Abstract
22 min readApoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFΔ1–120) exhibit NADH oxidase activity, whereas superoxide anion (O2−) is formed. AIFΔ1–120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFΔ1–120, which lacks FAD, has no NADH oxidase activity. However, native AIFΔ1–120, apoAIFΔ1–120, and the reconstituted (FAD-containing) holoAIFΔ1–120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFΔ1–120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFΔ1–120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function. Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFΔ1–120) exhibit NADH oxidase activity, whereas superoxide anion (O2−) is formed. AIFΔ1–120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFΔ1–120, which lacks FAD, has no NADH oxidase activity. However, native AIFΔ1–120, apoAIFΔ1–120, and the reconstituted (FAD-containing) holoAIFΔ1–120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFΔ1–120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFΔ1–120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function. apoptosis-inducing factor mitochondrial transmembrane potential 5.5′-dithiobis-(2-nitrobenzoate-(Nbs2) 2,2′-di-p-nitrophenyl-5–5′-diphenyl-3,3′ [3–3′-dimetoxy-4–4′difenilen]tetrazolium chloride superoxide dismutase dithiothreitol high performance liquid chromatography bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane polyacrylamide gel electrophoresis 2,6-dichlorophenolindophenol Mitochondria are considered as central players in apoptosis of mammalian cells (1Green D.R. Kroemer G. Trends Cell Biol. 1998; 8: 267-271Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 2Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 3Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar). Early during the apoptotic process, the outer mitochondrial membrane becomes permeabilized, and mitochondria release soluble proteins normally confined to the intermembrane space (4Patterson S. Spahr C.S. Daugas E. Susin S.A. Irinopoulos T. Koehler C. Kroemer G. Cell Death Differ. 2000; 7: 137-144Crossref PubMed Scopus (170) Google Scholar). Such apoptogenic proteins include the caspase activator cytochromec (5Budijardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2274) Google Scholar), procaspases 2, 3, and 9 (6Mancini M. Nicholson D.W. Roy S. Thornberry N.A. Peterson E.P. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 1998; 140: 1485-1495Crossref PubMed Scopus (373) Google Scholar, 7Krajewski S. Krajewska M. Ellerby L.M. Welsh K. Xie Z.H. Deveraux Q.L. Salvesen G.S. Bredesen D.E. Rosenthal R.E. Fiskum G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5752-5757Crossref PubMed Scopus (483) Google Scholar, 8Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Larochette N. Alzari P.M. Kroemer G. J. Exp. Med. 1999; 189: 381-394Crossref PubMed Scopus (637) Google Scholar), the inhibitor of apoptosis protein (IAP) inhibitor Smac/DIABLO (9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 10Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar), as well as AIF1 (11Susin 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). In contrast to cytochrome c and Smac/DIABLO, AIF is a caspase-independent death effector, which translocates via the cytosol to the nucleus, where it causes chromatin condensation and large scale (50 kilobase pairs) DNA fragmentation (12Daugas E. Susin S.A. Zamzami N. Ferri K. Irinopoulos T. Larochette N. Prevost M.C. Leber B. Andrews D. Penninger J. Kroemer G. FASEB J. 2000; 14: 729-739Crossref PubMed Scopus (713) Google Scholar, 13Susin S.A. Daugas E. Ravagnan L. Samejima K. Zamzami N. Loeffler M. Costantini P. Ferri K.F. Irinopoulou T. Prévost M.-C. Brothers G. Mak T.W. Penninger J. Earnshaw W.C. Kroemer G. J. Exp. Med. 2000; 192: 577-585Crossref Scopus (665) Google Scholar). Neutralization of the AIF protein by microinjection of a specific antibody into the cytoplasm of intact cells has revealed AIF to be rate-limiting for apoptotic chromatin condensation and, in some cases, for mitochondrial membrane permeabilization (11Susin 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, 14Susin S.A. Larochette N. Geuskens M. Kroemer G. Methods Enzymol. 2000; 322: 205-208Crossref PubMed Google Scholar, 15Ferri K.F. Jacotot E. Blanco J. Esté J.A. Zamzami A. Susin S.A. Brothers G. Reed J.C. Penninger J.M. Kroemer G. J. Exp. Med. 2000; 192: 1081-1092Crossref PubMed Scopus (207) Google Scholar). Conversely, microinjection of AIF may cause full-blown apoptosis with nuclear condensation, dissipation of the mitochondrial transmembrane potential, release of cytochromec, and exposure of phosphatidylserine on the outer plasma membrane leaflet (11Susin 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, 14Susin S.A. Larochette N. Geuskens M. Kroemer G. Methods Enzymol. 2000; 322: 205-208Crossref PubMed Google Scholar, 15Ferri K.F. Jacotot E. Blanco J. Esté J.A. Zamzami A. Susin S.A. Brothers G. Reed J.C. Penninger J.M. Kroemer G. J. Exp. Med. 2000; 192: 1081-1092Crossref PubMed Scopus (207) Google Scholar). The AIF precursor protein (612 amino acids) contains an N-terminal (first 100 amino acids) mitochondrial localization sequence. The protein is synthesized in cytoplasmic ribosomes and imported into the mitochondrial intermembrane space, where the mitochondrial localization sequence is cleaved off (11Susin 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). The C-terminal domain of AIF (last 485 amino acids) shares significant homology with oxidoreductases from other vertebrates (Xenopus laevis), non-vertebrate animals (Caenorhabditis elegans, Drosophila melanogaster), plants, fungi, eubacteria, and archaebacteria (16Lorenzo H.K. Susin S.A. Penninger J. Kroemer G. Cell Death Differ. 1999; 6: 516-524Crossref PubMed Scopus (426) Google Scholar). The mature AIF protein purifies as a flavoprotein, both from mitochondria and from Escherichia coli used to produce recombinant AIF (11Susin 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). This fact prompted us to investigate the putative electron transfer (redox) function of AIF, in relation to its apoptogenic activity. Indeed, apoptosis is accompanied by a general shift of the redox balance characterized by a depletion of NADH, NADPH, glutathione, as well as by an increase of free radicals, including superoxide anion, lipid peroxidation products (such as 4-hydroxynonenal), and oxidative damage of membranes and DNA (17Kroemer G. Petit P.X. Zamzami N. Vayssière J.-L. Mignotte B. FASEB J. 1995; 9: 1277-1287Crossref PubMed Scopus (967) Google Scholar). In several paradigms of apoptosis, culture in anoxic conditions, treatments with cell-permeable antioxidants, or overexpression of anti-oxidant enzymes (such as superoxide dismutase, glutathione peroxidase, catalase, the thioredoxin system) have profound inhibitory effects on cell death (18Zhang P. Liu B. Kang S.W. Seo M.S. Rhee S.G. Obeid L.M. J. Biol. Chem. 1997; 272: 30615-30619Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Bai J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 19241-19249Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 20Kampranis S.C. Damianova R. Atallah M. Toby G. Kondi G. Tsichlis P.N. Makris A.M. J. Biol. Chem. 2000; 275: 29207-29216Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). It appears that the respiratory chain is a prime source for the generation of oxygen radicals (presumably derived from uncoupling and/or interruption of electron transfer due to the release of cytochrome c) (21Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Moreover, mitochondrial targeting of anti-oxidant enzymes is particularly efficient in blocking apoptosis in several models (19Bai J. Cederbaum A.I. J. Biol. Chem. 2000; 275: 19241-19249Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 22Coulter C.V. Kelos G.F. Lin T.K. Smith R.A. Murphy M.P. Free Radic. Biol. Med. 2000; 15: 1547-1554Crossref Scopus (77) Google Scholar). Here we report the detailed biochemical characterization of the redox function of AIF, which turns out to be an FAD-containing oxidase capable of oxidizing NAD(P)H while generating superoxide anion. Interestingly, the electron transfer function of AIF can be dissociated from its apoptogenic activity, both in cell-free systems and in intact cells. HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mml-glutamine, 1 mm pyruvate, 100 mmHepes, 100 units/ml penicillin/streptomycin, and 10% decomplemented fetal calf serum (Life Technologies, Inc.). These cells were used for the purification of nuclei- and cell-free system experiments, as described (23Susin S.A. Zamzami N. Larochette N. Dallaporta B. Marzo I. Brenner C. Hirsch T. Petit P.X. Geuskents M. Kroemer G. Exp. Cell Res. 1997; 236: 397-403Crossref PubMed Scopus (75) Google Scholar). Rat-1 fibroblast cells were cultured as above and were used in microinjection experiments. AIF deletion mutant (Δ1–120) and AIF deletion mutant (Δ1–351) (11Susin 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) were expressed from a Novagen pET32 expression vector and purified from E. coli. The proteins were stored at −80 °C in 50 mm Hepes, pH 7.9, 100 mm NaCl, 2 mm EDTA, 1 mm DTT, and 10% glycerol. Molecular mass was measured by means of a matrix-assisted laser desorption ionization system from Applied Biosystems at the Services Cientifico-Técnicos, Barcelona University. Sinapinic acid was used as matrix, and bovine serum albumin was used as standard protein. For the experimental analysis, AIFΔ1–120 (1.8 mg/ml) was mixed with sinapinic acid (10 mg/ml in H2O:CH3CN 1:1 + 0.3% trifluoroacetic acid), using a 1:6 ratio. Gel filtration on a Superose 12 (Amersham Pharmacia Biotech) was also used for molecular weight determination. The protein was eluted using 0.3 ml/min of 50 mm Tris-HCl, pH 8, 2 mm EDTA, 1 mmDTT, and 200 mm NaCl. Semiquantitative detection of metals was performed in an ELAN 6000 system (PerkinElmer Life Sciences), and semiquantitative determination of calcium was performed in a multicanal Thermo Jarred Ash 61E Polyscan following standard protocols. Proteins were run in a Phast System from Amersham Pharmacia Biotech, following the manufacturer's instructions. Free thiols' content of AIFΔ1–120 preparations was determined by using a 50-fold molar excess of Ellman's reagent, 5.5′-dithiobis-(2-nitrobenzoate-(Nbs2) (DTNB) (24Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar). The total thiol content was confirmed in the presence of 8 m urea and excess NaBH4 (25Cavallini D. Graziani M.T. Drupe S. Nature. 1966; 212: 294-295Crossref PubMed Scopus (164) Google Scholar). AIF was quantified spectrophotometrically on the basis of the extinction coefficient calculated in this work. Absorption spectrometry studies were carried out using a Kontron Uvikon 860 spectrophotometer. The molar extinction coefficient of bound FAD at 450 nm was determined based on the absorption changes detected after releasing the bound FAD from the enzyme by heating (5 min at 90 °C) in 50 mm Tris-HCl, pH 8. An extinction coefficient of 11.3 mm−1 cm−1at 450 nm was assumed for the free FAD (26Whitby L.G. Biochem. J. 1953; 54: 437-442Crossref PubMed Scopus (308) Google Scholar). Reductive titrations were performed under anaerobic conditions at 25 °C. The anaerobic enzyme sample, in 50 mm Tris-HCl, pH 8, was prepared in an anaerobiosis cuvette by sequential air evacuation and re-equilibration with oxygen-free argon. Anaerobic NAD(P)H was prepared identically, introduced by means of the titration syringe. Identical amounts of NAD(P)H were added to the reference cuvette. HPLC for identification and quantification of FAD was performed using a C18 Vydac column. A linear gradient 0–100% of 0.1m ammonium acetate, pH 6, and methanol in 40 min was performed using 1 ml/min flow rate. FMN, riboflavin, and FAD were used as standards. 1 mg of AIFΔ1–120 in 1 ml of 50 mmTris-HCl, pH 8, maintained in the dark, was heated for 10 min at 90 °C. After centrifugation, aliquots of the supernatant were injected into the HPLC, and flavins were detected at 445 nm. Phosphorylated residues were determined by dot-blot analysis using mouse monoclonal anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine antibodies (Sigma) and an antimouse IgG alkaline phosphatase conjugate (Sigma). The method used was based on the procedure previously described (27Ternynck T.H. Avrameas S. Tecnicas de Inmunologia: Tecnicas Inmunoenzimaticas. Grupo Editorial Iberoamericana, Mexico City, Mexico1989Google Scholar), using polyvinylidene difluoride membranes (Immobilon-P from Millipore). Visible redox titrations of AIF were performed under anaerobic conditions. Appropriated mediators that covered a potential range from +11 mV to −450 mV (1 mm) (28Bes M.T. Parisini E. Inda L.A. Saraiva L.R. Peleato M.L. Sheldrick G. Structure. 1999; 7: 1201-1213Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) were added to the protein solution (10 μm) in 50 mm Bis-Tris for pH 6.5 or 100 mm Tris-HCl for pH values 7.5 and 9. Injection of small volumes of air-free sodium dithionite allowed the reduction of the protein. A Crison 2002 digital potentiometer was used, and spectra were recorded on a Shimadzu UV-260 spectrophotometer. The reduction potential was determined by following the absorbance changes at 450 nm. Potential involvement of cysteinyl redox centers in electron transfers was tested at 25 °C, under anaerobic conditions, following the NADH-DTNB oxidoreductase assay described by Ohnisni et al.(29Ohnisni K. Niimura Y. Yokoyama K. Hidaka M. Masaki H. Uchimura T. Suzuki K. Uozumi T. Kozaki M. Komagata K. Nishimo T. J. Biol. Chem. 1994; 269: 31418-31423Abstract Full Text PDF PubMed Google Scholar). Briefly, 18 μm AIFΔ1–120 in 50 mmsodium phosphate, pH 7, containing 0.5 mm EDTA was mixed with 0.5 mm NADH, 0.02% bovine serum albumin. The reaction was started adding 0.4 mm DTNB, and the nitrothiobenzoate anion production was monitored at 412 nm using an extinction coefficient of 13.6 mm−1cm−1. Apoprotein preparation and holoprotein reconstitution AIFΔ1–120 apoprotein was prepared following the protocol described by Chapman and Reid (30Zanetti G. Cidaria D. Curti B. Eur. J. Biochem. 1982; 126: 453-458Crossref PubMed Scopus (28) Google Scholar) by exhaustive dialysis against 0.1 m Hepes, 2.5m CaCl2, 1 mm DTT, 0.1 mm EDTA, 0.1 m guanidine chloride, 17% glycerol (v/v) at pH 7.5, concentration on Centricon 30 K (Amicon) membranes, and a second round of dialysis against 50 mmHepes, 100 mm NaCl, 2 mm EDTA, 1 mmDTT, 10% glycerol at pH 7.9. The yield of this preparation was ∼15%. Reconstitution of the holoprotein was performed by incubation with 1000-fold molar excess of FAD, acetone precipitation, and repeated ultracentrifugation on Centricon 30 K membranes to remove non-bound FAD. Initial velocity studies of the NAD(P)H oxidase activity of the flavoprotein followed assay procedures described previously (31Ahmed S.A. Claiborne A. J. Biol. Chem. 1989; 264: 19856-19863Abstract Full Text PDF PubMed Google Scholar). 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 air-saturated 50 mm Tris-HCl, pH 8, buffer. The reaction was initiated by the addition of the enzyme and was followed by the decrease in absorbance at 340 nm. Steady-state kinetic data were obtained by varying NADH concentration. One unit of activity is defined as the amount of protein required to catalyze the conversion of 1 μmol of NAD(P)H to NAD(P)+ per minute at 25 °C. NBT reduction and monodehydroascorbate reductase activity (32Pez-Huertas E.L. Corpas F.J. Sandalio L.M. Del Roeo L.A. Biochem. J. 1999; 337: 531-536Crossref PubMed Google Scholar, 33Lumper L. Schneider W. H. J. S. Biol Chem. Hoppe-Seyler. 1967; 348: 323-328Crossref Scopus (42) Google Scholar), superoxide formed in the reaction of AIFΔ1–120 with oxygen (34McCord J.M. Fridowich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar) and hydrogen peroxide production (35Arcari P. Masullo L. Masullo M. Cantazano F. Bocchini V. J. Biol. Chem. 2000; 275: 890-895Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) were quantified as described. This latter reaction was carried out coupled with NADH oxidase in a mixture containing 1 mm sodium phosphate buffer, pH 6.9, 2.34 mg/ml phenol, 1 mg/ml 4-aminoantipyrine, and 0.02 units of horseradish peroxidase in a total volume of 0.5 ml. The absorbance was measured at 505 nm, and the concentration of H2O2 was calculated from a calibration curve (36Green M.J. Hill A.O. Methods Enzymol. 1984; 105: 15-16Google Scholar). DCPIP (85 μm) or ferricyanide (2 mm) reduction were assayed as described (37Sancho J. Peleato M.L. Gomez-Moreno C. Edmondson D.E. Arch. Biochem. Biophys. 1988; 260: 200-207Crossref PubMed Scopus (58) Google Scholar). SOD inhibition was measured by adding the indicated units of enzyme to the reaction mixtures. The electron transfer between NADH-AIF-ferredoxin/adrenodoxin-cytochrome c was assayed as described (37Sancho J. Peleato M.L. Gomez-Moreno C. Edmondson D.E. Arch. Biochem. Biophys. 1988; 260: 200-207Crossref PubMed Scopus (58) Google Scholar), using AIFΔ1–120 instead of ferredoxin-NADP+ reductase and adrenodoxin instead of ferredoxin. Supernatants obtained from mitochondria undergoing permeability transition were subjected to a 10% SDS-PAGE and transferred (100 V, 75 min at room temperature) to a nitrocellulose membrane. AIF immunoblot analysis was performed using a rabbit antiserum generated against a mixture of three peptides derived from the mouse AIF amino acids 151–200 (11Susin 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). In situ detection of 2, 2′-Di-p-nitrophenyl-5–5′-diphenyl-3,3′ (3–3′-dimetoxy-4–4′difenilen) tetrazolium chloride (NBT) reduction on native-PAGE was done using the reaction mixture described by Pez-Huertas et al. (32Pez-Huertas E.L. Corpas F.J. Sandalio L.M. Del Roeo L.A. Biochem. J. 1999; 337: 531-536Crossref PubMed Google Scholar). Briefly, samples obtained from mitochondria undergoing permeability transition were loaded onto a 10% native-PAGE. The gel was incubated 20 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. Purified HeLa cell nuclei (103/μl) were exposed 90 min at 37 °C to AIFΔ1–120 preincubated (15 min at 37 °C) with or without NADH, NADPH, para-chloromercuriphenylsulfonic acid, superoxide dismutase, or diphenyleneiodonium. For the standard assessment of chromatin condensation, nuclei were stained with Hoechst 33342 (2 μm, 15 min, room temperature) and analyzed by fluorescence microscopy (Leica DM IRB). DNA content was determined by staining with propidium iodide (10 μg/ml) followed by analysis in a Vantage fluorescence-activated cell sorter (Becton-Dickinson). A minimum of 2500 events were scored. Rat-1 fibroblasts were microinjected using a computer-controlled microinjector (pressure 150 hPa; 3 s; Eppendorf) with buffer only, 7.5 μm AIFΔ1–120, apoAIFΔ1–120, FAD-reconstituted holoprotein, and AIFΔ1–351. After microinjection, cells were cultured at 37 °C for 180 min and stained with the ΔΨm-sensitive dye CMXRos (100 nm, 15 min) and the DNA-intercalating dye Hoechst 33342 (1.5 μm, 15 min) (11Susin 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). Microinjected viable cells (100/session, three independent sessions of injections) were identified by inclusion of 0.25% (w:v) FITC-dextran (green fluorescence) in the injectate. Only the blue and the red fluorescence were recorded. All chemical reagents used in this work were purchased from Sigma. Recombinant AIFΔ1–120 was found to elute as a single peak on a gel filtration column, with a calculated mass weight of 57 kDa, which corresponds to its theoretical molecular mass (57,046), indicating that, at near-physiological salt concentrations (200 mm NaCl), AIFΔ1–120 is a monomer. This result was confirmed by mass spectroscopy analysis (data not shown). It is in contrast with its apparent molecular mass determined by SDS-PAGE (67.5 kDa, about 10 kDa more than expected), a migration behavior reported for other proteins such as ferredoxins (38Böhme H. Schrautemeier S. Biochim. Biophys. Acta. 1987; 981: 1-7Google Scholar). AIFΔ1–120 does not contain significant amounts of metals including Ca2+, Co2+, Cu2+, Mg2+, Fe2+, Se2+, and Zn2+. However, it contains phosphogroups among serine and threonine residues, as revealed by immunoblotting (data not shown). AIFΔ1–120 has 3 cysteins in its sequence, and it was found to contain 3.2 accessible thiol groups per molecule, as determined by derivatization with the Ellman's reagent. Total thiol content was confirmed after unfolding in 8 m urea. This suggests that none of the three cysteines contained in the AIF amino acid sequence engages in disulfide links. The flavin moiety of AIFΔ1–120 is non-covalently bound and was identified by HPLC as FAD (data not shown). FAD is the only identified prosthetic group present in AIFΔ1–120, at a molar ratio of 1:1. The absorption spectrum of AIF (Fig. 1) shows the typical features of an oxidized FAD flavoprotein, with the visible maximum at 378 nm and 450 nm and a shoulder at 467 nm. The ratioA270 nm/A450 nm was 7 in pure preparations, and the extinction coefficient for oxidized AIF at 450 nm was calculated to be 12.12 mm−1cm−1. The addition of an equimolar amount of NADH to AIFΔ1–120 in anaerobic conditions leads to complete flavin reduction, without intermediate semiquinone formation (Fig. 2). Similar titration curves were obtained using NADPH as reductant, and in both cases the spectral changes are similar to the reduction of AIFΔ1–120 with dithionite (data not shown). The reduced form was stable over several hours, and admission of air to the sample did not lead to the immediate appearance of the oxidized AIF spectrum. Upon addition of an increasing molar excess of NADH or NADPH over AIFΔ1–120, the appearance of long wavelength absorbance bands was observed (Fig.3). These long wavelength absorbances of the reduced enzyme were stable at 25 °C for hours, even upon exposure to air. They exhibit the blue-green and green color described for other electron transfer complexes (31Ahmed S.A. Claiborne A. J. Biol. Chem. 1989; 264: 19856-19863Abstract Full Text PDF PubMed Google Scholar). By analogy to other enzymes (31Ahmed S.A. Claiborne A. J. Biol. Chem. 1989; 264: 19856-19863Abstract Full Text PDF PubMed Google Scholar), these long wavelength absorptions are likely to correspond to charge transfer complexes between the reduced FAD and tightly bound NAD+ or NADP+.Figure 3Anaerobic titration of AIF Δ1–120 with an excess of NAD(P)H. A, long wavelength absorbance changes observed in oxidized AIFΔ1–120 (22 μm) without (a) or after addition of NADH (b and c; molar excess NADH/AIF was 3:1 (b) and molar excess was 6:1 (c), with maxima at 637 nm and 774 nm, respectively). B, same asA using NADPH instead of NADH, with appearance of maxima at 740 nm (b, molar excess NADPH/AIF, 3:1) and 769 nm (c, molar excess 6:1).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The spectral titration of AIFΔ1–120 with dithionite revealed that the redox potential of AIF is strongly influenced by the pH (Fig. 4). Assuming a two-electron reduction step, midpoint redox potentials were determined to be −264 ± 15 mV at pH 6.5 (Fig. 4 A), −308 ± 15 mV at pH 7.5 (Fig. 4 B), and −373 ± 15 mV at pH 9.0 (Fig. 4 C). Neither semiquinone formation nor long wavelength absorbing bands were detected upon reduction by dithionite. A plot of AIF's redox potential (FAD/FADH2) versus pH has a slope of −44 mV (data not shown). This slope deviates from that expected for a two-electron reduction involving two protons (58 mV/pH unit) or one proton (29 mV/pH unit). The deviation from theoretical values indicates the possible presence of other dissociable groups whose pKa values are linked to the redox state of the enzyme. Even though titrations with dithionite and NAD(P)H showed an uptake of two electrons, it was considered important to discard the involvement of cysteinyl residues as active redox acceptors; in the NADH:DTNB oxidoreductase assay, no involvement of cysteinyl residues in redox transferences was detected (data not shown). AIFΔ1–120 was found to exhibit NADH and NADPH oxidase activities (Fig.5 A). NAD(P)H oxidation in presence of AIF was followed measuring initial rates of ΔA340 nm. The apparent Kmfor NADH was calculated as 99.4 ± 10 μm and the turnover number 2.09 min−1. When NADPH was used as electron donor, the apparent Km was 52.9 ± 12 μm and the turnover number 2.8 min−1 (Table I). These kinetic parameters are very similar to previous values described for other superoxide forming NADH oxidases (39Glass G.A. DeLisle D.M. DeTogni P. Gabig T.G. Magee B.H. Marker M. Babior B.M. J. Biol. Chem. 1986; 261: 13247-13251Abstract Full Text PDF PubMed Google Scholar), and the steady-state kinetic data may be interpreted taking into account the possible formation of relatively stable charge-transfer complexes. Addition of exogenous FAD did not stimulate the NADH oxidase activity of AIFΔ1–120 (data not shown), in contrast to several NADH oxidases from bacteria (29Ohnisni K. Niimura Y. Yokoyama K. Hidaka M. Masaki H. Uchimura T. Suzuki K. Uozumi T. Kozaki M. Komagata K. Nishimo T. J. Biol. Chem. 1994; 269: 31418-31423Abstract Full Text PDF PubMed Google Scholar, 35Arcari P. Masullo L. Masullo M. Cantazano F. Bocchini V. J. Biol. Chem. 2000; 275: 890-895Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 40Niimura Y. Yokoyama K. Ohnisni K. Massey V. Biosci. Biotechnol. Biochem. 1994; 58: 2310-2311Crossref Scopus (5) Google Scholar, 41Niimura Y. Poole L.B. Massey V. J. Biol. Chem. 1995; 270: 25645-25650Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Toomey D. Mayhew S.G. Eur. J. Biochem. 19
Discussion(0)
No comments yet. Be the first to comment.