using wild-type and mutated XIAPs showed that 3 forms a 1:2 stoichiometric complex with XIAP containing the BIR3-only domain. However, it forms a 1:1 stoichiometric complex with XIAP containing both BIR2 and BIR3 domains, and both BIR domains are involved in the binding. Compound 3 efficiently antagonizes inhibition of XIAP in a cell-free functional assay and is >200 times more potent than its corresponding monovalent compound 2. Determination of the crystal structure of 3 in complex with the XIAP BIR3 domain confirms that 3 induces homodimerization of the XIAP BIR3 domain and provides a structural basis for the cooperative binding of one molecule of compound 3 to two XIAP BIR3 molecules. On the basis of this crystal structure, a binding model of XIAP containing both BIR2 and BIR3 domains and 3 was constructed, which sheds light on the ability of 3 to relieve the inhibition of XIAP with not only caspase-9 but also caspase-3/-7. Compound 3 is cell-permeable, effectively activates caspases in whole cells, and potently inhibits cancer cell growth. Compound 3 is a useful biochemical and pharmacological tool for further elucidating the role of XIAP in regulation of apoptosis and represents a promising lead compound for the design of potent, cell-permeable Smac mimetics for cancer treatment.     

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.     

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.     

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.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Monomerization of Cytosolic Mature Smac Attenuates Interaction with IAPs and Potentiation of Caspase Activation

The four residues at the amino-terminus of mature Smac/DIABLO are an IAP binding motif (IBM). Upon exit from mitochondria, mature Smac interacts with inhibitor of apoptosis proteins (IAPs), abrogating caspase inhibition. We used the ubiquitin fusion model to express mature Smac in the cytosol. Transiently expressed mature Smac56-239 (called Smac56) and Smac60-239 (called Smac60), which lacks the IBM, interacted with X-linked inhibitor of apoptosis protein (XIAP). However, stable expression produced wild type Smac56 that failed to homodimerize, interact with XIAP, and potentiate caspase activation. Cytosolic Smac60 retained these functions. Cytosolic Smac56 apparently becomes posttranslationally modified at the dimer interface region, which obliterated the epitope for a monoclonal antibody. Cytosolic Smacδ, which has the IBM but lacks amino acids 62–105, homodimerized and weakly interacted with XIAP, but failed to potentiate apoptosis. These findings suggest that the IBM of Smac is a recognition point for a posttranslational modification(s) that blocks homodimerization and IAP interaction, and that amino acids 62–105 are required for the proapoptotic function of Smac.     

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.     

Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by removing the resistance-mediating block of effector caspase maturation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-kappaB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-kappaB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.     

Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation

X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.     

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.