Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.     

The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3- and caspase 9-interacting sites

The X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. partial differential RING point mutants of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation.     

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.     

Mitochondrial fission leads to Smac/DIABLO release quenched by ARC

Apoptosis plays a critical role for the development of a variety of cardiac diseases. Cardiomyocytes are enriched in mitochondria, while mitochondrial fission can regulate apoptosis. The molecular mechanism governing cardiomyocyte apoptosis remain to be fully elucidated. Our results showed that Smac/DIABLO is necessary for apoptosis in cardiomyocytes, and it is released from mitochondria into cytosol in response to apoptotic stimulation. Smac/DIABLO release is a consequence of mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Upon release Smac/DIABLO binds to X-linked inhibitor of apoptosis protein (XIAP), resulting in the activation of caspase-9 and caspase-3. Their activation is a prerequisite for the initiation of apoptosis because the administration of z-LEHD-fmk and z-DQMD-fmk, two relatively specific inhibitors for caspase-9, and caspase-3, respectively, could significantly attenuate apoptosis. Smac/DIABLO release could not be blocked by these caspase inhibitors, indicating that it is an event upstream of caspase activation. ARC (apoptosis repressor with caspase recruitment domain), an abundantly expressed apoptotic repressor in cardiomyocytes, could inhibit mitochondrial fission and Smac/DIABLO release. Our data reveal that Smac/DIABLO is a target of ARC in counteracting apoptosis.     

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

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.     

Adding facets to TNF signaling. The JNK angle

In the October 3 issue of Cell, Deng et al. (2003) report that a JNK-dependent mitochondrial signal, the release of Smac/DIABLO, is required for TNF-induced caspase 8 activation and apoptosis. This process is interceded by a novel processed form of the BH3 only protein Bid.     

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.     

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.     

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.     

Caspase pathway of elaidic acid (9t-C18:1)-induced apoptosis in human umbilical vein endothelial cells

Although TFAs (trans fatty acids) do have effects on many endothelial functions, systemic inflammation and immune disorders, only limited experimental evidence is available that TFAs participate in the pathogenesis of endothelial cell apoptosis. HUVEC (human umbilical vein endothelial cells) were grown in medium with elaidic acid (9t-C18:1) at 50, 100, 200 and 400 μmol/l for 24 h. Apoptosis was measured by flow cytometry, and caspase 3, 8 and 9 activities by colorimetric assay and their mRNA expression by qRT-PCR (quantitative real-time PCR). Results showed that 9t-C18:1 induced apoptosis of HUVEC in a dose-dependent manner. The activities and mRNA expression of caspases 8, 9 and 3 were significantly increased compared with that of the control. Z-IETD-FMK and Z-LEHD-FMK inhibited the activation of caspase 3 and apoptosis induced by 9t-C18:1. Also Z-IETD-FMK inhibited the activation of caspase 9. mRNA expressions of Bid and Smac (second mitochondria-derived activator of caspase)/DIABLO [direct IAP (inhibitor of apoptosis)-binding protein with low pI] were also significantly elevated. We conclude that 9t-C18:1 induces apoptosis of HUVEC through activating caspases 8, 9 and 3. The death receptor pathway and the mitochondrial pathway both participated in the apoptosis course induced by 9t-C18:1.     

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.     

The membrane-associated inhibitor of apoptosis protein, BRUCE/Apollon, antagonizes both the precursor and mature forms of Smac and caspase-9

Smac/DIABLO, HtrA2/Omi, and caspase-9 play key roles in the initiation of apoptosis. The inhibitor of apoptosis proteins (IAPs) are believed to bind to the N-terminal IAP binding motifs of the mature (proteolytically processed) forms of Smac, HtrA2, and caspase-9. However, we show here that BRUCE/Apollon, a 528-kDa IAP whose degradation promotes apoptosis, associates with their precursors as well as the mature forms by binding to regions in addition to the IAP binding motif. Through these associations, BRUCE promotes the degradation of Smac and inhibits the activity of caspase-9 but not the effector caspase, caspase-3. In response to apoptotic stimuli, BRUCE is degraded by proteasomes and/or cleaved by caspases and HtrA2 depending on the specific stimulus and the cell type. These results suggest that the ability of BRUCE to antagonize both the precursor and mature forms of Smac and caspase-9 is an important mechanism for the prevention of apoptosis under normal conditions.     

Bid truncation, bid/bax targeting to the mitochondria, and caspase activation associated with neutrophil apoptosis are inhibited by granulocyte colony-stimulating factor

Neutrophil apoptosis constitutes a way of managing neutrophil-mediated reactions. It allows coping with infections, but avoiding overt bystander tissue damage. Using digitonin-based subcellular fractionation and Western blotting, we found that spontaneous apoptosis of human neutrophils (after approximately 20 h of culture) was associated with translocation of two proapoptotic Bcl-2 homologues, Bid and Bax, to the mitochondria and truncation of Bid, with subsequent release of Omi/HtrA2 and Smac/DIABLO into the cytosol. These events were accompanied by processing and increased enzymatic activity of caspase-8, -9, and -3. A G-CSF-mediated reduction in apoptosis coincided with inhibition of all these reactions. The G-CSF-induced effects were differentially dependent on newly synthesized mediators. Whereas inhibition of Bax targeting to the mitochondria and inhibition of caspase activation by G-CSF were dependent on protein synthesis, Bid truncation and redistribution were prevented by G-CSF regardless of the presence of the protein synthesis inhibitor cycloheximide. Apparently, the observed Bid changes were dispensable for neutrophil apoptosis. Although the regulators of the inhibitor of apoptosis proteins (IAPs), Omi/HtrA2 and Smac/DIABLO, were released into the cytosol during apoptosis, we did not observe cleavage of X-linked IAP, which suggests that another mechanism of IAP deactivation is involved. Together our results support an integrative role of the mitochondria in induction and/or amplification of caspase activity and show that G-CSF may act by blocking Bid/Bax redistribution and inhibiting caspase activation.     

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.     

Differentiation-induced HL-60 cell apoptosis: A mechanism independent of mitochondrial disruption?

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.     

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells

Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)-induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase-direct IAP binding protein with low pl (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 +/- 0.3 degrees C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and alphaB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.     

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.