凋亡诱导因子与细胞凋亡

凋亡诱导因子 (apoptosisinducefactor,AIF)是定位于线粒体膜间隙中的一种氧化还原酶 ,含有线粒体定位信号和核定位信号序列 ,具有很强的促凋亡活性 ,在类胚体成腔和胚胎早期分化过程中具有重要作用。在死亡信号或细胞胁迫的刺激下 ,线粒体通透性转变孔开放 ,释放AIF及细胞色素c至细胞质溶质中 ,具有核定位信号序列的AIF便进入细胞核内 ,引起染色质的初步凝集和DNA大规模断片化 (约 5 0kb) ,进而引发不依赖于胱冬肽酶的细胞凋亡途径 ;线粒体膜间隙释放出来的细胞色素c则可引起染色质的进一步凝集和DNA的寡核小体断片化 ,从而引发依赖于胱冬肽酶的细胞凋亡途径 ;与此同时 ,从线粒体膜间隙释放出来的AIF又可反馈放大线粒体通透性转变孔的渗透性 ,引起AIF与细胞色素c的进一步释放从而加快细胞死亡的进程。此外 ,细胞胁迫还可激活由多聚 (ADP 核糖 )聚合酶 1(PARP 1)所引发的细胞凋亡途径 ,通过AIF和细胞色素c引发细胞凋亡。最新研究结果表明 ,AIF同源线粒体关联死亡诱导者 (AIF homologousmitochondria associatedinducerofdeath ,AMID)与p5 3应答基因的编码产物 (p5 3 responsivegene 3,PRG3)均为AIF的同源蛋白质 ,可直接诱导人类细胞的凋亡。线虫的凋亡诱导因子WAH 1所诱导的细胞凋亡途径依赖于胱冬肽酶     

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.     

Steroid receptor coactivator-interacting protein (SIP) inhibits caspase-independent apoptosis by preventing apoptosis-inducing factor (AIF) from being released from mitochondria

Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis.     

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.     

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.     

Nondegradative ubiquitination of apoptosis inducing factor (AIF) by X-linked inhibitor of apoptosis at a residue critical for AIF-mediated chromatin degradation

Apoptosis inducing factor (AIF) is a mediator of caspase-independent cell death that is also necessary for mitochondrial energy production. How these seemingly opposite cellular functions of AIF are controlled is poorly understood. X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspases that also regulates several caspase-independent signaling pathways. The RING domain of XIAP possesses E3 ubiquitin ligase activity, though the importance of this function to signal regulation remains incompletely defined. XIAP binds and ubiquitinates AIF, and in this study, we determined the functional consequences of XIAP-mediated AIF ubiquitination. Unlike canonical ubiquitination, XIAP-dependent AIF ubiquitination did not lead to proteasomal degradation of AIF. Experiments using ubiquitin mutants demonstrated that the XIAP-dependent ubiquitin linkage was not formed through the commonly used lysine 48, suggesting a noncanonical ubiquitin linkage is employed. Further studies demonstrated that only lysine 255 of AIF was a target of XIAP-dependent ubiquitination. Using recombinant AIF, we determined that mutating lysine 255 of AIF interferes with the ability of AIF not only to bind DNA but also to degrade chromatin in vitro. These data indicate that XIAP regulates the death-inducing activity of AIF through nondegradative ubiquitination, further defining the role of XIAP in controlling AIF and caspase-independent cell death pathways.     

Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria

Apoptosis-inducing factor (AIF) is a phylogenetically ancient mitochondrial intermembrane flavoprotein endowed with the unique capacity to induce caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation when added to purified nuclei. In addition to its apoptogenic activity on nuclei, AIF can also participate in the regulation of apoptotic mitochondrial membrane permeabilization and exhibits an NADH oxidase activity. Under normal circumstances, AIF is secluded behind the outer mitochondrial membrane. However, upon apoptosis induction AIF translocates to the cytosol and the nucleus. Injection of anti-AIF antibodies or knockout of the AIF gene have demonstrated that AIF may be required for cell death occurring in response to some stimuli. In particular, inactivation of AIF renders embryonic stem cells resistant to cell death following growth factor withdrawal. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies, the very first wave of (caspase-independent) cell death indispensable for mouse morphogenesis. We have recently found that AIF is neutralized by heat-shock protein (HSP) 70, in a reaction that appears to be independent of ATP or the ATP-binding domain (ABD) of HSP70 and thus differs from the previously described Apaf-1/HSP70 interaction (which requires ATP and the HSP70 ABD). Intriguingly, HSP70 lacking ABD (HSP70 Delta ABD) inhibits apoptosis induced by serum withdrawal, staurosporin, and menadione, three models of apoptosis which are also affected by micro-injection of anti-AIF antibody or genetic ablation of AIF. Altogether, these data suggest that AIF plays a role in the regulation of caspase-independent cell death.     

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.     

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.     

AIF Downregulation and Its Interaction with STK3 in Renal Cell Carcinoma

Apoptosis-inducing factor (AIF) plays a crucial role in caspase-independent programmed cell death by triggering chromatin condensation and DNA fragmentation. Therefore, it might be involved in cell homeostasis and tumor development. In this study, we report significant AIF downregulation in the majority of renal cell carcinomas (RCC). In a group of RCC specimens, 84% (43 out of 51) had AIF downregulation by immunohistochemistry stain. Additional 10 kidney tumors, including an oxyphilic adenoma, also had significant AIF downregulation by Northern blot analysis. The mechanisms of the AIF downregulation included both AIF deletion and its promoter methylation. Forced expression of AIF in RCC cell lines induced massive apoptosis. Further analysis revealed that AIF interacted with STK3, a known regulator of apoptosis, and enhanced its phosphorylation at Thr180. These results suggest that AIF downregulation is a common event in kidney tumor development. AIF loss may lead to decreased STK3 activity, defective apoptosis and malignant transformation.     

Characterization of apoptotic pathway associated with caspase-independent excision of DNA loop domains

Excision of chromatin loop domains and internucleosomal DNA fragmentation are widely considered as consecutive stages of chromatin disassembly during apoptosis. We report here on apoptosis induced by staurosporine in NB-2a neuroblastoma cells, which was accompanied by excision of chromatin loop domains, but proceeded without internucleosomal DNA cleavage. In contrast to apoptosis associated with internucleosomal DNA fragmentation, the apoptotic pathway associated with excision of chromatin loop domains was largely caspase independent. We identify here MAPK family member, p38/JNK, mitochondria, and topoisomerase II as the components of this caspase-independent apoptotic pathway. While caspase-independent excision of chromatin loop domains was a predominant mechanism of DNA disintegration in staurosporine-treated neuroblastoma, both caspase-dependent internucleosomal DNA fragmentation and caspase-independent excision of chromatin loop domains accompanied staurosporine-induced apoptosis of promyelocytic leukemia cells. Our results suggest that caspase-independent excision of chromatin loop domains represents a separate cell death pathway, which operates either in parallel or independently from caspase-dependent internucleosomal DNA fragmentation.     

Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.     

Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells

Parkinson's disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.     

Mitochondrial regulation of cell death: Processing of apoptosis-inducing factor (AIF)

Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.     

Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of caspase-dependent and -independent pathways

Cadmium is a widely used heavy metal that causes severe damage to many organs including liver, kidney and lung. Cadmium toxicity has been described as in vitro and in vivo apoptosis but its molecular mechanisms are not fully understood. In this study, we used the human lymphoblastoid cell line Boleth to characterise cadmium-induced apoptosis further, using sub-lethal (10 microM) and lethal (IC50: 350 microM) doses. At lethal concentration, we observed features of apoptosis between 6 and 8 h after treatment: maturation of caspases 3 and 8, poly(ADP-ribose)polymerase (PARP) cleavage and DNA fragmentation. In order to determine the role of the MAPKs in this process, we investigated p38, ERK1/2 and c-Jun NH2-terminal kinases (JNK) phosphorylation: at lethal concentration, all these pathways were rapidly activated, but no decrease in the apoptotic rate was seen on inhibition of these kinases with drugs. Chemical inhibitors of caspases 3 and 8 blocked cleavage of PARP but not cell death, suggesting the existence of a caspase-independent death. We found that cadmium depolarised membrane potential in less than 1 h, as determined with DiOC6 dye. Interestingly, mitochondrial alteration led to the translocation of apoptosis-inducing factor (AIF) to the nucleus, where we observed chromatin condensation and possibly DNA fragmentation. These results suggest that cadmium-induced apoptosis can occur in the Boleth cell line through caspase-dependent and -independent pathways, independently of activation of major MAPKs.     

AIF 的生物特性与功能

凋亡诱导因子(AIF)是一种具有氧化还原酶活性的黄素蛋白,在参与线粒体组成和能量代谢方面发挥重要作用。此外,AIF是一种不依赖caspase的凋亡因子,在凋亡刺激影响下,AIF经蛋白酶水解形成57 kD的成熟AIF,释放到胞质中,通过自身的NLS及CypA的辅助作用转位入核,引起染色质凝集和DNA的大片段化,导致细胞凋亡。AIF的这种双功能性是由其结构决定的,特有的插入序列构像的改变可影响其诱导细胞凋亡的活性。本文主要对AIF的结构及其功能作用进行综述。     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

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.