Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. However, its inhibitory mechanism and subcellular localization remain controversial. In this report, we provided evidence for the first time that Survivin physically interacts with Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin fail to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis. The N-terminal of mature Smac/DIABLO is absolutely required for Survivin.Smac complex formation. Subcellular distributions of Survivin and Smac/DIABLO showed that they co-localized within the cytosol during interphase. In addition, Survivin was found to be incapable of binding to caspase. We also identified that the co-presence of Smac/DIABLO and XIAP was required for Survivin to inhibit caspase cleavage in a cell-free system. In conclusion, our results provide the first evidence that the interaction between Smac/DIABLO and Survivin is an essential step underling the inhibition of apoptosis induced by Taxol.     

INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.     

Hip2 interacts with and destabilizes Smac/DIABLO

Hip2 is a ubiquitin-conjugating enzyme that is involved in the cell cycle and suppression of cell death. To understand its role further, we tried to identify proteins that interact with Hip2. Using the immunoprecipitation technique and one-dimensional gel electrophoresis, we identified Smac/DIABLO, a proapoptotic molecule, as a protein that interacts with Hip2. The interaction of Hip2 and Smac was confirmed through in vivo and in vitro experiments. Hip2 promoted degradation of mature Smac through the ubiquitin proteasome pathway. As a result, Hip2 significantly blocked cell death induced by staurosporine and Smac. This study suggests that Hip2 might be involved in the regulation of Smac-mediated apoptosis.     

Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells

Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.     

TRAF3 interacts with Smac/DIABLO and enhances the proapoptotic effect of Smac/DIABLO in cytoplasm

Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding proteinwith low PI) is a 29 kDa mitochondrial precursor protein,which is proteolytically processed in mitochondriainto a 23 kDa mature protein.It is released from the mitochondrial intermembrane space to cytosol after anapoptotic trigger.Smac/DIABLO acts as a dimer and it contributes to caspase activation by sequestering theinhibitor of apoptosis proteins (IAPs).In order to further investigate the mechanism of Smac/DIABLOaction,we used the mature form of Smac/DIABLO as a bait and screened proteins that interact with matureSmac/DIABLO in human liver cDNA library using the yeast two-hybrid system.Forty-two colonies wereobtained after 5.8x 10~6 colonies were screened by nutrition limitation and X-galactosidase assay.After DNAsequence analysis and homology retrieval,one of the candidate proteins was identified as TRAF domain ofthe TNF receptor associated factor 3 (TRAF3).The interaction site between TRAF3 and Smac/DIABLOwas identified by β-galactosidase test. The interaction between TRAF3 and Smac/DIABLO via TRAF domainwas identified in vivo by co-immunoprecipitation in HepG2 cells,and the direct interaction between TRAF3and Smac/DIABLO in vitro was identified by GST-pull down assay.Co-expression of TRAF3 and matureSmac/DIABLO in 293 cells could enhance the Smac/DIABLO-mediated apoptosis.These results suggestedthat TRAF3 interacted with Smac/DIABLO via TRAF domain,leading to an increased proapoptotic effectof Smac/DIABLO in cytoplasm.     

Structural basis for recognition of H3T3ph and Smac/DIABLO N-terminal peptides by human Survivin

Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.     

Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.     

Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.     

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.     

Smac/DIABLO is required for effector caspase activation during apoptosis in human cells

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.     

Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype

BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢 (H2 O2 )所致C2 C12 肌原细胞凋亡中的作用 ,采用Hoechst 3 3 2 58染色 ,观察H2 O2 (0 5mmol/L)处理C2 C12 肌原细胞不同时间后 ,细胞核形态学改变并计算凋亡核百分率 ,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带 ,利用细胞成分分离后蛋白质印迹分析H2 O2 是否导致Smac/DIABLO从线粒体释放 ,采用Caspase检测试剂盒及蛋白质印迹分析Caspase 3和Caspase 9的活化 ,转染Smac/DIABLO基因 ,观察Smac/DIABLO过表达对H2 O2 所致的C2 C12 肌原细胞凋亡的影响 .结果表明 :H2 O2 处理 1h后 ,Smac/DIABLO从C2 C12 肌原细胞线粒体释放入胞浆 ,2h更明显 ;H2 O2 处理 4h后 ,Caspase 3和Caspase 9活化 ,12h达高峰 ;H2 O2 处理 2 4h后 ,C2 C12 肌原细胞显示特征性的凋亡形态改变 ,凋亡核百分率明显升高 ,DNA电泳出现明显“梯状”条带 .与单纯过氧化氢损伤组相比 ,Smac/DIABLO高表达的C2 C12 肌原细胞经过氧化氢损伤组的Caspase 3和Caspase 9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显 .结果表明 ,H2 O2 可导致Smac/DIABLO从C2 C12 肌原细胞线粒体释放 ,促进Caspase 9和Caspase 3的活化而促进细胞凋亡的发生     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

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.     

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

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

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.