X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.     

Apoptosis-inducing factor is a target for ubiquitination through interaction with XIAP

X-linked inhibitor of apoptosis (XIAP) is an inhibitor of apoptotic cell death that protects cells by caspase-dependent and independent mechanisms. In a screen for molecules that participate with XIAP in regulating cellular activities, we identified apoptosis-inducing factor (AIF) as an XIAP binding protein. Baculoviral IAP repeat 2 of XIAP is sufficient for the XIAP/AIF interaction, which is disrupted by Smac/DIABLO. In healthy cells, mature human AIF lacks only the first 54 amino acids, differing significantly from the apoptotic form, which lacks the first 102 amino-terminal residues. Fluorescence complementation and immunoprecipitation experiments revealed that XIAP interacts with both AIF forms. AIF was found to be a target of XIAP-mediated ubiquitination under both normal and apoptotic conditions, and an E3 ubiquitin ligase-deficient XIAP variant displayed a more robust interaction with AIF. Expression of either XIAP or AIF attenuated both basal and antimycin A-stimulated levels of reactive oxygen species (ROS), and when XIAP and AIF were expressed in combination, a cumulative decrease in ROS was observed. These results identify AIF as a new XIAP binding partner and indicate a role for XIAP in regulating cellular ROS.     

Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP)

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3beta. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.     

Down-regulation of apoptosis-inducing factor protein by RNA interference inhibits UVA-induced cell death

Apoptosis-inducing factor (AIF) is a caspase-independent apoptosis effector. UVA-induced Raji cell death was not completely inhibited by pan-caspase inhibitor zVAD.fmk. Moreover, AIF translocated from its normal location, the mitochondrial intermembrane space, into the nucleus, and induced peripheral chromatin condensation during the early stage of UVA-inducing cell death. Enforced expression of AIF can induce Raji cell death in a caspase-independent manner. Down-regulation of AIF protein level by RNA interference (RNAi) can reduce UVA-induced Raji cell death, but the combination of down-regulation of AIF and zVAD.fmk almost completely inhibited UVA-induced Raji cell death. All these suggest that caspase and AIF are two independent pathways and that UVA-induced Raji cell death is dependent on caspase and AIF.     

Ubiquitin-dependent protein processing controls radiation-induced apoptosis through the N-end rule pathway

The ubiquitination of nuclear proteins activated in human lymphocytes undergoing radiation-induced apoptosis and the subsequent downstream proteasomal protein processing, shown to be involved in apoptotic death control, may be dependent on an amino-terminal sequence identity of ubiquitin target proteins, the "N-end rule" pathway. Here we report that this selective pathway controls radiation-induced apoptosis and that it is involved in the initiation of this type of cell death. Dipeptide competitors of protein ubiquitination/processing dependent solely on the basic amino-terminal residues (type I) efficiently inhibited the radiation-induced apoptotic death phenotype, indicating that only the substrates of ubiquitination with basic NH2-terminal amino acids are involved in apoptotic death control. This selective inhibition was followed by an early, overall but also target-specific inhibition of ubiquitination and by an activation and stabilization of poly(ADP-ribose) polymerase (PARP) that occurs through inhibition of ubiquitination of its cleaved form (85 kDa). Interestingly, caspases-3 and -7 were not activated following irradiation, further suggesting that PARP cleavage may be regulated by an N-end rule pathway in a caspase-independent manner. These results highly suggest involvement of this subset of the ubiquitin system in the apoptotic death control and in the specific regulation of PARP activity.     

Fibroblast apoptosis induced by Porphyromonas gingivalis is stimulated by a gingipain and caspase-independent pathway that involves apoptosis-inducing factor

Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z-VAD-fmk, had little effect. Moreover, P. gingivalis downregulated caspase-3 mRNA levels and caspase-3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor-alpha-induced apoptosis, which is caspase-3-dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis-induced translocation of apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H-89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis-induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase-3.     

An IAP-IAP complex inhibits apoptosis

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

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.     

TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death

Emerging evidence suggests that ubiquitin mediated post translational modification is a critical regulatory process involved in diverse cellular pathways including cell death. During ubiquitination, E3 ligases recognize target proteins and determine the topology of ubiquitin chains. Recruitment of E3 ligases to targets proteins under stress conditions including oxidative stress and their implication in cell death have not been systemically explored. In the present study, we characterized the role of TRIM32 as an E3 ligase in regulation of oxidative stress induced cell death. TRIM32 is ubiquitously expressed in cell lines of different origin and form cytoplasmic speckle like structures that transiently interact with mitochondria under oxidative stress conditions. The ectopic expression of TRIM32 sensitizes cell death induced by oxidative stress whereas TRIM32 knockdown shows a protective effect. The turnover of TRIM32 is enhanced during oxidative stress and its expression induces ROS generation, loss of mitochondrial transmembrane potential and decrease in complex-I activity. The pro-apoptotic effect was rescued by pan-caspase inhibitor or antioxidant treatment. E3 ligase activity of TRIM32 is essential for oxidative stress induced apoptotic cell death. Furthermore, TRIM32 decreases X-linked inhibitor of apoptosis (XIAP) level and overexpression of XIAP rescued cells from TRIM32 mediated oxidative stress and cell death. Overall, the results of this study provide the first evidence supporting the role of TRIM32 in regulating oxidative stress induced cell death, which has implications in numerous pathological conditions including cancer and neurodegeneration.     

Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis

Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells.     

Involvement of C-jun NH2-terminal kinase and apoptosis induced factor in apoptosis induced by deglycosylated bleomycin in laryngeal carcinoma cells

In our previous studies, we demonstrated that the deglycosylation of bleomycin-A2 (BLM-A2) does not affect the capacity of this drug to induce cell death by apoptosis in a caspase-independent manner in laryngeal cancer cells (HEp-2), but suppresses the ability of BLM-A2 to induce ROS formation. We have now investigated the consequence of BLM-A2 deglycosylation in terms of the involvement of apoptotic pathways in HEp-2 cells. Apoptosis induced by bleomycin-A2 and deglyco-BLM-A2 is associated with the release of cytochrome c and AIF. Only Bax was oligomerized with BLM-A2-induced HEp-2 cell death. BLM-A2 and deglyco-BLM-A2-induced apoptosis depended on JNK activation but was independent of death receptors expression. In contrast, both of these drugs would sensitize HEp-2 cells to death receptor ligand-induced cell death. These observations indicate that the deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway by the release of AIF responsible for mitochondrial permeability and chromatin condensation independent of caspases activation.     

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.     

X-linked Inhibitor of Apoptosis Protein promotes the degradation of its antagonist, the pro-apoptotic ARTS protein

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system.     

Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation

The physiological activity of Notch is a function of its ability to increase survival in many cell types. Several pathways have been shown to contribute to the survival effect of Notch, but the exact mechanism of Notch action is not completely understood. Here we identified that the regulation of cell survival by Notch intracellular domain could partly be attributed to a selective increase of X-linked inhibitor of apoptosis protein (XIAP). We further found that Notch intracellular domain inhibited the degradation of XIAP during apoptosis. The transactivation domain of Notch interacted directly with the RING region of XIAP to block the binding of E2 and prevent the in vivo and in vitro ubiquitination of XIAP. This antiapoptotic activity of Notch was abolished when XIAP was knocked down. Our results reveal a novel mechanism for Notch-selective suppression of apoptosis through an increase in the stability of a key antiapoptotic protein, XIAP.     

DNA binding is required for the apoptogenic action of apoptosis inducing factor

The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.     

The Proteasome Inhibitor MG-132 Induces AIF Nuclear Translocation Through Down-Regulation of ERK and Akt/mTOR Pathway

Anticancer activity of proteasome inhibitors has been demonstrated in various cancer cell types. However, mechanisms by which they exert anticancer action were not fully understood. The present study was undertaken to examine the effect of the proteasome inhibitor MG-132 and the underlying mechanism in glioma cells. MG-132 caused alterations in mitochondrial membrane potential and apoptosis-inducing factor (AIF) nuclear translocation. MG-132 induced reduction in ERK and Akt activation. The transient transfection of constitutively active forms of MEK, an upstream of ERK, and Akt blocked the MG-132-induced cell death. Similarly to down-regulation of Akt, expression levels of mTOR were inhibited by MG-132. Addition of rapamycin, an inhibitor of mTOR, caused stimulation of the MG-132-induced cell death. There were no significant changes in levels of XIAP, survivin, and Bax. Overexpression of constitutively active forms of MEK and Akt blocked the MG-132-induced AIF nuclear translocation. These findings indicate that MG-132 induces AIF nuclear translocation through down-regulation of ERK and Akt/mTOR pathways. These data suggest that proteasome inhibitors may serve as potential therapeutic agents for malignant human gliomas.     

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.     

α7 Nicotinic receptor activation reduces β-amyloid-induced apoptosis by inhibiting caspase-independent death through phosphatidylinositol 3-kinase signaling

The neurotoxicity of amyloid-β (Aβ) involves caspase-dependent and -independent programmed cell death. The latter is mediated by the nuclear translocation of the mitochondrial flavoprotein apoptosis inducing factor (AIF). Nicotine has been shown to decrease Aβ neurotoxicity via inhibition of caspase-dependent apoptosis, but it is unknown if its neuroprotection is mediated through caspase-independent pathways. In the present study, pre-treatment with nicotine in rat cortical neuronal culture markedly reduced Aβ(1-42) induced neuronal death. This effect was accompanied by a significant reduction of mitochondrial AIF release and its subsequent nuclear translocation as well as significant inhibition of cytochrome c release and caspase 3 activation. Pre-treatment with selective α7nicotinic acetylcholine receptor(nAChR) antagonist (methyllycaconitine), but not the α4 nAChR antagonist (dihydro-β-erythroidine), could prevent the neuroprotective effect of nicotine on AIF release/translocation, suggesting that nicotine inhibits the caspase-independent death pathway in a α7 nAChR-dependent fashion. Furthermore, the neuroprotective action of nicotine on AIF release/translocation was suppressed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Pre-treatment with nicotine significantly restored Akt phosphorylation, an effector of PI3K, in Aβ(1-42) -treated neurons. These findings indicate that the α7 nAChR activation and PI3K/Akt transduction signaling contribute to the neuroprotective effects of nicotine against Aβ-induced cell death by modulating caspase-independent death pathways.