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Originally published In Press as doi:10.1074/jbc.M601751200 on April 27, 2006

J. Biol. Chem., Vol. 281, Issue 27, 18507-18518, July 7, 2006
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Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity*

Cécile Delettre1, Victor J. Yuste2, Rana S. Moubarak3, Marlène Bras4, Nadine Robert, and Santos A. Susin5

From the Apoptose et Système Immunitaire, CNRS-URA 1961, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France

Received for publication, February 23, 2006 , and in revised form, April 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis-inducing factor (AIF)6 is a flavoprotein, with significant homology to bacterial and plant oxidoreductases, located in the mitochondrial intermembrane space (1–3). Under physiological conditions, AIF is a NADH oxidase that plays a role in oxidative phosphorylation (4–6). Moreover, AIF plays a major role in cell death (7). Indeed, after a cellular insult, AIF is cleaved by calpains and/or cathepsins (8, 9) and translocates from mitochondria to cytosol and nucleus where it causes chromatin condensation (10) and large scale (~50 kb) DNA fragmentation in a caspase-independent fashion (1). This apoptogenic function of AIF seems essential in some types of cell death. In fact, AIF was described as a central mediator of relevant experimental models of cell death such as As2O3-induced cell death in human cervical cancer cells (11), DNA damage-mediated p53 activation (12, 13), Sulindac-induced PCD in colon cancer cells (14), geldanamycin-mediated PCD in human glioma cells (15), staurosporine-induced PCD in neuroblastoma cells (10), caspase-independent apoptosis induced by Survivin in melanoma cells (16), hexaminolevulinate-mediated photodynamic therapy in human leukemia cells (17), or poly(ADP-ribose) polymerase-mediated cell death (18, 19). In addition, blockage of the AIF signal transduction pathway seems to be implicated in the chemoresistance of non-small cell lung carcinomas (20) and other human cancers (21). On the other hand, in human colon cancer cells, AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells (22). Thus, AIF is a bifunctional protein with a vital role, via its redox activity in mitochondria, and a lethal role, via its translocation to the nucleus.

AIF comprises 16 exons and locates on chromosome X, region q25–26, and A6 in humans and mice, respectively (23). AIF is expressed as a precursor of 67 kDa. This form is addressed and compartmentalized in mitochondria by two-mitochondrial localization sequences (MLS) located in the N-terminal prodomain. Once in mitochondria, the full-length AIF is processed and the prodomain removed, yielding a mature form of ~57 kDa (1). This form comprises three structural domains: FAD-binding domain, NADH-binding domain, and the C-terminal domain (2). The first two domains compose the oxidoreductase part of AIF, which confers an electron transfer activity to the protein (4). The C-terminal part of AIF seems to be the pro-apoptotic domain (24). One splice variant of AIF, AIF-exB, was reported soon after the initial description of AIF (25). This variant contained an alternative exon 2b instead of the original exon 2. Although the function of this alternatively spliced variant is not yet deciphered, it is established that the alternative exon 2b usage does not affect AIF-exB mitochondrial import and function (25). More recently, our group identified a third AIF isoform, AIFsh (24). This isoform results from an alternate transcriptional start site located at intron 9 of AIF. As a result, AIFsh lacks the AIF N-terminal oxidoreductase domain. Intriguingly, AIFsh, which is a cytosolic protein, provokes the same effects as AIF: chromatin condensation and large-scale (50 kb) DNA fragmentation (24). Therefore, it seems that the oxidoreductase part of AIF is not necessary for the induction of cell death. However, some work suggest that, through the production of reactive oxygen species, the oxidoreductase portion of AIF could play an additional role in caspase-independent apoptosis (4, 22, 26).

In the present article, we report the identification of a novel exon in AIF, which generates two new AIF mRNA transcripts in human (AIFsh2 and AIFsh3) and one in mouse (mAIFsh2). Through biochemical, cellular, and molecular biology approaches, we localized the AIFsh2 resulting protein in mitochondria from mouse liver or human HeLa cells. In contrast, these same approaches failed to detect the AIFsh3 protein. In addition, we explored the expression pattern of AIFsh2 and its biochemical properties and showed that this novel AIF isoform, which is absent in normal brain tissue, exhibited NADH oxidase but no nuclear proapoptotic activity. Together with our previous results (24), our present data completes the knowledge of AIF and helps to better understand the mechanisms regulating life and death AIF-dependent functions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Northern Blotting—First choice Human Blot I membrane containing total RNA from 10 tissues (Ambion) was hybridized as described previously (24) with a 330-bp 32P-labeled exon 9b fragment generated by PCR with forward 5'-TGGAGACTTTAGAAGCTTGGAG-3' and reverse 5'-TTCTGTGCCCAAGGCTCGAGAGGACA-3' primers.

Rapid Amplification of cDNA End (RACE) Analysis—5'- and 3'-RACE were performed using the Marathon Ready cDNA library from human kidney (Clontech) according to the manufacturers recommendation. For the 5'-RACE, the first round of PCR amplification was done using the adaptor primer 1 and reverse primer HCR1 (5'-CCTCGGTGCTCAAGTTCATTTGTAACGC-3'). The nested PCR was done with the nested primer adaptor primer 2 and HAIFshR (5'-CATAAGTGCTTTGCAACCTCTGAATAGGA-3'). For the 3'-RACE PCR the product was amplified using the adaptor primer 1 and the forward primer: I9F (5'-GCGTTACAAATGAACTTGAGCACCGAGG-3').

ARN Extraction and RT-PCR—Total RNA from human tissues was from Stratagene. RNAs from human cancer cell lines and mouse kidney were purified using the RNeasy mini kit (Qiagen). For reverse transcription, 1 µg of total RNA was mixed with 200 units of Superscript II reverse transcriptase (Invitrogen) and 150 ng of random primers (Promega). As a control, reverse transcription was performed without reverse transcriptase. Human AIFsh and AIFsh2 isoforms were amplified simultaneously using the primers I9F (5'-GCGTTACAAATGAACTTGAGCACCGAGG-3') and R4 (5'-CACCAACTGTGGGCAAACTACT-3'). Human AIFsh3 was amplified using primers sh3F1 (5'-AGAGGAACCGGCTCCCAGGCCTA-3') and HCR1 (5'-CCTCGGTGCTCAAGTTCATTTGTAACGC-3'). GAPDH was amplified using primers GAPDHF (5'-ACCACAGTCCATGCCATCAC-3') and GAPDHR (5'-TCCACCACCCTGTTGCTGTA-3'). Mouse AIFsh2 isoform was amplified using primers mAIFsh2F (5'-GCCGAAATGTTCCGGTGTGGA-3') and mAIFsh2R (5'-GCAAGACAGCCTTAATGGCTACGCTTCT-3'). In a set of experiments, PCR products were quantified using the Fluor-S MultiImager from Bio-Rad.

For RNAi experiments AIF was amplified using primers HE7F (5'-GTACAGCTGGATGTGAGAGACAAC-3') and R4 (5'-CACCAACTGTGGGCAAACTACT-3'), AIFsh was amplified using the specific primers I9F2 (5'-TCATGCCCACTGTCCTGTAAGT-3') and R4 (5'-CACCAACTGTGGGCAAACTACT-3'), and AIFsh2 was amplified using the specific primers Hsh2F (5'-TCATGCCCACTGTCCTCTCGAGCCTT-3') and R4 (5'-CACCAACTGTGGGCAAACTACT-3'). The conditions used for the PCR were 94 °C for 2 min, 30 cycles for GAPDH, 35 cycles for AIF, and 40 cycles for AIFsh, AIFsh2, and AIFsh3 of 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. All PCR products were separated by electrophoresis in a 1% agarose gel.

Vector Constructions—Mammalian expression vectors for AIF, AIFsh2, and AIFsh3 were carried out by PCR amplification of the corresponding cDNA fragments and subsequently cloned into cytomegalovirus promoter-based vectors pcDNA3 (Invitrogen), pEGFP-N1 (Stratagene), or C-terminal p3xFLAG (Sigma). Final constructs are referred to as pcDNA3-AIFsh2, pcDNA3-AIFsh3, pEGFP-AIF, pEGFP-AIFsh2, or p3xFLAG-AIFsh2. Empty vectors were used as controls.

Cell Culture, Transfections, and Cell Death Induction—HeLa cells were cultured in complete culture medium (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin, Invitrogen). Cell cultures were maintained at 37 °C with 5% CO2. HeLa cells were seeded at a concentration of 2 x 105 into 6-well plates. Transient transfections were performed using Lipofectamine 2000 in Opti-MEM (Invitrogen) according to the manufacturers protocol. 16 h after pcDNA3-AIFsh2, pcDNA3-AIFsh3, or pcDNA3-empty vector transfection, AIFsh2 and AIFsh3 protein expression was assessed by immunobloting.

For RNAi assays, HeLa cells were transfected with specific siRNA double-stranded oligonucleotides designed against human AIF (I = 5'-GCAGAAAGGCTCGAGCCTT-3'), human AIFsh (II = 5'-GAATGTTTCATGGCAACTA-3'), human AIFsh2 (III = 5'-GGCAGAAAGGATATATGAA-3'), or a common siRNA against human AIF, AIFsh, and AIFsh2 (IV = 5'-GCATGCTTCTACGATATAA-3'). As a control, we used an irrelevant siRNA oligonucleotide (Co. = 5'-GCGATAAGTCGTGTCTTAC-3'). 48 h after the indicated transfection of HeLa cells, AIF, AIFsh, or AIFsh2 mRNA expression was assessed by RT-PCR as described above. In these RNAi transfections we used Oligofectamine (Invitrogen) instead Lipofectamine 2000. Apoptosis was induced by treatment of HeLa cells for 8 h with staurosporine (STS) (1 µM, Sigma), etoposide (50 µM, Sigma), camptothecin (5 µM, Sigma), thapsigargin (10 µM, Sigma), TNF/CHX (25 ng/ml + 2 µg/ml, Sigma), and beta-lapachone (20 µM, Sigma).

Mitochondrial Purification, HeLa Cell Fractionation, and Assessment of AIF/AIFsh2 Release—Mitochondria from HeLa cells or mouse liver were isolated as previously described (9) and resuspended in a buffer containing 10 mM Hepes/KOH, pH 7.2, 250 mM sucrose, and 5 mM EGTA. Mitochondrial protein concentration was determined by the Bio-Rad DC Protein Assay® method. 300 µg of mitochondria were left untreated, treated with Atractyloside (5 mM), or pretreated for 15 min at 4 °C with the broad cysteine protease inhibitor leupeptin (10 µM, Sigma), the cathepsin inhibitor Z-FA-fmk (10 µM, Sigma), or the calpain inhibitor calpeptin (10 µM, Calbiochem) before addition of atractyloside. The mixture was incubated for 35 min at 25 °C and centrifuged (30 min, 4 °C, 13,400 x g) to obtain a mitochondrial pellet (P) or supernatant containing proteins released from mitochondria (SN). Supernatants were further re-clarified (90 min, 4 °C, 100,000 x g) to produce a soluble fraction.

In pcDNA3-AIFsh3 overexpression assays, the cytosolic purification was performed with 2 x 106 HeLa cells. Cells left untreated or treated with STS, etoposide, camptothecin, thapsigargin, TNF/CHX, or beta-lapachone were resuspended in buffer containing 220 mM mannitol, 70 mM sucrose, 50 mM Hepes/KOH (pH 7.2), 10 mM KCl, 5 mM EGTA, 2 mM MgCl2, and 0.025% digitonin, and kept on ice for 5 min. Cells were centrifuged (16,000 x g, 5 min at 4 °C) and the supernatant was retained as the cytosolic fraction. Protein content was quantified by the Bio-Rad DC Protein Assay® method.

Immunofluorescence—For viewing the localization of pEGFP-AIF, pEGFP-AIFsh2, and pEGFP-N1, transfected HeLa cells seeded on coverslips were washed with phosphate-buffered saline three times and stained with Mitotracker Red® (20 nM, Invitrogen) for 15 min at room temperature. Cells were mounted and red (mitochondrial) and green (GFP) fluorescence was observed in a Nikon Eclipse TE2000-U microscope and analyzed using Nikon ACT-1 software.


Figure 1
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  		FIGURE 1.
Schematic representation of the AIF gene structure, and resulting protein isoforms. A, genomic organization of AIF and resulting AIF, AIF-exB, AIFsh, AIFsh2, and AIFsh3 mRNA transcripts (gray line). Translation start (ATG) and stop (TGA/TAA) codons are indicated, and the predicted protein product is shown at the right. Numbers in AIF designate exons and, in predicted proteins, amino acids. MLS, Pyr-redox, nuclear localization sequence (NLS), and C-terminal domains are indicated. I9 indicates intron 9. AIF, AIF-exB, and AIFsh isoforms are described elsewhere (1, 24, 25). The inclusion of the 203-bp exon 9b produces AIFsh2 and AIFsh3, which encodes 324- and 237-amino acid proteins, respectively. AIFsh2 contains the AIF mitochondrial localization sequences and the oxidoreductase domain, but lacks the C-terminal amino acids of AIF. AIFsh3 has a similar structure as AIFsh2 with the splicing of exon 2, which leads to the loss of MLS. B, structure of the novel alternatively spliced exon 9b. AIFsh2 retains a 203-bp long portion of intron 9 as a novel exon, termed exon 9b (nucleotides 28309–28511 of the published sequence of AIF: GenBank accession number NT_011786). Conserved 5' donor site (GT) and 3' acceptor site (AG) that can be recognized by spliceosome are indicated by an asterisk. Y corresponds to C or T nucleotide, N corresponds to any nucleotide.

 
Recombinant Proteins—N-terminal His-tagged AIF, AIFsh, and AIFsh2 human recombinant proteins were produced from a Novagen pET28b expression vector, purified from Escherichia coli extracts on a nickel-nitrilotriacetic acid affinity matrix and stored in 50 mM Hepes, pH 7.9, 100 mM NaCl, 1 mM dithiothreitol, and 10% glycerol until use.

Reconstitution of AIFsh2 and Redox Activity—AIF, AIFsh, and AIFsh2 were reconstituted with FAD following the protocol described by Miramar et al. (4). In situ detection of nitro blue tetrazolium (NBT) reduction on native-PAGE was done using the reaction mixture described elsewhere (27). Briefly, samples were loaded onto a 8% native-PAGE. The gel was incubated 60 min in the dark with 2 mM NBT solution. Then, 1 mM NADH was added to reduce NBT and the reaction was stopped with water after the appearance of the blue band. NAD(P)H oxidase activity of AIFsh2 was performed following procedures described previously (4). Briefly, NAD(P)H oxidase activity was measured at 25 °C in a total volume of 0.5 ml containing 0.25 mM NAD(P)H in 50 mM Tris-HCl, pH 8, buffer. The reaction was initiated by the addition of AIFsh2 and was followed by decrease in absorbance at 340 nm. NBT reduction and superoxide formed in the reaction of AIFsh2 with oxygen were quantified as described (4).


Figure 2
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  		FIGURE 2.
AIF, AIFsh2, and AIFsh3 sequence comparison. A, alignment of amino acid sequences of human AIF, AIFsh2, and AIFsh3. Note that AIFsh2 mimics the N-terminal domain of AIF and lacks amino acids 323–613 at the C terminus of AIF. The AIFsh3 theoretical protein lacks both the mitochondrial localization sequence and the C-terminal domain of AIF. Black boxes indicate identical residues. Gray boxes highlight the two common AIF/AIFsh2 mitochondrial localization sequences. B, alignment of amino acid sequences of mouse AIF and AIFsh2. In this case, mAIFsh2 lacks amino acids 322–612 at the C terminus of mouse AIF. The AIFsh3 mRNA transcript was not detected in mice. Black boxes indicate identical residues.

 
Immunoblot Analysis—For immunoblot analysis, 5µg of purified mitochondria or 20 µg of total or cytosolic cell extract were subjected to a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Millipore), which was blocked and probed with an anti-AIF N-terminal (1/1,000, N-19, Sta Cruz Biotechnology), an anti-AIF C-terminal (1/10,000, epitope being aa 593–613, Sigma), or an anti-FLAG antibody (1/100,000, Sigma) and incubated with a secondary antibody conjugated with peroxidase (1/10,000, Sigma). In transient transfection essays, AIFsh3 was detected with a N-terminal anti-AIF antibody designed against an AIF peptide (1/5000, amino acids 186–216). As substrates for immunodetection, we used the ECL Western blotting analysis system (Amersham Biosciences).

Cell-free System—For the study of chromatin condensation, nuclei from HeLa cells were purified as described by Susin et al. (28). In standard conditions, nuclei (103 nuclei/µl) were cultured in the presence of AIF, AIFsh, or AIFsh2 for 90 min at 37 °C. Nuclei were stained with Hoechst 33342 (0.5 µg/ml, Sigma) and examined by fluorescence microscopy. Nuclei presenting chromatin with a translucid aspect were considered apoptotic. Alternatively, nuclei were stained with propidium iodide (0.5 µg/ml) followed by cytofluorometric analysis (28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Novel Exon in the Human AIF Gene, Which Results in Two Novel AIF Isoforms: AIFsh2 and AIFsh3—Recently, by 3'- and 5'-RACE, we have identified a new AIF transcript that results from an alternate transcriptional start site located in intron 9 of AIF (24). This new transcript encoded for a new protein called AIFsh (24). By a similar 3'- and 5'-RACE approach, we identified here two new AIF cDNA species in a human kidney cDNA Marathon library: AIF short 2 (AIFsh2) and AIF short 3 (AIFsh3). AIFsh2 and AIFsh3 differed from the three previously described AIF forms, AIF, AIF-exB, and AIFsh (1, 24, 25), by alternative usage of a new exon of 203 bp: exon 9b (Fig. 1, A and B). This new exon was located in intron 9 of AIF, between exons 9 and 10 (nucleotides 28309–28511 of AIF, GenBankTM accesion number NT_011786, Gene ID 9131). Analysis of the flanking genomic sequences of exon 9b showed that it contained a consensus for splicing signals (29) (Fig. 1B).

In AIFsh2, exon 9b encoded for only two amino acids (Asp and Ile), which are followed by a stop codon (Fig. 2). The resulting 324 amino acid protein did not contain the AIF C-terminal domain (Figs. 1A and 2A). The AIFsh3 transcript included exon 9b but lacked AIF exon 2 (Fig. 1A). Here, the absence of exon 2 provoked a change in the open reading frame that generates a stop codon. As a result, the translation initiation site for AIFsh3 was located in exon 3 of AIF (Fig. 1A). The resulting putative AIFsh3 protein (237 amino acids) lacked both the mitochondrial localization sequences and the C-terminal domain of AIF (Figs. 1A and 2A).

We next searched for a mouse ortholog of AIFsh2 and AIFsh3. An in silico analysis of the mouse AIF nucleotide sequence failed to detect an equivalent of exon 9b. Although, a RT-PCR assessment, performed between exon 1 and intron 9 (see "Experimental Procedures"), led us to amplify an mRNA transcript (mouse AIFsh2, mAIFsh2) containing the murine ortholog sequence for exon 9b. This exon encoded three amino acids (Cys, Glu, and Tyr), which were followed by a stop codon suppressing the C-terminal domain of mouse AIF (Fig. 2B). The resulting mAIFsh2 protein contained 324 amino acids (Fig. 2B). A similar approach failed to detect the AIFsh3 mouse ortholog.


Figure 3
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  		FIGURE 3.
Subcellular distribution of AIFsh2 and expression pattern of AIFsh3. A, mitochondria from mouse liver or HeLa cells were purified in a Percoll® gradient as described under "Experimental Procedures" and mitochondrial fractionation quality was verified by Western blot analysis of the mitochondrial AIF protein, the lysosomal LAMP1 marker, and the ERK cytosolic protein (left panel). AIFsh2 was immunodetected in liver or HeLa mitochondrial-enriched fractions either with an anti-AIF N-terminal antibody or an anti-AIF C-terminal antibody. Note that only the anti-AIF N-terminal antibody recognizes AIFsh2. A total lysate from HeLa cells transiently transfected with the expression plasmids pcDNA3-empty vector or pcDNA3-AIFsh2 was used as a positive control (right panels). B, expression vectors encoding GFP, AIF-GFP, and AIFsh2-GFP were transfected in HeLa cells. Six hours after transfection, cells were stained with the mitochondrial specific dye Mitotracker Red (20 nM, 15 min). Mitotracker Red and GFP fusion proteins were determined by fluorescent microscopy. Note that, like AIF, AIFsh2 colocalizes with Mitotracker Red in mitochondria. C, HeLa cells were transiently transfected with expression plasmids pcDNA3-empty vector or pcDNA3-AIFsh3. AIF and AIFsh3 were immunodetected with an anti-AIF N-terminal antibody designed against an AIF peptide (amino acids 186–216). Note the absence of AIFsh3 in pcDNA3-empty vector-transfected cells. Right panels showed the expression pattern of AIFsh3 mRNA in a variety of cancer cell lines and human tissues. AIFsh3 expression was analyzed by RT-PCR with forward and reverse primers described under "Experimental Procedures." GAPDH was used as a loading control.

 
Altogether, our findings design a new map for AIF. Indeed, this gene includes a new exon, exon 9b, and codes for, at least, five different transcripts in human: AIF, AIF-exB, AIFsh, AIFsh2, and AIFsh3. In mice, the AIFsh3 mRNA is absent.

AIFsh2 Localizes in Mouse and Human Mitochondria—We next focused on the detection of the AIFsh2 and AIFsh3 mRNA resulting proteins. In this way, given that AIF is confined in mitochondria (1), we checked whether AIFsh2 and/or AIFsh3 were also localized in this organelle. Thus, we first separated mitochondria from other organelles and debris on a discontinuous density gradient. The use of specific antibodies against AIF, LAMP1, and ERK, three markers of the mitochondrial, lysosomal, or cytoplasmic compartments, confirmed the mitochondrial enrichment of our preparations (Fig. 3A, left panel). For AIFsh2 protein detection, we used two complementary approaches:

  1. An immunoblotting assessment performed with an N-terminal anti-AIF antibody in the mitochondrial-enriched fractions obtained from mouse liver or HeLa cells. This approach led us to recognize AIF and a second band of ~35 kDa (Fig. 3A). The molecular mass of this band was compatible with the theoretical molecular mass of AIFsh2, suggesting that AIFsh2 existed and was localized in mitochondria. In this way, immunoblotting of mitochondrial extracts using an antibody against a C-terminal epitope of AIF did not reveal the ~35-kDa band, indicating the specificity of AIFsh2 detection. Indeed, as the C-terminal domain of AIF was absent in AIFsh2, with this antibody we detected AIF but failed to detect AIFsh2 (Fig. 3A). The apparent molecular mass of mitochondrial AIFsh2 was consistent with the molecular mass of the main product resulting from AIFsh2 overexpression assays (Fig. 3A, right panels), further establishing the specificity of the mitochondrial AIFsh2 detection.
  2. In a second immunofluorescent approach, we generated expression plasmids encoding AIF-GFP and AIFsh2-GFP fusion proteins. Upon transfection into HeLa cells AIF-GFP and AIFsh2-GFP distribution patterns were assessed. As depicted in Fig. 3B, in contrast to the cytosolic GFP protein, AIF-GFP and AIFsh2-GFP exhibited a filamentous pattern 6 h after transfection, which colocalized with the specific mitochondrial marker Mitotracker Red. These data corroborated that, like AIF, AIFsh2 localized on mitochondria. It is important to mention that the location of AIFsh2 in mitochondria is in line with the presence of the two MLS previously described for AIF in the AIFsh2 mRNA (Fig. 1A) (1).

Similar cellular subfractionation and immunoblotting approaches failed to identify the AIFsh3 protein in whole cell, cytosolic or mitochondrial extracts from human HeLa cells (data not shown). This negative result was also confirmed in AIFsh3 overexpression assays. Indeed, this approach helped to visualize a ~27-kDa protein in HeLa-transfected cells, which was absent in whole extracts from control cells (Fig. 3C). Thus, it seems that, in this cell line, the human AIFsh3 mRNA is absent or not translated.

To examine whether AIFsh3 mRNA is expressed in HeLa and other commonly used cancer cell lines, a specific RT-PCR approach was developed (Fig. 3C). In this assessment, the GAPDH mRNA amplification was used as a control. Primers sh3F1 and HCR1 were used to detect the AIFsh3 mRNA as a 920-bp product. We amplified AIFsh3 mRNA only in OV10 and 293T cell lines (Fig. 3C). A similar RT-PCR method was used to determine the AIFsh3 mRNA expression pattern in a panel of tissues. In this case, AIFsh3 mRNA was detected as a faint band only in kidney, brain, and colon (Fig. 3C). These results indicate that the AIFsh3 transcript displays different tissue expression both in normal and transformed cells. In HeLa cells, the AIFsh3 mRNA is absent.


Figure 4
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  		FIGURE 4.
AIFsh2 mRNA expression pattern. A, Northern blot analysis of AIFsh2 expression. Northern blot purchased from Ambion was hybridized with an exon 9b probe to detect a single band (~2.5 kb). Note that the AIFsh2 mRNA is absent in the brain tissue. B, expression of AIFsh and AIFsh2 mRNA in a panel of human tissues. Expression of AIFsh and AIFsh2 was analyzed by RT-PCR using a forward primer located in exon 9b and a reverse primer located in exon 14 of AIF. In the lower panel, GAPDH was used as an internal control to ensure that an equal amount of the template was used. AIFsh2 and AIFsh mRNA were quantified in a Fluor-S MultiImager and expressed in a histogram as AIFsh2/AIFsh ratio. C, expression pattern of AIFsh and AIFsh2 mRNA in human cancer cells using the same primers as in B. AIFsh2 and AIFsh were quantified as above.

 
Expression of AIFsh2 mRNA in Human Normal Tissues and Cancer Cells—The tissue distribution and function of AIFsh2 were analyzed further in detail. First, through a Northern blot assessment performed with a specific probe located on exon 9b, we sought to determine its expression pattern in tissues. This approach revealed a single ~2.5-kb mRNA transcript in kidney, skeletal muscle, heart, liver, and colon. Surprisingly, this transcript seemed to be absent in brain (Fig. 4A). To further confirm AIFsh2 distribution, we complemented our Northern blot by a RT-PCR approach. We used total RNA from a panel of tissues (Fig. 4B) and I9F and R4 as primers (described under "Experimental Procedures"). These two primers are designed to simultaneously amplify the AIFsh and AIFsh2 transcripts (as 736- and 599-bp products, respectively), allowing us to compare the expression of these two mRNAs at the same time. GAPDH amplification was used as an internal control. We observed that AIFsh2 mRNA was expressed in all tested tissues except brain (Fig. 4B), thereby confirming the Northern blot result. Interestingly, a different expression pattern was detected between AIFsh and AIFsh2 (Fig. 4B). In fact, quantification of the RT-PCR result allowed us to calculate that, in thyroid, the expression level of AIFsh2 was about 4 times higher than that of AIFsh. A similar expression of the two AIF isoforms was measured in lung and, finally, a more abundant AIFsh transcript was detected in kidney, ovary, muscle, liver, heart, and colon tissues (Fig. 4B).


Figure 5
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  		FIGURE 5.
Different modulation of AIF, AIFsh, and AIFsh2 transcripts. A, schematic representation of the four siRNA double-stranded oligonucleotides used in RNAi assays. siRNA I was AIF specific, siRNA II was AIFsh specific, siRNA III was AIFsh2 specific, and siRNA IV interferes with the expression of AIF, AIFsh, and AIFsh2. B, HeLa cells were transfected with a scramble siRNA double-stranded oligonucleotide (Co.), siRNA I, siRNA II, siRNA III, or siRNA IV. 48 h after the indicated transfection, expression of AIF, AIFsh, or AIFsh2 was analyzed by RT-PCR. GAPDH was used as a loading control.

 
Transcription of AIFsh2 was also investigated, by a similar RT-PCR approach, in 12 human cancer cell lines (Fig. 4C). AIFsh2 transcript was amplified in all cancer cell lines tested with a lower expression in SH-SYSY, IMR5, U937, IMR32, and HeLa cells (Fig. 4C). In contrast, AIFsh2 was highly expressed (8 times more than AIFsh) in Ramos. Note that even if the AIFsh2 mRNA is absent from brain tissue (Fig. 4, A and B), it is detected in neuroblastoma-derived cell lines (e.g. SH-SYSY, IMR5, and IMR32) (Fig. 4C). Thus there seems to be a different regulation in normal and tumor tissues from the brain lineage. In any case, as it happens in normal tissues, AIFsh and AIFsh2 are differentially expressed in tumor cells.

Our above RT-PCR data strongly suggested that AIFsh2 and AIFsh expression are regulated separately. To confirm this possibility, we used an RNAi approach (Fig. 5A). Indeed, if AIFsh2 and AIFsh transcripts are regulated independently, it should be possible to interfere with the expression of one mRNA without disturbing the transcription of the other. In this way, using the RNAi approach depicted in Fig. 5A, we fully confirmed this outcome (Fig. 5B). Interestingly, a similar result was found in RNAi assays designed to independently down-regulate AIF, AIFsh, or AIFsh2 (Fig. 5B). Thus, there seems to be three individual AIF, AIFsh, and AIFsh2 mRNAs, which could be autonomously regulated.

AIFsh2 Redox Activities—Under physiological conditions, AIF is a mitochondrial NADH oxidase that plays a role in oxidative phosphorylation (4, 5). In fact, the oxidoreductase part of AIF, which is conserved in AIFsh2, confers an electron transfer activity to the protein (4–6). Thus, we generated the human AIFsh2 recombinant protein to investigate whether AIFsh2 presents the same NADH oxidase activity as AIF. AIFsh, which lacks the AIF oxidoreductase domain, was used as a negative control. AIF, AIFsh, and AIFsh2 recombinant proteins were synthesized in E. coli and the FAD moiety was reconstituted by external addition. As depicted in Fig. 6A, the absorption spectrum of reconstituted AIFsh2 showed the typical features of an oxidized flavoprotein, with visible maximums at 381 and 453 nm and a shoulder at 478 nm. These absorbance characteristics are similar to the spectrum obtained with AIF. In contrast, reconstituted AIFsh showed no visible maximums (Fig. 6A). Thus, AIF and AIFsh2 proteins associated FAD, whereas, in contrast, AIFsh did not incorporate this flavin. Consequently, AIF- and AIFsh2-reconstituted holoproteins showed NADH oxidase activity measured by in situ NBT detection (Fig. 6A, inset), whereas the reconstituted AIFsh lacked any detectable NADH oxidase-NBT reductase activity (Fig. 6A). This fact suggests that, like AIF, AIFsh2 exhibits an oxidoreductase activity.

We next investigated the characteristics of the AIFsh2 redox function and showed that AIFsh2 features NADH and NADPH oxidase activities (Fig. 6B). NAD(P)H oxidation in the presence of AIFsh2 was followed by measuring the initial rates of A340 nm. The apparent Km for NADH was calculated as 102.6 ± 7 µM and the turnover number 2.7 min–1. When NADPH was used as electron donor, the apparent Km was 45.3 ± 9 and the turnover number 3.5 min–1. These kinetic parameters are very similar to previous values described for AIF (4), indicating that AIFsh2 has major affinity for NADH. When oxidizing NADH or NADPH, AIFsh2 catalyzed the reduction of NBT (Fig. 6C). This was due to the AIFsh2(FADH2)-mediated reduction of O2 to Formula (Fig. 6D). In this way, the reduction of NBT was abolished by 10 units/ml of the Formula scavenger superoxide dismutase (data not shown). In summary, our data fully confirms that AIFsh2 exhibits a similar NADH oxidase activity to AIF (4).


Figure 6
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  		FIGURE 6.
Redox activity of AIFsh2. A, absorption spectrum of FAD and reconstituted recombinant AIF, AIFsh, and AIFsh2. The inset shows the recovery of the NADH oxidase activity of recombinant proteins after FAD reconstitution measured by in situ NBT detection. B, NAD(P)H oxidation by AIFsh2. NADH (•) and NADPH ({circ}) oxidation were measured following absorbance variation at 340 nm, after addition of different amounts of NAD(P)H. AIFsh2 was added at a concentration of 3 mM. C, AIFsh2 induced NBT reduction with NADH (•) and NADPH ({circ}) as electron donors. AIFsh2 was added at a concentration of 100 nM. D, NADH (•) and NADPH ({circ}) oxidase activities of AIFsh2 result in generation of superoxide anion. AIFsh2 concentration was 100 nM.

 


Figure 7
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  		FIGURE 7.
AIFsh2 release from the intermembrane space of mitochondria. A, purified mitochondria were left untreated (Mit.), treated with Atractyloside alone (Control,5mM), or pretreated with the broad cysteine protease inhibitor leupeptin (Leup., 10 µM), the cathepsin inhibitor Z-FA-fmk (10 µM), or the calpain inhibitor calpeptin (Calpep., 10 µM) before addition of Atractyloside. After incubation, the mixture was centrifuged to separate mitochondrial pellet (P) or supernatants (SN) containing proteins released from mitochondria. Atractyloside treatment provokes both AIF cleavage into tAIF and mitochondrial release of the AIF soluble form. Like AIF, AIFsh2 is cleaved and released from mitochondria after Atractyloside treatment. Leupeptin, Z-FA-fmk, and calpeptin significantly alleviated AIF/AIFsh2 cleavage and release. B, STS, etoposide, camptothecin, thapsigargin, TNF/CHX, or beta-lapachone proapoptotic treatment provokes AIFsh2 redistribution from mitochondria to cytosol. HeLa cells were transfected with p3xFLAG-AIFsh2. 16 h after transfection total cell lysates were subjected to a double immunoblot analysis performed with a N-terminal anti-AIF antibody or an anti-FLAG antibody. As shown in the left panels, the anti-FLAG antibody specifically recognizes AIFsh2-FLAG. In a similar set of experiments, 16 h after transfection HeLa cells were left untreated (Co.) or treated 8 h with STS (1 µM), etoposide (Etop., 50 µM), camptothecin (Ctp., 20 µM), thapsigargin (Thap., 10 µM), TNF/CHX (25 ng/ml + 2 µg/ml), or beta-lapachone (beta-lap., 20 µM) and subjected to subcellular fractionation. Cytoplasmic fractions were blotted for immunodetection of the flagged protein (right panel). Note the presence of tAIFsh2-FLAG in the cytosol of STS, etoposide, camptothecin, thapsigargin, TNF/CHX, or beta-lapachone-treated cells. It is important to underline that each proapoptotic treatment produces the loss of 75% in cell viability measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). Cytosolic fractionation quality and protein loading were verified by the distribution of the specific subcellular marker ERK. The mitochondrial COX IV protein was used to confirm the purity of the cytosolic fractions.

 
In Pro-apoptotic Conditions, AIFsh2 Is Released from Mitochondria to Cytosol in a Calpain/Cathepsin-dependent Manner—After a cellular insult, AIF is released from mitochondria and translocated to cytosol and nucleus, where it achieves a caspase-independent pro-apoptotic function (1, 12, 19, 28, 30, 31). It is well established that, to be released from mitochondria AIF is cleaved into tAIF by proteolytic processing at position Gly102-Leu103 of the mouse AIF amino acid sequence (9). This processing is accomplished by µ-calpain or cathepsins B, L, or S (8, 9).

AIFsh2 mimics the N-terminal sequence of AIF, which includes the calpain/cathepsin cleavage site. Thus, accepting the complexity of cell death regulation and execution (32), we could assume that AIFsh2 can be cleaved into tAIFsh2 in a µ-calpain-dependent or cathepsin-dependent fashion. To verify this possibility, we used two independent tools: (i) purified mitochondria. If AIF and AIFsh2 are similarly released from mitochondria, it should be possible to detect tAIF and tAIFsh2 in supernantants from mitochondria treated with Atractyloside, an agent highly effective in inducing mitochondrial AIF liberation (9); and (ii) AIFsh2 overexpression in HeLa cells. With this approach, we searched whether AIFsh2 was released from mitochondria to cytosol after classical proapoptotic insults, such as the tyrosine kinase inhibitor STS, the topoisomerase II blocker etoposide, the topoisomerase I inhibitor camptothecin, the inhibitor of the endoplasmic reticulum Ca2+-ATPase thapsigargin, the death receptor TNF, and the 1,2-naphthoquinone beta-lapachone. In this assay, each proapoptotic treatment was designed to produce, after 8 h of injury, the loss of 75% cell viability (measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, data not shown). Using the first approach, we confirmed that it is possible to detect tAIFsh2 in supernatants from Atractyloside-treated mitochondria (Fig. 7A). Thus, like AIF, AIFsh2 is also cleaved and released from the intermembrane space of mitochondria. Interestingly, AIFsh2 cleavage and release was precluded by the broad cysteine protease inhibitor leupeptin, the cathepsin inhibitor Z-FA-fmk, or the calpain inhibitor calpeptin. Thus it seems that like AIF, AIFsh2 is cleaved into tAIFsh2 and released from mitochondria in a calpain or a cathepsin-dependent fashion. Our second approach, the detection of AIFsh2 in cytosol purified from STS, etoposide, camptothecin, thapsigargin, TNF/CHX, or beta-lapachone-treated HeLa cells, fully confirmed that, like AIF, AIFsh2 is released from the mitochondria to cytosol after an apoptotic insult (Fig. 7B).


Figure 8
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  		FIGURE 8.
Effects of AIFsh2 on isolated nuclei. A, purified HeLa nuclei were incubated in the absence (control) or presence of human recombinant proteins AIF, AIFsh, or AIFsh2 at 7 µg/ml and stained by Hoescht 33342. Chromatin condensation was determined by fluorescent microscopy. B, in addition, nuclei were stained with propidium iodide and the percentage of hypoploid nuclei was measured by flow cytometry. Numbers indicate the % of hypoploid nuclei. C, dose response of the apoptogenic effect of AIF, AIFsh, and AIFsh2 on nuclei. This experiment has been done three times, yielding lower interexperimental variability (<5%).

 
Effect of AIFsh2 on Isolated Nuclei—We previously demonstrated that the C-terminal domain of AIF contains the information to induce nuclear apoptosis (24). However, is there an additional role of the oxidoreductase part of AIF, which is mimicked by AIFsh2, in nuclear cell death? Using a cell-free in vitro system in which the recombinant AIFsh2 protein was confronted to purified HeLa nuclei, we answered this question (30). AIF and AIFsh were used as positive controls. After a 90-min incubation, nuclear morphology was assessed by fluorescent and cytofluorometric approaches (33). As expected, recombinant AIF and AIFsh caused peripheral chromatin condensation and loss of DNA content. In contrast, recombinant AIFsh2 yielded negative results (Fig. 8, A–C). Addition of NADH or NADPH failed to disclose apoptogenic activity on AIFsh2. Moreover, AIFsh2 was unable to provoke large scale DNA fragmentation or the "ladder type" of chromatin digestion in purified HeLa nuclei (data not shown), confirming that AIFsh2 was not able to reproduce the nuclear features characterizing AIF/AIFsh action on nuclei (1, 24). Altogether these results indicate that the reactive oxygen species produced by AIF/AIFsh2 do not induce nuclear apoptosis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The identification of the new exon 9b illustrates a continuously increasing complexity of the human AIF gene. There are now five variants described for AIF: AIF, AIF-exB, AIFsh, AIFsh2, and AIFsh3. The first two isoforms differ in the alternative use of exon 2 or exon 2b (25) and are targeted to the mitochondrial intermembrane space. We recently described a third variant of AIF, AIFsh, which results from an alternate transcriptional start site located at intron 9 of AIF (24). In the present study we identified exon 9b, an additional exon located between exons 9 and 10 of AIF. This novel exon leads to two new AIF cDNA species: AIFsh2 and AIFsh3. We failed to detect the AIFsh3 protein in human HeLa cells. However, AIFsh2 mRNA transcript yields a novel mitochondrial form of AIF composed by 324 amino acids, which include the two MLS and the pyridine nucleotide-disulfide oxidoreductase (Pyr-redox) domain of AIF. In this context, AIFsh2 is able to incorporate FAD and exhibit NADH oxidase activity. In fact, like AIF, AIFsh2 reacts rapidly to O2 forming Formula.

AIFsh and AIFsh2 differ in a main feature: AIFsh2 lacks the C-terminal domain of AIF and has no apoptogenic activity on purified nuclei. In contrast, AIFsh lacks the N-terminal domain of AIF and has no oxidoreductase activity (Figs. 1A and 6A and Ref. 24). These complementary results confirm that AIF is a bifunctional protein with dissociable redox and apoptogenic moieties. Indeed, contrary to other bifunctional proapoptotic proteins (e.g. cytochrome c) (34, 35), AIF yields an independent genetic regulation of its two different functions. This is an important issue to understand the physiological role of AIF.

Although AIFsh2 mitochondrial function remains unknown, the presence of the Pyr-redox domain and oxidoreductase activity suggests that, like other Pyr-redox proteins (36, 37), AIFsh2 may be involved in a variety of redox-dependent processes such as antioxidant defense and regulation of the cellular redox state. Moreover, because AIFsh2 shares similar redox functions to AIF, we may speculate that these two proteins have similar functions in mitochondria. In this sense, the different expression levels of AIF and AIFsh2 tested in a variety of tissues and cancer cell lines indicated that the two variants are differentially regulated. This was confirmed by an RNAi approach. It is important to remark that our RNAi approach provides new instruments to evaluate the specific role of AIF, AIFsh, and AIFsh2 in cell death and in oxidative phosphorylation. Indeed, from now on it should be possible to avoid generation of AIF and AIFsh, two caspase-independent death effectors. Moreover, it would be possible to obtain, for the first time, a cellular system in which AIF, AIFsh, and AIFsh2 isoforms are down-regulated. In any case, it is possible that AIFsh2 substitutes the redox function of AIF in some particular conditions (e.g. normal versus tumor cells). The different AIFsh2 expression observed in brain normal/tumor tissue suggests this possibility.

AIF redox activity was described as having a protective function in some cell types on the Harlequin mutant mouse, which has an 80% reduction in AIF. This mutant mouse appeared normal except for premature neurodegeneration and increased peroxide sensitivity in specific subsets of neurons in adult animals (38). The precise mechanism of action of AIF in free radical scavenging and counteracting oxidative stress in this mouse model remains to be established. Therefore, a particular study of AIFsh2 in these animals could greatly help to understand AIF/AIFsh2 function. More recently, it has also been shown that a redox-active domain of AIF and reduced glutathione are required for the inhibition of cytoplasmic stress granule formation under conditions of chemical stress, suggesting that AIF is involved in an adaptive response (39). Moreover, AIF deficiency compromises oxidative phosphorylation by inhibiting respiratory chain complex I in vitro and in vivo, revealing a "life" function for AIF (5, 6). Finally, AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function (22). Unfortunately, the mechanistic relations between the redox activity of AIF, complex I, oxidative phosphorylation, and cell survival remain unclear. Moreover, in these systems, the genetic suppression of the AIF transcript will lead to AIFsh2 ablation. Thus, it is difficult to evaluate whether, like AIF, AIFsh2 may play a role in electron transfer and have a function in the defense against oxidative damage.

We demonstrated here that the cysteine protease mechanism implicated in mitochondrial AIF cleavage and release provokes a similar outcome in AIFsh2. Thus, it is possible that, in apoptotic conditions, deficiencies in oxidative phosphorylation may not only be related to AIF mitochondrial loss. The AIFsh2 ectopic presence could contribute to the defects in oxidative phosphorylation detected in apoptotic cells. Obviously, a mutant mouse specifically suppressing AIF or AIFsh2 could greatly help in the understanding of the mitochondrial function of these two proteins.

In summary, and despite the functional similarities between AIFsh2 and AIF (e.g. electron transfer activity), AIFsh2 has three different features. First, AIFsh2 mRNA is not expressed in brain. Second, AIFsh2 lacks the C-terminal domain of AIF. Third, AIFsh2 lacks the proapoptotic activity of AIF. These interesting features definitely confirm that the C-terminal domain of AIF is necessary and sufficient to induce AIF-dependent nuclear apoptosis. Indeed, by using the recombinant protein AIFsh2, we have demonstrated that the first 323 amino acids of the N-terminal domain in AIF are not required for its nuclear proapoptotic activity. Thus, AIF does not induce chromatin condensation and 50-kb DNA fragmentation through the production of the Formula reactive oxygen radical associated with this part of the protein.

In conclusion, our present study completes the knowledge of AIF. The identification and characterization of a novel AIF variant, lacking its apoptotic domain, provides a "natural mutant" that would greatly help in the understanding of the functional role of AIF in mitochondria and, by extension, in the understanding of the bifunctional role of AIF in life and death.


   FOOTNOTES
 
* This work was supported in part by grants from Fondation de France and Association pour la Recherche sur le Cancer contract number 4812. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) DQ016498, DQ016500, and DQ016499 for the human AIFsh2, AIFsh3 and mAIFsh2, respectively. Back

1 Supported by a postdoctoral fellowship from Fondation de France. Back

2 Supported by a Marie Curie Intra-European fellowship within the 6th European Community Framework Programme under contract MEIF-2003-501887. Back

3 Supported by a Ph.D. fellowship from the Fondation Hariri. Back

4 Supported by a Ph.D. fellowship from Caisse Nationale d'Assurance Maladie des Professions Indépendantes (CANAM)-Pasteur. Back

5 To whom correspondence should be addressed: Apoptose et Systeme Immunitaire, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France. Tel.: 33-1-40-61-31-84; Fax: 33-1-40-61-31-86; E-mail: susin{at}pasteur.fr.

6 The abbreviations used are: AIF, apoptosis inducing factor; AIFsh2, apoptosis inducing factor short 2; AIFsh3, apoptosis inducing factor short 3; CHX, cycloheximide; GFP, green fluorescent protein; NBT, nitro blue tetrazolium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAIFsh2, mouse apoptosis inducing factor short 2; MLS, mitochondrial localization sequence; RACE, rapid amplification of cDNA ends; RT, reverse transcription; TNF, tumor necrosis factor; RNAi, RNA interference; siRNA, small interfering RNA; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; ERK, extracellular signal-regulated kinase; STS, staurosporine. Back


   ACKNOWLEDGMENTS
 
We thank Martine Cohen-Salmon and Marcela Segade for critical comments and review of the manuscript and Sophie Laine for p3xFLAG-AIFsh2 plasmid construction.



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 ABSTRACT
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 DISCUSSION
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