SciELO - Scientific Electronic Library Online

vol.42 número2Effects of substrate water potential in root growth of Agave salmiana Otto ex Salm-Dyck seedlingsFe de Erratas índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados


Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.42 n.2 Santiago  2009 

Biol Res 42: 249-260, 2009

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal



1  State Key Laboratory of Cancer Biology, Departments of Immunology, the Fourth Military Medical University, Xi'an, China.

2  Department of Medical Oncology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.

3 Biochemistry and Molecular Biology, the Fourth Military Medical University, Xi'an, China.

4 Preclinical Medical Teaching Lab Center, the Fourth Military Medical University, Xi'an, China.


Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

Key terms: apoptosis inducing factor (AIF), apoptosis, cytochrome c, mitochondria, HER2.



The apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein that mediates both NADH-oxidizing and caspase--independent apoptosis (Lorenzo et al., 1999, Susin et al., 2000, Cande et al., 2002). The mammalian AIF precursor consists of an N-terminal mitochondrial localization sequence (MLS, amino acid (aa) 1-100), a spacer sequence (aalOl-121),

an FAD-binding domain (aal22-262 and aa400-477), an NADH-binding domain (aa263-399), and a C-terminal domain (aa478-613) (Mate et al., 2002). It has been reported that the C-terminal domain is responsible for the proapoptotic activity of AIF (Ye et al., 2002). AIF is normally confined to the mitochondrial intermembrane space, where it probably acts as an NADH-oxidoreductase and catalyzes electron transfer in the mitochondrial respiratory chain (Susin et al., 1999). In response to the apoptosis signal, AIF is translocated into the cytosol and then, the nucleus. The AIF proteins bind to DNA and trigger peripheral chromatin condensation, large-scale DNA fragmentation (approximately 50 kb), and apoptosis in the nucleus (Zhang et al., 2002). Studies have suggested that the oxidoreductase activity of AIF is not essential for its proapoptotic activity (Delettre et al., 2006, Miramar et al., 2001). The strongly positive electrostatic potential present on the surface of the AIF protein is responsible for its DNA-binding activity, which is independent of the DNA sequence and is crucial for inducing apoptosis (Ye et al., 2002). The mechanisms of AIF-mediated chromatin condensation and DNA degradation in apoptosis have not been completely elucidated. The possible mechanisms include the following: (1) AIF may bind to DNA and increase the susceptibility of the DNA to some nucleases; (2) AIF may recruit proteases and nucleases that subsequently cause chromatin condensation and fragmentation; and (3) AIF may itself possesses some protease and nuclease activities.

Once released from the mitochondria, AIF can initiate and mediate caspase-independent apoptosis, a process that does not require the participation of cytosolic factors and that cannot be prevented by intrinsic caspase inhibitors or antiapoptotic proteins, e.g., bcl-2 (Lorenzo et al., 1999). Moreover, knockout of the AIF gene is lethal in mouse embryos, indicating the indispensable role played by critical caspase-independent apoptosis during the early stages of mammalian development (Cecconi et al., 1998). AIF-mediated apoptosis is also involved in the physiological and pathological death of most eukaryocytes (Boujrad et al., 2007). The accumulation of AIF in the extramitochondrial compartment, particularly in the nucleus, leads to caspase-independent apoptosis (Loeffler et al., 2001). Susin et al. have confirmed that AIFΔ1-120, an MLS-truncated AIF, is located in the cytoplasm and has proapoptotic activity comparable to that of wild-type AIF (Susin et al., 1999). Yu et al. generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A (PEA) translocation domain, and AIFΔ1-120 and demonstrated that the antibody/AIF fusion protein efficiently triggered apoptosis of HER2-overexpressing tumor cells (Yu et al., 2006). Recently, alternatively spliced variants of AIF have been identified. AIFsh, which contains aa353-613 of AIF, is a proapoptotic isoform and another new isoform AIFsh2 possesses intact NADH oxidase activity (Delettre et al., 2006,).

Here, we generated a truncated AIF, namely, AIFΔ1-480, which comprised aa481-613 of the wild-type protein, and demonstrated that AIFΔ 1-480 effectively induced apoptosis in transfected cells a process that involves the release of cytochrome c from the mitochondria and possibly the activation of caspase-9. We also generated the genes of a chimeric proteins, designated immuno-AIFΔ 1-480, in which AIFΔ 1-480 were fused sequentially at the N-terminus to a cell translocation domain PEA, an anti-HER2 single chain antibody and a signal peptide. The chimeric proteins were expected to selectively kill HER2-overexpressing tumor cells.


DNA constructs

The cDNAs that encode AIFΔ1-480 and AIF360-480 were amplified by polymerase chain reaction (PCR) from pcDNA3-AIFΔl-120 with the corresponding primers (Table). The resulting fragments, which contained the coding sequence of a FLAG tag at the 3'-end, were cloned into the pcDNA3 vector (Fig 1). To inhibit the expression of caspase-9 by siRNA, a 19-nt target sequence in the coding region of the human caspase-9 gene was empirically chosen. Synthesized oligodeoxyribonucleotides encoding the corresponding siRNA were designed as follows. The 19-nt target sequence was used as the sense strand, and this was followed by a 9-nt spacer and complementary antisense strand with 5 repeat Ts as the termination signal. For the negative control, a double-stranded oligonucleotide, which contained a 19-nt sequence that did not match any known human gene, was introduced into the pSUPER.puro vector (Table). The genes of recombinant immuno-AIFAl-480 was generated by sequential fusion of the coding sequences of a signal peptide (Met-Lys-His-Leu-Trp-Phe-Phe-Leu-Leu-Leu-Val-Ala-Ala-Pro-Arg-Trp-Val-Leu-Ser-), a signal-chain HER2 antibody (sFv23e), a PEA translocation domain (aa253-358) and AIFΔ1-480. The immuno-AIFΔl-480 gene was cloned into a pCMV plasmid respectively. All constructs were confirmed by DNA sequencing.

Cell culture and transfection

Human cervical carcinoma cell line HeLa, human breast tumor cell line SKBR-3, human gastric cancer cell line SGC-7901, human umbilical vein endothelial cell line ECV304, human laryngeal tumor cell line Hep2 and human lymphoma cell line Jurkat were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% C02. At 24 h before transfection, the cells were seeded in 12-well plates at a density of 1 x 105 cells per well. Transfection was performed at 70-80% cell confluence by using LipofectAMINE 2000 (Invitrogen) using the standard procedure. For the transient expression of truncated AIF, the cells were harvested at the indicated time after transfection. For stable expression, transfected cells were selected using puromycin (50 mg/ml) or G418 medium (800 mg/ml) for 3-4 weeks.

Semiquantitative RT-PCR analysis

Total RNA was extracted, and cDNA was prepared from HeLa cells stably transfected with pSUPER.puro-siC9 (expressing shRNA targeting caspase-9) and pSUPER.puro-NC (negative control) after puromycin selection. Using C9U as the upstream primer and C9D as the downstream primer (corresponding to bp 98-793 of the caspase-9 cDNA), the caspase-9 gene was amplified from the cDNA templates. PCR amplification of (3-actin using primers BU and BD was used as the control. The primers used for PCR amplification are as follows. C9U 3'-agctgtt caggccccata-3'; C9D, 5'-tcacaatcttctcgaccg-3'; BU, 5'-tggcaccacaccttctacaa-3'; BD, 5'-gcagctcgtagctcttctcc-3'.

Western blot analysis

The lysates of the AIFAl-480-transfected cells were concentrated and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were transferred to polyvinylidene difluoride membranes (Amersham Biosciences) and incubated sequentially with mouse anti-FLAG primary antibody (1: 2000) and a biotin-conjugated secondary antibody. The bands were developed using 3, 3' -diaminobenzidine tetrahydrochloride (DAB) western blot detection reagents. Mouse monoclonal caspase-9 antibody (Abeam) (1: 2000) and the secondary antibodies ZB2305  anti-mouse immunoglobulin horseradish peroxidase-linked F(ab)2 fragments from goat (Zhongshan Goldenbridge Ltd.) were used for western blot analysis of caspase-9, and the bands were developed with enhanced chemiluminescence (ECL) western blotting detection reagents (Pierce Biotechnology Inc.).

Laser confocal microscopy

HeLa cells cultured on coverslips were fixed, permeabilized, and stained for AIF-FLAG. A polyclonal anti-FLAG antibody (Sigma) was used to detect the expression of truncated AIF, and a SABS-cy3-conjugated rabbit anti-mouse IgG was used to amplify and visualize the staining. The nuclei were stained by 4', 6-diamidino-2-phenylindole (DAPI). The expression of truncated AIF-Flag (red) in transfected cells was assessed by observation using laser confocal microscopy.

Annexin V/PI staining

The transfected HeLa cells were collected, washed, and stained using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). To quantify the apoptosis, flow-cytometric analysis was performed using an ELITE ESP Flow Cytometer (Beckman-Coulter).

Electron microscopy

The transfected HeLa cell pellets were fixed for 2 h at 4°C with 2.5% glutaraldehyde in 0.1 mol/1 sodium phosphate buffer (pH 7.4). The pellets were washed 3 times and fixed in 1% osmic acid in phosphate buffer, followed by scraping, dehydration, and embedding. Ultrathin sections were mounted on a 200-mesh grid and examined under a GEM-2000EX electron microscope.

Cell viability assay

The cells transfected with truncated AIF constructs were cultured in 12-well plates for 48 h. Cells were trypsinized and resuspended in an appropriate volume of pre-warmed growth medium to give a cell density of 106 cells/ml. Add 0.5 ml 0.4% (w/v) trypan blue and 0.3 ml PBS to 0.1 ml of the cell suspension. Count the stained and unstained cells using a hemocytometer. The percent viable cells was determined as follows: (number of unstained cells/total number of cells)xl00%. Each assay was performed in triplicate by conducting at least 3 independent experiments.

For the methyl-thiazol-tetrazolium (MTT) assay, the cells transfected with truncated AIF constructs were cultured in 96-well plates for 24-96 h. The cells were then incubated with 20 ml of 1.5 mg/ml MTT per well for 4 h, followed by the addition of 150 \i\ dimethyl sulfoxide (DMSO). The OD at 490 nm was determined using a Sunrise microplate reader (Tecan).

Indirect immunofluorescence staining

The HeLa cells cultured on coverslips were fixed, permeabilized, and stained for AIF. A polyclonal anti-Cyt C antibody (Santa Cruz) was used to detect the release of cytochrome c, and a rabbit anti-goat IgG conjugated to biotin and SABS-Cy3 were used to amplify and visualize the staining.

In vitro cytotoxicity of immuno-AIFAl-480 proteins

HER2-overexpressing SKBR-3 and SGC-7901 cells, as well as ECV-304 and Hep2 cells that rarely express HER2 were seeded into 12-well plates at a density of lxl05cells/well, and 0.5 ml/well supernatants prepared from 2xl06 modified Jurkat cells were added every 12 h. Viable cells were counted by trypan blue exclusion at the indicated times after cocultivation. The percentages of cell killing were determined as follows: (1-the number of cells cocultured with the supernatant of Jurkat-immuno-AIFs or Jurkat-pCMV-sFv23e-PEA/the number of cells cocultured with the supernatant of untransfected Jurkat cell)xl00%. Each assay was performed in triplicate by conducting at least 3 independent experiments.

Statistical analysis

Statistical analyses were performed with the SPSS 11.5 software package for Windows. Statistical significance was based a p-value of 0.05 (p<0.05).


Ectopic expression and subcellular location of truncated AIFs in HeLa cells

HeLa cells were transfected with the pcDNA3 constructs of FLAG-tagged truncated AIF. At 24 h after transfection, the expressions of AIFΔ1-120, AIFΔ1-480, and AIF360-480 were confirmed by western blotting using a FLAG antibody (Fig. 2A). Unlike AIFΔ1-120, which was detected in both the cytoplasm and the nucleus, confocal microscopy revealed that AIFΔ1-480 and AIF360-480 were distributed exclusively in the cytoplasm (Fig. 2B).

Expression of AIFAl -480 induced apoptosis in transfected HeLa cells

The proapoptotic activity of truncated AIFs in HeLa cells was assessed. Annexin V/PI staining revealed that the percentages of apoptotic cells in the transfectants of AIFΔl-120 and AIFΔ 1-480 were 25.1% and 30.4%, respectively, which was in contrast to 2.0% and 2.4% apoptotic cells observed in the parental HeLa cells and AIF360-480-transfected cells, respectively (Fig. 3A). These data suggested that AIFΔ1-480 could efficiently trigger apoptosis and that cytoplasmic AIF play a role in mediating apoptotic signals.

The typical features of apoptotic cells as detected by electron microscopy were plasma membrane blebbing, peripheral chromatin condensation, and nuclear shrinkage (Fig. 3B). Cell viability assay showed decreased viable cells in AIFΔl-120 and AIFΔ1-480 transfectants when compared with AIF360-480-transfected and the parental HeLa cells (Fig. 3C). Thus, the apoptosis-inducing ability of the N-terminal-truncated AIF, AIFΔ 1-480, which lacks FAD- or NADH-binding activity and nuclear translocation ability, was almost unaffected.

The expression of AIFΔ1-480 induced cytochrome c release from the mitochondria

Since the AIFΔ1-480 protein was located in the cytoplasm of transfected cells, the direct binding of truncated AIF to chromosomal DNA was prevented, implying that the cytoplasmic targets and signaling of truncated AIF are responsible for the initiation of apoptosis. Therefore, we examined the release of cytochrome c in the transfected cells. In fact, indirect immunofluorescence staining revealed that the expression of AIFΔ 1-480 in HeLa cells induced cytochrome c release from the mitochondria, which is a process also observed in AIFAl-120-transfected cells but not in the parent cells or cells transfected with AIF360-480 constructs (Fig. 4).

Caspase-9 is required for the proapoptotic activity of AIFΔl-480

In cells undergoing typical apoptosis, the release of cytochrome c from the mitochondria leads to the activation of caspase-9, and then, caspase-3 (Yang et al., 1997). This involves the formation of an apoptosome, which is a multiprotein complex comprising cytochrome c, Apaf-1, pro-caspase-9, and ATP (Lorenzo et al., 2007). To test whether caspase-9 participates in the apoptotic pathway mediated by AIFΔ1-480, we knocked down the expression of the caspase-9 gene in HeLa cells by using RNAi. The stable expression of caspase-9-targeted siRNA from a pSUPER.puro vector resulted in a decrease of approximately 70% in caspase-9 expression (Fig. 5A and B). The cell viability assay demonstrated that the proapoptotic activity of AIFΔ 1-480 was inhibited by caspase-9 knockdown (Fig. 5C). These results suggest that the proapoptotic activity of AIFΔ1-480 is mediated by caspase-9-dependent cytoplasmic signaling events.

Secreted immuno-AIFΔl-480 from modified Jurkat cells selectively kill HER2-overexpressing tumor cells

To further probe whether the AIF C-terminal domain is structurally and functionally independent and whether it maintains a proapoptotic activity when covalently incorporated into heterogeneous peptides, we generated the genes of a chimeric proteins, designated immunoAIFΔ 1-480. Human lymphoma Jurkat cells stably transfected with pCMV-immuno-AIFΔ 1-480 or previously reported pCMV-immuno-AIFA1-120 were modified to express and secrete the corresponding chimeric proteins. Western blot analysis confirmed the expression and the secretion of immuno-AIFΔ1-480 and immuno-AIFΔl-120 proteins, as shown in Figure 6A.

The secreted immuno-AIFΔ 1-480 and immuno-AIFΔl-120 proteins show significant cytotoxic effects on both HER2-overexpressing cells lines, but not on HER2-negative cells (Fig. 6B). These data indicate that immuno-AIFΔl-480 molecules secreted from lymphocytes specifically recognize and kill HER2-overexpressing tumor cells, but not normal or tumor cells lacking HER2 expression in vitro.


AIF is a multifunctional mitochondrial protein, which mediates both oxidoreductase activity (via its FAD- and NADH-binding domains) and caspase-independent apoptosis (partially by its DNA-binding activity) (Mate et al., 2002, Miramar et al., 2001). Studies on the crystal structures of mouse and human AIF have provided substantial information regarding its role in the cell (Mate et al., 2002), which have extended our understanding of molecular mechanisms underlying the proapoptotic activity of AIF; thus, making it a potential candidate for cancer gene therapy (Lorenzo et al., 2007). However, a previous study has shown that the C-terminal domain, consisting of 136 amino acid residues, is responsible for the proapoptotic activity of AIF protein, and Susin et al. demonstrated that AIFΔ 1-120 could induce cell apoptosis and that further truncation impaired the proapoptotic activity of AIF (Susin et al, 1999). Ce' cile Delettre et al. found a novel AIF proapoptotic isoform AIFsh in many cancer cells. AIFsh contains the C-terminal amino acids from 353-613, and a study has demonstrated that AIFsh triggers chromatin condensation and DNA fragmentation of isolated nuclei (Delettre et al., 2006). In our study, we deleted the N-terminal l-480aa of the AIF protein and demonstrated that this truncated variant, despite lacking NADH-binding and nuclear translocation abilities, induces remarkable apoptosis of HeLa cells in cytochrome c- and caspase-9-dependent manners.

The cell viability assay showed that the proliferation of AIFΔ1-480- and AIFΔ1-120-transfected HeLa cells was significantly inhibited. We also detected the cell cycle of transfected cells. Compared to the parent or negative cells, the cell cycle of AIFΔl-480-transfected cells was not obviously affected (data not shown). Together, these results demonstrate the effectiveness of AIFΔ 1-480 in inhibiting cell survival and growth via the triggering of apoptotic signal pathways.

A previous study has shown that AIF induces apoptosis in a caspase-independent manner as this process is not inhibited by the pan-caspase inhibitor z-VAD-fmk (Daugas et al., 2000, Pérez-Galán et al., 2002). By introducing serially truncated AIF into HeLa cells, we observed significantly inhibited cell growth and increased apoptosis following the expression of AIFΔ1-120 or AIFΔ1-480. Unlike AIFΔ1-120, the ectopic expression of AIFΔ 1-480 did not affect cell cycle, suggesting a diversity of mechanisms through which AIF induces apoptotic cell death. Cytochrome c is a key regulator of apoptosis (Zou et al., 1999, Yoshida et al., 1998, Li et al., 2000). The release of cytochrome c from the mitochondria leads to the activation of caspase-9 and subsequently of caspase-3. This involves the formation of an apoptosome, which is a multiprotein complex comprising cytochrome c, Apaf-1, pro-caspase-9, and ATP (Genini et al., 2000, Jiang et al., 2000). In our study, we observed that cytochrome c was released from the mitochondria in AIFA 1-480- and AIFΔ1-120-transfected cells. These data suggest a crosstalk between the classical AIF apoptotic signaling and caspase pathways. Although the mechanism by which cytoplasmic AIF causes the release of cytochrome c from the mitochondria remains to be elucidated, the caspase-9-dependent signaling may represent an alternative apoptotic pathway of AIF and a conserved approach through which AIF joins the mainstream cellular apoptotic machinery.

Chimeric proteins comprising antibodies and cytotoxic molecules hold out great promise in targeted elimination of cells undergoing pathological alterations, e.g. carcinogenesis (Yu et al., 2006, Jia et al., 2003, Wang et al., 2007). Yu et al. generated a fusion protein, immuno-AIFΔl-120, which consisted of a HER2 antibody, a PEA translocation domain, and AIFΔ1-120. The chimeric protein could be internalized via endocytosis and undergoes a furin-mediated cleavage between Arg279 and Gly280 of the original PEA peptide, releasing its C-terminal peptide into the cytosol. This peptide, which comprises a PE sequence and AIF1-120, then induces apoptosis (Yu et al., 2006). However, only a minority of the truncated AIF could be released into the cytosol probably due to a large molecular weight of AIFΔ1-120. Given that we have demonstrated a similar proapoptotic activity of AIFΔ 1-480 with AIFΔ1-120, we developed a smaller immuno-AIF variant, immuno-AIFΔ 1-480. Indeed, immuno-AIFΔl-480 exhibited a significantly higher efficiency than immuno-AIFΔl-120 in specifically killing HER2-overexpressing tumor cells. Our finding that AIF exerts a proapoptotic effect independent of its large FAD- and NADH-binding domains has implications in the development of tumor-targeted therapeutics with simplified constituents but enhanced cytotoxicity.


This work was supported by grants from the National High Technology Research and Development Program of China (2001AA217101), the National Basic ResearchProgram  of China

(2004CB518805), National Natural Science Foundation of China (30672430) and the Program for Changjiang Scholars and Innovative Research Teams in Universities (PCSIRT) from the Ministry of Education of People's Republic of China (IRT0459).


BOUJRAD H, GUBKINA O, ROBERT N, KRANTIC S, SUSIN SA (2007) AIF-mediated programmed necrosis: a highly regulated way to die. Cell Cycle 6: 2612-9        [ Links ]

CANDE C, COHEN I, DAUGAS E, RAVAGNAN L, LAROCHETTE N, ZAMZAMI N, LOEFFLER M, COSTANTINI P, FERRI KF, IRINOPOULOU T, PRÉVOST MC, BROTHERS G, MAK TW, PENNINGER J, EARNSHAW WC, KROEMER G (2002) Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie 84: 215-22        [ Links ]

CECCONI, F, ALVAREZ-BOLADO, G, MEYER, BI, ROTH KA, GRUSS P (1998) Apafl (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94: 727-37        [ Links ]

DAUGAS E, SUSIN SA, ZAMZAMI N, FERRI KF, IRINOPOULOU T, LAROCHETTE N, PRÉVOST MC, LEBER B, ANDREWS D, PENNINGER J, KROEMER G (2000) Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 14: 729-39        [ Links ]

DELETTRE C, YUSTE VJ, MOUBARAK RS, BRAS M, LESBORDES-BRION JC, PETRES S, BELLALOU J, SUSIN SA (2006) AIFsh, a novel apoptosis-inducing factor (AIF) pro-apoptotic isoform with potential pathological relevance in human cancer. J Biol Chem 281: 6413-27        [ Links ]

DELETTRE C, YUSTE VJ, MOUBARAK RS, BRAS M, ROBERT N, SUSIN SA (2006) Identification and characterization of AIFsh2, a mitochondrial apoptosis-inducing factor (AIF) isoform with NADH oxidase activity. J Biol Chem 281: 18507-18        [ Links ]

GENINI D, BUDIHARDJO I, PLUNKETT W, WANG X, CARRERA CJ, COTTAM HB, CARSON DA, LEONI LM (2000) Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway. J Biol Chem 275: 29-34        [ Links ]

JIA LT, ZHANG LH, YU CJ, ZHAO J, XU YM, GUI JH, JIN M, JI ZL, WEN WH, WANG CJ, CHEN SY, YANG AG (2003) Specific tumouricidal effect of a novel secreted pro-apoptotic fusion protein consisting of caspase-3 and anti-erbB2/HER2 antibody. Cancer Res 63: 3257-62        [ Links ]

JIANG X, WANG X (2000) Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-l.J Biol Chem 275: 31199-203        [ Links ]

LI K, LI Y, SHELTON JM, RICHARDSON JA, SPENCER E, CHEN ZJ, WANG X, WILLIAMS RS (2000) Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101: 389-99        [ Links ]

LI P, NIJHAWAN D, BUDIHARDJO I, SRINIVASULA SM, AHMAD M, ALNEMRI ES, WANG X (1997) Cytochrome c and dATP-dependent formation of Apaf-l/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479-89        [ Links ]

LOEFFLER M, DAUGAS E, SUSIN SA, ZAMZAMI N, METIVIER D, NIEMINEN AL, BROTHERS G, PENNINGER JM, KROEMER G (2001) Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 15: 758-67        [ Links ]

LORENZO HK, SUSIN SA (2007) Therapeutic potential of AIF-mediated caspase-independent programmed cell death. Drug Resist Updat 10: 235-55        [ Links ]

LORENZO HK, SUSIN SA, PENNINGER J, KROEMER G (1999) Apoptosisinducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 6: 516-24        [ Links ]

MATE MJ, ORTIZ-LOMBARDIA M, BOITEL B, HAOUZ A, TELLO D, SUSIN SA, PENNINGER J, KROEMER G, ALZARI PM (2002) The crystal structure of the mouse apoptosis-inducing factor AIF. Nat Struct Biol 9: 442-6        [ Links ]

MIRAMAR MD, COSTANTINI P, RAVAGNAN L, SARAIVA LM, HAOUZI D, BROTHERS G, PENNINGER JM, PELEATO ML, KROEMER G, SUSIN SA (2001) NADH-oxidase activity of mitochondrial apoptosis inducing factor (AIF). J Biol Chem 276: 16391 -8        [ Links ]

PÉREZ-GALÁN P, MARZO I, GIRALDO P, RUBIO-FÉLIX D, LAS IERRA P, LARRAD L, ANEL A, NAVAL J (2002) Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia. Leukemia 16: 2106-14        [ Links ]

SUSIN SA, DAUGAS E, RAVAGNAN L, SAMEJIMA K, ZAMZAMI N, LOEFFLER M, Costantini P,. Ferri KF, Irinopoulou T, Prévost MC, Brothers G, Mak TW, Penninger J, Earnshaw, WC, Kroemer G (2000) Two distinct athways leading to nuclear apoptosis. J Exp Med 192: 571-9        [ Links ]

SUSIN SA, LORENAO HK, ZAMZAMI N, MARZO I, SNOW BE, BROTHERS GM, MANGION J, JACOTOT E, COSTANTINI P, LOEFFLER M, LAROCHETTE N, GOODLETT DR, AEBERSOLD R, SIDEROVSKI DP, PENNINGER JM, KROEMER G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441-6        [ Links ]

WANG T, ZHAO J, REN JL, ZHANG L, WEN WH, ZHANG R, QIN WW, JIA LT, YAO LB, ZHANG YQ, CHEN SY, YANG AG (2007) Recombinant Immunoproapoptotic Proteins with Furin Site Can Translocate and Kill HER2-Positive Cancer Cells. Cancer Res 67: 11830-9        [ Links ]

YANG J, LIU X, BHALLA K, KIM CN, IBRADO AM, CAI J, PENG TI, JONES DP, WANG X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129-32        [ Links ]

YE H, CANDE C, STEPHANOU NC, JIANG S, GURBUXANI S, LAROCHETTE N, DAUGAS E, GARRIDO C, KROEMER G, WU H (2002)DNA binding is required for the apoptogenic action of apoptosis inducing factor. Nat Struct Biol 9: 680-4        [ Links ]

YOSHIDA H, KONG YY, YOSHIDA R, ELIA AJ, HAKEM A, HAKEM R, PENNINGER JM, MAK TW (1998) Apafl is required for mitochondrial pathways of apoptosis and brain development. Cell 94: 739-50        [ Links ]

YU CJ, JIA LT, MENG YL, ZHAO J, ZHANG Y, CHUN XC, XU YM, WENG WH, YAO LB, FAN DM, JIN BQ, CHEN SY, YANG AG (2006) Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2. Gene Ther 13: 313-20        [ Links ]

ZHANG X, CHEN J, GRAHAM SH, DU L, KOCHANEK PM, DRAVIAM R, GUO F, NATHANIEL PD, SZABÓ C, WATKINS SC, CLARK RS (2002) Intranuclear localization of apoptosis inducing factor (AIF) and large scale dna fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite. J Neurochem 82: 181-91        [ Links ]

ZOU H, LI Y, LIU X, WANG X (1999) An Apaf-1 .cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274: 11549-56.        [ Links ]

* These authors contributed equally to this work.

** Corresponding Authors. Dr. An-Gang Yang, Department of Immunology, Dr. Lin-Tao Jia, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, 17 Changle West Road, 710032, Xi'an, China. E-mail address: Tel. 86-29-84774528, Fax. 86-29-83253816

Received: January 12, 2009 In Revised form: June 3, 2009. Accepted: June 8, 2009

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons