Molecular Cloning and Characterization of a Human AIF-like Gene with Ability to Induce Apoptosis
2005; Elsevier BV; Volume: 280; Issue: 20 Linguagem: Inglês
10.1074/jbc.m409517200
ISSN1083-351X
AutoresQiang Xie, Tianxin Lin, Yan Zhang, Jianping Zheng, Joseph A. Bonanno,
Tópico(s)ATP Synthase and ATPases Research
ResumoIn this study, we cloned and characterized a human gene homologous to the apoptosis-inducing factor (AIF), which is named AIF-like (AIFL). Human AIFL has 598 amino acids, with a characteristic Rieske domain and a pyridine nucleotide-disulfide oxidoreductase domain (Pyr_redox). AIFL shares 35% homology with AIF, mainly in the Pyr_redox domain. Reverse transcriptase-PCR analysis showed the expression of AIFL mRNA in all tissues tested, i.e. brain, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, placenta, small intestine, and testis. We developed antibodies against human AIFL using fusion proteins as antigens. The antibodies specifically recognized the antigen and heterologously expressed AIFL proteins. The expression of AIFL proteins in human tissues was also ubiquitous, demonstrated by immunohistochemistry in tissue array slides. Subcellular fractionation and immunofluorescence staining studies revealed that AIFL is predominantly localized to the mitochondria. Similar to AIF, overexpression of AIFL induced apoptosis, as shown by increased cytoplasmic nucleosomes and subdiploid cell populations in AIFL-transfected cells. The segment 1–190 containing the Rieske domain induced apoptosis, whereas the segment containing the Pyr_redox domain did not contribute to the pro-apoptotic function. The mitochondrial membrane potential of cells transfected with AIFL was significantly more depolarized than that of the control. AIFL transfection-induced cytochrome c release and cleavage of caspase 3. Furthermore, the pan-caspase inhibitor Z-VAD-fmk inhibited AIFL induced apoptosis. In summary, AIFL induces apoptosis in a caspase-dependent manner when heterologously expressed. In this study, we cloned and characterized a human gene homologous to the apoptosis-inducing factor (AIF), which is named AIF-like (AIFL). Human AIFL has 598 amino acids, with a characteristic Rieske domain and a pyridine nucleotide-disulfide oxidoreductase domain (Pyr_redox). AIFL shares 35% homology with AIF, mainly in the Pyr_redox domain. Reverse transcriptase-PCR analysis showed the expression of AIFL mRNA in all tissues tested, i.e. brain, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, placenta, small intestine, and testis. We developed antibodies against human AIFL using fusion proteins as antigens. The antibodies specifically recognized the antigen and heterologously expressed AIFL proteins. The expression of AIFL proteins in human tissues was also ubiquitous, demonstrated by immunohistochemistry in tissue array slides. Subcellular fractionation and immunofluorescence staining studies revealed that AIFL is predominantly localized to the mitochondria. Similar to AIF, overexpression of AIFL induced apoptosis, as shown by increased cytoplasmic nucleosomes and subdiploid cell populations in AIFL-transfected cells. The segment 1–190 containing the Rieske domain induced apoptosis, whereas the segment containing the Pyr_redox domain did not contribute to the pro-apoptotic function. The mitochondrial membrane potential of cells transfected with AIFL was significantly more depolarized than that of the control. AIFL transfection-induced cytochrome c release and cleavage of caspase 3. Furthermore, the pan-caspase inhibitor Z-VAD-fmk inhibited AIFL induced apoptosis. In summary, AIFL induces apoptosis in a caspase-dependent manner when heterologously expressed. Apoptosis is an important cellular process occurring in both normal development and pathological conditions in all metazoans (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar, 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar, 3Jiang X. Wang X. Annu. Rev. Biochem. 2004; 73: 87-106Crossref PubMed Scopus (1138) Google Scholar). Generally speaking, there are two pathways for apoptosis: intrinsic and extrinsic. The extrinsic pathway is initiated upon death receptor activation (4Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar). The intrinsic pathway is mainly through mitochondria (5Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar, 6Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar, 7Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar). Importantly, mitochondria is one of the integrators in initiating and executing apoptosis. 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In particular, apoptosis can still happen in cells that lack cytochrome c, Apaf-1, or caspases (20Li K. Li Y. Shelton J.M. Richardson J.A. Spencer E. Chen Z.J. Wang X. Williams R.S. Cell. 2000; 101: 389-399Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 21Yoshida H. Kong Y.Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar). AIF and EndoG can cause apoptotic changes independent of caspase activity (10Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3464) Google Scholar, 12Li L.Y. Luo X. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1404) Google Scholar). 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In addition, AIF was also shown to act as a free radical scavenger (28Klein J.A. Longo-Guess C.M. Rossmann M.P. Seburn K.L. Hurd R.E. Frankel W.N. Bronson R.T. Ackerman S.L. Nature. 2002; 419: 367-374Crossref PubMed Scopus (519) Google Scholar) and play a role in normal mitochondrial oxidative phosphorylation (29Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (521) Google Scholar). Upon stimulus by some death signals, AIF is released from mitochondria and translocates into the nucleus (10Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. 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A gene homologous to the human AIF, known as AIF-homologous mitochondrion-associated inducer of death (AMID) (27Wu M. Xu L.G. Li X. Zhai Z. Shu H.B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 34Wu M. Xu L.G. Su T. Tian Y. Zhai Z. Shu H.B. Oncogene. 2004; 23: 6815-6819Crossref PubMed Scopus (38) Google Scholar) and p53-responsive gene 3 (PRG3) (35Ohiro Y. Garkavtsev I. Kobayashi S. Sreekumar K.R. Nantz R. Higashikubo B.T. Duffy S.L. Higashikubo R. Usheva A. Gius D. Kley N. Horikoshi N. FEBS Lett. 2002; 524: 163-171Crossref PubMed Scopus (83) Google Scholar), has also been reported to induce apoptosis when heterologously expressed. By blasting the human genome with AIF, we find a third gene that shares sequence homology to AIF, which we call AIF-like gene (AIFL). Because both AIF and AMID/PRG3 are involved in apoptosis, we hypothesized that AIFL may also play a role in apoptosis. The purpose of this study was to clone the human AIFL gene and characterize its expression and function. RT-PCR and immunohistochemistry revealed the ubiquitous expression of AIFL in human tissues. Heterologous expression of AIFL protein in HEK 293 cells induced apoptosis in a caspase-dependent manner. Mitochondrial membrane potential (ΔΨm) was more depolarized in AIFL-transfected cells than the control. Taken together, we cloned the AIFL gene with pro-apoptotic activities when heterologously expressed. Bioinformatics—Gene blasting was performed using the BLAST server (www.ncbi.nlm.nih.gov/BLAST) (36Zhang J. Madden T.L. Genome Res. 1997; 7: 649-656Crossref PubMed Scopus (274) Google Scholar). Domain structure was analyzed by the SMART program (smart.embl-heidelberg.de) (37Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3029) Google Scholar). The mitochondrial localization algorithm was analyzed by the Mitopred program (mitopred.sdsc.edu) (38Guda C. Guda P. Fahy E. Subramaniam S. Nucleic Acids Res. 2004; 32: W372-W374Crossref PubMed Scopus (67) Google Scholar, 39Guda C. Fahy E. Subramaniam S. Bioinformatics. 2004; 20: 1785-1794Crossref PubMed Scopus (115) Google Scholar). Multiple sequence alignment was carried out using ClustalW (www.ebi.ac.uk/clustalw/#) (40Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). Primers were designed by the Vector NTI software (Invitrogen, Carlsbad, CA). RT-PCR Cloning of Human AIFL—We designed PCR primers (see Supplemental Materials Table I) to amplify the hypothetical gene FLJ30473. Using RT-PCR, we amplified the full-length sequence of human AIFL from pooled human small intestine and colon cDNAs (Clontech, Palo Alto, CA). The PCR was carried out in a total volume of 50 μl using the high-fidelity TaqDNA polymerase according to the manufacturer's instructions (Roche Diagnostics). Thirty-five PCR cycles (94 °C for 30 s, 56 °C for 45 s, and 68 °C for 3 min) were performed after an initial denaturation at 94 °C for 3 min followed with a final extension at 68 °C for 10 min. Sequencing of the PCR product revealed a cDNA sequence identical to the hypothetical gene FLJ30473. RT-PCR Analysis of the Tissue Distribution of AIFL Gene Expression—RT-PCR was performed using human cDNAs from Clontech. The PCR were carried out in a total volume of 50 μl using the high-fidelity TaqDNA polymerase according to the manufacturer's instructions (Roche). Thirty-five PCR cycles (95 °C for 30 s, 61 °C for 30 s, and 68 °C for 30 s) were performed after denaturation at 95 °C for 2 min followed with a final extension at 68 °C for 10 min. PCR primers are listed in supplemental materials Table I. All RT-PCR products were subjected to 2% agarose gel electrophoresis, stained by ethidium bromide, and visualized by ultraviolet illumination. The band was cut and purified using the Qiagen gel extraction kit (Qiagen, Valencia, CA). The purified PCR products were subcloned into the pcDNA3.1-V5-His-TOPO vector (Invitrogen, Carlsbad, CA). The inserted PCR products were sequenced using the T7 primer. Plasmid Constructs—The AIFL full-length cDNA contains a BamHI site at 1789–1794 bp (just before the stop codon). To facilitate cloning of AIFL into different vectors, the 1791 base (T) was substituted with A to eliminate the BamHI site without changing the amino acid sequence of the protein (supplemental materials Table I). For expression of AIFL in mammalian cells, AIFL was cloned into the EcoRI and XhoI sites of pcDNA3 after PCR amplification of the full-length cDNA by primers with the EcoRI site on the 5′ end and the SalI site on the 3′ end (the SalI site is compatible with the XhoI site of the pcDNA3 vector) because AIFL has an intrinsic XhoI site. For expression of AIFL-YFP fluorescent protein, AIFL was cloned into the BamHI and XhoI sites of a pcDNA3-YFP vector. All truncation mutants of AIFL were constructed similarly. To make MBP fusion proteins, the cDNA sequences of AIFL-(1–82) and –(191–598) were PCR amplified and inserted into the BamHI and SalI sites of the pMAL6H vector. All plasmids were sequenced by the Indiana University School of Medicine or University of Southern California Comprehensive Cancer Center DNA sequencing facilities. All primers used in plasmid construction are listed in supplemental materials Table I. Generation and Purification of the Anti-AIFL Antibody—The fusion proteins MBP-AIFL-(1–82) and MBP-AIFL-(191–598) were induced by isopropyl 1-thio-β-d-galactopyranoside (0.5 mm) for 4 h at 30 °C. Bacteria were centrifuged at 4000 × g for 30 min at 4 °C. Bacteria were suspended, sonicated on ice, and centrifuged. Both fusion proteins were soluble and purified by affinity chromatography using amylose resin (New England Biolabs, Beverly, MA). The purity of proteins was examined by Coomassie staining. Briefly, the SDS-PAGE gel was soaked in Coomassie solution (40% methanol, 7% acetic acid, 0.25% Coomassie Blue) and destained in the destaining solution (40% methanol, 7% acetic acid). The purified proteins MBP-AIFL-(1–82) and MBP-AIFL-(191–598) were injected into New Zealand White rabbits (Covance, Philadelphia, PA) and the resultant antisera were named anti-AIFL-(82) and anti-AIFL-(191–598), respectively. The antisera were further purified by affinity chromatography. Briefly, purified MBP, MBP-AIFL-(1–82) and MBP-AIFL-(191–598) were covalently coupled to CNBr Sepharose beads (Amersham Biosciences, Piscataway, NJ). The sera were first purified by protein A and G (Sigma) and then by the MBP-CNBr beads to absorb anti-MBP IgGs. MBP-AIFL-(1–82) and MBP-AIFL-(191–598) CNBr beads were then used to purify the remaining IgGs. Finally, purified antibodies were eluted with 0.2 m glycine (pH 2.8) and neutralized with 1 m Tris-HCl (pH 8.0). The concentration of anti-AIFL-(1–82), AIFL-(191–598), and preimmune sera was 1:1000 in Western blotting. Transfection—HEK 293 cells, DU145, and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Invitrogen, Carlsbad, CA). Transfection was performed using the Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, 1 day before transfection, HEK 293 cells were subcultured in antibiotic-free medium supplemented with 10% fetal calf serum (Invitrogen). DNA and Lipofectamine 2000 were mixed for 30 min before transfection. After transfection for 1 h in OPTI-MEM I medium (Invitrogen), fresh antibiotic-free medium with 10% fetal calf serum was added. Subcellular and Submitochondrial Fractionation—Plasma membrane (PM) proteins were extracted using the biotinylation approach (41Planes C. Blot-Chabaud M. Matthay M.A. Couette S. Uchida T. Clerici C. J. Biol. Chem. 2002; 277: 47318-47324Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). After washing the cells 5 times with buffer A (20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 250 mm sucrose and Protease Inhibitors; Sigma), the cells were biotinylated using sulfo-NHS-biotin (Pierce, Rockford, IL) at room temperature for 30 min. Excessive biotin was quenched and washed off by buffer A plus 100 mm glycine. Cells were lysed in RIPA buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor mixture (Sigma), and centrifuged at 10,000 × g to pellet cell debris. 100 μl of streptavidin beads (Pierce) were added to the supernatant and rotated for 2 h at room temperature to pull down biotinylated protein. The biotinylated proteins were plasma membrane proteins. Other subcellular fractionations were carried out similarly as described in Nijhawan et al. (42Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (520) Google Scholar) and Zhu et al. (43Zhu H. Larade K. Jackson T.A. Xie J. Ladoux A. Acker H. Berchner-Pfannschmidt U. Fandrey J. Cross A.R. Lukat-Rodgers G.S. Rodgers K.R. Bunn H.F. J. Biol. Chem. 2004; 279: 30316-30325Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cultured cells were collected and suspended in PBS. After centrifugation at 1,000 × g for 10 min, the cell pellet was resuspended in buffer A on ice for 20 min. Then the cells were passed through a 23-gauge needle 25 times. The resulting broken cells were centrifuged in three sequential steps: 1,000, 10,000, and then 100,000 × g. The 10,000 × g pellet was the mitochondrial fraction and the 100,000 × g supernatant was the cytosol. The 100,000 × g pellet was the microsomal membrane fraction, which includes endoplasmic reticulum (ER) membranes. The 100,000 × g supernatant was the cytosolic fraction. Nuclear proteins were extracted from the 1,000 × g pellet using the nuclear extract kit (Active Motif, Carlsbad, CA). Antibodies for organelle markers were tubulin (1:2000, Developmental Studies Hybridoma Bank (DHSB), Iowa City, IA), calreticulin (1:2000, Sigma), porin (1:2000, Molecular Probes), sodium potassium ATPase (1:1000, DHSB, Iowa City, IA), and poly(ADP-ribose) polymerase (1:2000, Calbiochem, San Diego, CA). Submitochondrial fractions were extracted according to the methods of Milon et al. (44Milon L. Meyer P. Chiadmi M. Munier A. Johansson M. Karlsson A. Lascu I. Capeau J. Janin J. Lacombe M.L. J. Biol. Chem. 2000; 275: 14264-14272Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), Valgardsdottir et al. (45Valgardsdottir R. Ottersen O.P. Prydz H. Exp. Cell Res. 2004; 299: 294-302Crossref PubMed Scopus (8) Google Scholar), and Liu and Spremulli (46Liu M. Spremulli L. J. Biol. Chem. 2000; 275: 29400-29406Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, an equal volume of fresh 1.2% digitonin solution was mixed with the above mitochondria fraction. The suspension was diluted 1:4 by buffer A and centrifuged at 9,500 × g for 15 min. The supernatant contained outer membrane and intermembrane space fractions and was further centrifuged at 165,000 × g for 20 min. The resultant pellet was the outer membrane and the supernatant was the intermembrane space fraction. The pellet after 9,500 × g for 15 min, containing the mitoplasts, was washed once with buffer A. The mitoplast pellet was resuspended in fractionation buffer (20 mm HEPES-KOH, pH 7.6, 40 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol, protease inhibitor mixture (Sigma), and homogenized in a Dounce homogenizer. The sample was sonicated and centrifuged at 500 × g for 45 min. The supernatant was collected and centrifuged for 20 min at 165,000 × g. The resultant pellet was the inner membrane fraction and the supernatant was the matrix fraction. The antibodies for compartment markers are porin (1:2000, Molecular Probes), cytochrome c (1:2000, Calbiochem), cytochrome c oxidase IV subunit IV (COX IV) (1:2000, Molecular Probes), and heat shock protein 60 (1:2000, StressGen Biotechnologies, Victoria, BC, Canada). Western Blotting—After transfection, cells were washed twice with PBS and sonicated briefly in RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor mixture (Sigma). Protein concentrations were determined with the Bio-Rad Protein DC assay kit (Bio-Rad). Protein samples were separated by SDS-PAGE as described previously (47Sun X.C. Bonanno J.A. Jelamskii S. Xie Q. Am. J. Physiol. 2000; 279: C1648-C1655Crossref PubMed Google Scholar). 10 μg of protein from transfected cells was loaded in each lane. The samples were then transferred to polyvinylidene difluoride membranes, which were blocked with 5% nonfat dry milk in PBST for 1 h at room temperature or overnight at 4 °C, incubated with primary antibodies (AIFL-(1–82) and AIFL-(191–598) or pre-immune serum, 1:1000) containing 5% dry milk for an additional hour, washed with PBST, and then incubated with a secondary antibody at 1/10,000 dilution in PBST containing 5% dry milk. The secondary antibodies were peroxidase-conjugated goat anti-rabbit or mouse IgG (Sigma) and the blots were detected with an enhanced West-Pico chemiluminescence kit (ECL) as described by the manufacturer (Pierce). The rabbit anti-green fluorescent protein antibodies were obtained from Molecular Probes (Eugene, OR). Immunohistochemistry—Immunohistochemistry was performed as described previously (48Zheng J. Rudra-Ganguly N. Miller G.J. Moffatt K.A. Cote R.J. Roy-Burman P. Am. J. Pathol. 1997; 150: 2009-2018PubMed Google Scholar). The Histostain-Plus Kits and MaxArray™ Human Normal Tissue Microarray Slides (Zymed Laboratories Inc.) were used for the tissue array study. The tissue array contains 30 different human samples. Sections of 4 μm thick, paraffin-embedded tissue microarrays were deparaffinized and rehydrated. The slides were then treated in 3% H2O2 in methanol for 10 min and blocked with serum blocking solution for 10 min to reduce nonspecific background. The affinity-purified anti-AIFL-(191–598) rabbit polyclonal antibody (1:50) was applied to the slides and incubated at room temperature for 1 h. The slides were then incubated with biotinylated goat anti-rabbit secondary antibody (kit supply) at room temperature for 10 min followed by streptavidin peroxidase conjugate for 10 min. Aminoethyl carbazole was added as a peroxidase substrate and incubated for 15 min. Cell nuclei were counterstained by hematoxylin to give a blue background contrast to red color of the positive reaction. The sections were cover-slipped and viewed under light microscope. Immunofluorescence Staining—Two days after transfection with pcDNA3-AIFL, HeLa cells on coverslips were washed with PBS three times and stained by Mitotracker Red (Molecular Probes) for 10 min. After fixation by 2% paraformaldehyde for 15 min, the coverslips were blocked by
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