From the a Departamento de Bioquímica y Biología Molecular y Celular. Universidad de Zaragoza, Plaza San Francisco s/n 50009 Zaragoza, Spain, c CNRS, UMR1599, Institut Gustave Roussy, 39 rue Camille Desmoulins, F-94805 Villejuif, France, the f Instituto de Tecnología Química y Biológica, Universidade Nova de Lisboa, rua da Quinta Grande, 6 Apartado 127, 2780 Oeiras, Portugal, and the g Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M5G 2C1, Canada
Received for publication, November 20, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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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 (AIF Mitochondria are considered as central players in apoptosis of
mammalian cells (1-3). Early during the apoptotic process, the outer
mitochondrial membrane becomes permeabilized, and mitochondria release
soluble proteins normally confined to the intermembrane space (4). Such
apoptogenic proteins include the caspase activator cytochrome
c (5), procaspases 2, 3, and 9 (6-8), the inhibitor of
apoptosis protein (IAP) inhibitor Smac/DIABLO (9, 10), as well
as AIF1 (11). 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 (12, 13). 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 (11, 14, 15). Conversely, microinjection of AIF may
cause full-blown apoptosis with nuclear condensation, dissipation of
the mitochondrial transmembrane potential, release of cytochrome
c, and exposure of phosphatidylserine on the outer plasma
membrane leaflet (11, 14, 15).
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 (11). 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 (16).
The mature AIF protein purifies as a flavoprotein, both from
mitochondria and from Escherichia coli used to produce
recombinant AIF (11). 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 (17). 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 (18-20). 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) (21). Moreover, mitochondrial
targeting of anti-oxidant enzymes is particularly efficient in blocking
apoptosis in several models (19, 22).
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.
Cells and Culture Conditions--
HeLa cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 2 mM
L-glutamine, 1 mM pyruvate, 100 mM
Hepes, 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 (23). Rat-1 fibroblast cells were cultured as above and were
used in microinjection experiments.
Recombinant AIF Proteins--
AIF deletion mutant ( Mass Spectroscopy, HPLC, and Elemental Analysis--
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, AIF SDS-PAGE--
Proteins were run in a Phast System from Amersham
Pharmacia Biotech, following the manufacturer's instructions.
Free thiols' content of AIF
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
HPLC for identification and quantification of FAD was performed
using a C18 Vydac column. A linear gradient 0-100% of 0.1 M 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 AIF
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 (27), 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
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. (29). Briefly, 18 µM AIF
Apoprotein preparation and holoprotein reconstitution AIF Determination of AIF Redox Activities--
Initial velocity
studies of the NAD(P)H oxidase activity of the flavoprotein followed
assay procedures described previously (31). 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 (32, 33), superoxide formed
in the reaction of AIF AIF Western Blot--
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 (11).
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. (32).
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.
Cell-free Systems of Nuclear Apoptosis--
Purified HeLa cell
nuclei (103/µl) were exposed 90 min at 37 °C to
AIF Microinjection--
Rat-1 fibroblasts were microinjected using a
computer-controlled microinjector (pressure 150 hPa; 3 s;
Eppendorf) with buffer only, 7.5 µM AIF
All chemical reagents used in this work were purchased from Sigma.
Properties of Recombinant AIF Protein--
Recombinant
AIF Electron Transfer Reactions from NADH and NADPH to AIF--
The
addition of an equimolar amount of NADH to AIF Redox Potential of AIF--
The spectral titration of AIF AIF Redox Activities--
AIF
AIF
AIF
In summary, AIF exhibits NADH oxidase activity and is able to transfer
one electron (to molecular oxygen and ferricyanide) or two electrons
(to DCPIP), as summarized in Table I.
AIF Is the Dominant NADH Oxidase Released through the Mitochondrial
Outer Membrane--
Purified mitochondria can be induced to undergo
the so-called permeability transition, a manipulation that leads to
osmotic swelling of the matrix and physical rupture of the outer
membrane, causing the release of soluble intermembrane proteins (4). Upon induction of permeability transition with Ca2+ or
arsenite, immunodetectable AIF was found in the mitochondrial supernatant. The release of AIF was inhibited by cyclosporin A, a
specific inhibitor of the permeability transition pore (Fig. 8). Separation of proteins via native
PAGE, followed by the in situ detection of an NADH oxidase
activity causing the reduction of NBT, yielded one single blue band
(Fig. 8A). This band also reacted with a specific anti-AIF
antiserum and co-migrated with recombinant AIF Apoptotic Versus Oxidoreductase Activity of AIF--
The FAD
moiety of AIF
Altogether, these data show that the apoptogenic and oxidase functions
of AIF can be dissociated.
The results from this work indicate that AIF has a marked NADH
oxidase activity. According to the classification by Massey (46), AIF
may belong to the electron-transferase class of NADH reductases,
because it reacts rapidly with oxygen, forming
O Several NADH oxidases from bacterial sources have been isolated and
characterized (31, 35). The putative role of those enzymes is to
maintain the cellular redox balance under aerobic conditions, by
converting NADH to NAD+ (42, 47). Additionally, several
poorly characterized superoxide (O AIF protein is present in the mitochondria of all mouse tissues
that have been assessed and has also been found in a panel of 60 human
cancer cell lines (53), suggesting that AIF may fulfill important
metabolic functions. However, based on the present data, it is
difficult to understand what the physiological function of AIF in
normal (non-apoptotic) conditions may be. Since AIF is the only NADH
oxidase detected in the intermembrane space, it is tempting to
speculate that AIF accounts for the mitochondrial superoxide anion or
hydrogen peroxide-generating NADH oxidase activity (54, 55), which is
lost from mitochondria, once cells have been induced to die (55).
Clearly, an NADH oxidase activity causing the collateral generation of
superoxide anion radicals would be of no advantage for the cell. It
thus may be speculated that the true, yet-to-be-discovered substrates
of the AIF oxidoreductase compete for endogenous NADH and/or that AIF
is normally inactivated by local inhibitory factors within the
intermembrane space. If AIF acted as a superoxide-generating NADH
reductase outside of mitochondria, after its apoptotic release, what
might be the contribution of this enzymatic activity to the apoptotic
process? Apoptosis is notoriously associated with a massive depletion
of NADH/NADPH (56), as well as an increase in the generation of
superoxide anions (57), at both mitochondrial and extramitochondrial
localizations (17). Furthermore, it should be mentioned that, in
isolated mitochondria, the permeability transition pore complex is
tightly regulated by the oxidation-reduction state of the pyridine
nucleotide pool, with oxidation causing an increase in the pore opening
probability (58). Although these changes in the redox potential may be
explained by a variety of factors, including uncoupling/blockade of the respiratory chain (21) and activation of poly(ADP)ribose polymerase (59), it will be interesting to study the contribution of AIF to this
process, for instance in embryonic stem cells in which the AIF gene is ablated.
It appears that the known apoptogenic functions of AIF and its
novel oxidoreductase activity can be dissociated from each other, based
on three arguments. First, removal of the prosthetic FAD group (which
obviously abolishes the oxidoreductase function of AIF) does not
curtail the apoptogenic effects of AIF on mitochondria and nuclei of
microinjected cells. Second, addition of NADH, addition of SOD, or
covalent derivatization of FAD failed to modulate the capacity of AIF
to induce nuclear apoptosis in a cell-free system. Third, inhibition of
the apoptogenic effect of AIF by means of para-chloromercuriphenylsulfonic acid failed to affect its
NADH oxidase activity. These data are similar to those obtained for cytochrome c in the sense that the apoptogenic activity of
cytochrome c does not depend on its redox status. Exchange
of the Fe2+ by Co2+ within the heme prosthetic
group of cytochrome c (a manipulation that abolishes the
electron transfer function of heme) fails to alter its
caspase-activatory functions (60), whereas certain amino acid
substitutions that do not affect its redox function do abrogate
cytochrome c-mediated caspase activation (60, 61). In
conclusion, both cytochrome c and AIF thus appear to be
bifunctional molecules with clearly dissociable redox and apoptogenic activities.
1-120) exhibit NADH
oxidase activity, whereas superoxide anion
(O
1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol
of protein. ApoAIF
1-120, which lacks FAD, has no NADH oxidase
activity. However, native AIF
1-120, apoAIF
1-120, and the
reconstituted (FAD-containing) holoAIF
1-120 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 AIF
1-120 assessed on purified nuclei in a cell-free system.
Conversely, blockade of the apoptogenic function of AIF
1-120 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-120)
and AIF deletion mutant (
1-351) (11) 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.
1-120 (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 mM
DTT, 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.
1-120 preparations was determined by
using a 50-fold molar excess of Ellman's reagent,
5.5'-dithiobis-(2-nitrobenzoate-(Nbs2) (DTNB) (24). The total thiol
content was confirmed in the presence of 8 M urea and
excess NaBH4 (25). AIF was quantified
spectrophotometrically on the basis of the extinction coefficient
calculated in this work.
1 cm
1
at 450 nm was assumed for the free FAD (26). 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.
1-120 in 1 ml of 50 mM
Tris-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.
450 mV (1 mM) (28) 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.
1-120 in 50 mM
sodium 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
1
cm
1.
1-120
apoprotein was prepared following the protocol described by Chapman and
Reid (30) by exhaustive dialysis against 0.1 M Hepes, 2.5 M 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 mM
Hepes, 100 mM NaCl, 2 mM EDTA, 1 mM
DTT, 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.
1-120 with oxygen (34) and hydrogen peroxide
production (35) 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 (36). DCPIP (85 µM) or ferricyanide (2 mM) reduction were assayed as described (37). 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 (37), using AIF
1-120 instead of
ferredoxin-NADP+ reductase and adrenodoxin instead of ferredoxin.
1-120 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.
1-120,
apoAIF
1-120, FAD-reconstituted holoprotein, and AIF
1-351. After
microinjection, cells were cultured at 37 °C for 180 min and stained
with the
m-sensitive dye CMXRos (100 nM,
15 min) and the DNA-intercalating dye Hoechst 33342 (1.5 µM, 15 min) (11). 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-120 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),
AIF
1-120 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 (38). AIF
1-120 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).
AIF
1-120 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 AIF
1-120 is
non-covalently bound and was identified by HPLC as FAD (data not
shown). FAD is the only identified prosthetic group present in
AIF
1-120, 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 ratio A270 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
1
cm
1.
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Fig. 1.
Absorption spectrum of recombinant
AIF 1-120. The concentration used was 13 µM.
1-120 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 AIF
1-120 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 AIF
1-120, 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 (31). By analogy to other enzymes
(31), these long wavelength absorptions are likely to correspond to
charge transfer complexes between the reduced FAD and tightly bound
NAD+ or NADP+.
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Fig. 2.
NADH titration of
AIF 1-120. The top
line shows the spectrum of the oxidized enzyme (47 µM) before (a) and after addition of 0.12 (b), 0.35 (c), 0.59 (d), 0.83 (e), 1.07 (f), 1.31 (g), 1.57 (h), and 1.79 (i) equivalents of NADH/FAD. The
inset shows the absorbance at 450 nm versus added
NADH equivalents.
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Fig. 3.
Anaerobic titration of
AIF 1-120 with an excess of NAD(P)H.
A, long wavelength absorbance changes observed in oxidized
AIF
1-120 (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 as
A 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).
1-120
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. 4A),
308 ± 15 mV at pH 7.5 (Fig. 4B), and
373 ± 15 mV at pH 9.0 (Fig. 4C). 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).
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Fig. 4.
pH effect on the redox potential of
AIF 1-120. Titration curves obtained
following absorbance variations at 450 nm of oxidized AIF
1-120 (10 µM) upon dithionite addition. Solid
curves calculated from a dielectronic Nernst equation of
264 mV at pH 6.5 (A),
308 mV at pH 7.5 (B)
and
373 mV at pH 9.0 (C).
1-120 was found to exhibit NADH
and NADPH oxidase activities (Fig.
5A). NAD(P)H oxidation in
presence of AIF was followed measuring initial rates of
A340 nm. The apparent Km for 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 (39), 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
AIF
1-120 (data not shown), in contrast to several NADH oxidases
from bacteria (29, 35, 40-42). When oxidizing NADH or NADPH, AIF
catalyzed the reduction of the tetrazolium salt NBT (Fig.
5B). This was due to the AIF(FADH2)-mediated
reduction of O2 to O
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Fig. 5.
NAD(P)H oxidation by
AIF 1-120. NADH (
) and NADPH (
)
oxidation measured following absorbance variation at 340 nm, after
addition of different amounts of NAD(P)H. AIF was added at a
concentration of 3 µM (A). AIF
1-120
induced NBT reduction with NADH (
) and NADPH (
) as electron
donors. AIF was added at a concentration of 95 nM
(B).
Steady-state kinetic parameters of AIF1-120
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Fig. 6.
NADH ( ) and NADPH (
) oxidase activities
of AIF result in generation of superoxide anion (A)
and hydrogen peroxide (B). AIF concentration was
95 nM in A and 3 µM in
B.
1-120 was also found to possess a monodehydroascorbate reductase
activity with a specific activity of 8.8 units/mg and a
kcat of 0.505 min
1, a
feature that is common to several AIF's homologous described in plants
(16). The AIF-monodehydroascorbate reductase activity was inhibited by
SOD, indicating that the reaction occurs via superoxide radicals (data
not shown). Cytochrome c reductase activity was also
measured, with a Km of 0.46 mM for NADH
and a kcat of 21.76 min
1 (Fig.
7A). Again, SOD inhibited the
cytochrome c reductase activity with an IC50 of
~10 units/ml, indicating that the AIF-catalyzed cytochrome
c reduction occurs via O
1.
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Fig. 7.
A, cytochrome c reduction
mediated by superoxide anion, using NADH as electron donor.
B, specific inhibition of superoxide anion-mediated
cytochrome c reduction by SOD. C, adrenodoxin
enhances cytochrome c reduction. AIF concentrations were 72 nM in A and B, and 90 nM
in C. NADH concentration was 3.4 mM in
B and C.
1-120 was found to transfer electrons at low rate from NADH (but
not from NADPH) to DCPIP or ferricyanide, two typical electron
acceptors of NAD(P)H dehydrogenases. From NADH, a two-electron donor,
AIF
1-120 was able to reduce DCPIP, a two-electron acceptor, with a
kcat of 21.6 min
1 and
a calculated Km for the NADH of 3.4 ± 0.4 mM (Table I). SOD did not affect the electron transfer
rate, indicating that the diaphorase activity of AIF is not mediated by
O
1-120 (Fig.
8B). Altogether, these data confirm that natural
(mitochondrial) AIF possesses a NADH oxidase/NBT reductase activity and
indicate that AIF is the quantitatively most important NADH oxidase/NBT
reductase contained in the mitochondrial intermembrane space.
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Fig. 8.
AIF is the dominant NADH oxidase released
from mitochondria undergoing permeability transition.
A, AIF release induced by 100 µM
Ca2+ and 2 mM sodium arsenite measured by
in situ NBT detection. B, AIF detection by
immunoblot of mitochondrial intermembrane proteins. As a control of
permeability transition induction, mitochondria were pretreated with 1 µM cyclosporine A (CycloA) to inhibit the
release of the intermembrane mitochondrial proteins.
1-120 can be removed by dialysis in the presence of
guanidine chloride (a manipulation that generates apoAIF
1-120) and
may be reconstituted by external addition of FAD (a manipulation that
generates reconstituted holoAIF
1-120). ApoAIF lacked any detectable
NADH oxidase-NBT reductase activity, whereas the reconstituted holoAIF
had a NADH oxidase-NBT reductase activity indistinguishable from native
AIF
1-120 (Fig. 9A).
Unmanipulated AIF
1-120, apoAIF
1-120, and reconstituted
holoAIF
1-120 were micro-injected into the cytoplasm of Rat-1
fibroblasts. All three protein preparations induced a similar level of
nuclear chromatin condensation, as well as a dissipation of the
mitochondrial transmembrane potential (Fig. 9, B and
C). A deletion mutant of AIF
1-351, which lacks part of
the oxidoreductase domain, yielded negative results in this system
(Fig. 9C). The nuclear effects of AIF were recapitulated in
a cell-free system, in which AIF
1-120 was added to purified HeLa
nuclei. In such a system, AIF
1-120 caused marked peripheral chromatin condensation (Fig.
10A) as well as a loss in
DNA content (Fig. 10B). Addition of NADH or NADPH failed to
enhance the apoptogenic activity of AIF
1-120 (Fig. 10C).
Moreover, inhibition of the oxidoreductase activity by external
addition of SOD or diphenyleneiodonium, an inhibitor of
flavonoid-containing enzymes covalently reacting with FAD (43-45),
failed to modify the apoptogenic activity of AIF (Fig. 10D).
In contrast, addition of para-chloromercuriphenylsulfonic acid, a thiol-reactive agent, did abolish the apoptogenic activity of
AIF
1-120, yet did not affect its NTB reductase activity (Fig. 10D).
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[in a new window]
Fig. 9.
Apoptotic activity of
apoAIF 1-120 and reconstituted
holoAIF
1-120 in a cell-free system and in
microinjection assays. Spectral characteristics of apoAIF
and reconstituted holoAIF are shown in A. The
inset shows the recovery of the NADH oxidase activity of
apoAIF after FAD reconstitution measured by in situ NBT
detection (as in Fig. 8A). Rat-1 cells were microinjected
with the indicated protein (7.5 µM AIF
1-120,
apoAIF
1-120, and reconstituted holoAIF
1-120), and stained with
Hoechst 33342 (blue fluorescence) and the
m-sensitive dye CMXRos (red fluorescence).
Representative phenotypes obtained 3 h after injection are shown
in B. Quantification of nuclear apoptosis and
m reduction induced by microinjection (100-150 Rat-1
cells/session) of AIF
1-120, apoAIF
1-120, reconstituted
holoAIF
1-120, or AIF
1-351 (determined as in B)
(C; x ± S.E. of three experiments).
View larger version (50K):
[in a new window]
Fig. 10.
Independence between the redox and apoptotic
activities of AIF: apoptotic nuclear features induced by
AIF 1-120 or
AIF
1-120+NADH in a cell-free system.
A, HeLa nuclei were exposed 120 min to 100 µg/ml
AIF
1-120 and/or 2 mM NADH followed by staining with
Hoechst 33342. B, in addition, nuclei were stained with
propidium iodide and the percentage of hypoploid nuclei was measured by
flow cytometry. C, dose response of the apoptogenic effect
of AIF
1-120 in the presence or absence of 2 mM NADH or
NADPH measured as in B. D, comparison of AIF
redox and apoptotic activity obtained after pretreatment of
AIF
1-120 (100 µg/ml, 15 min at 37 °C) with NADH (2 mM), NADPH (2 mM), SOD (40 units/ml),
diphenyleneiodonium (DPI, 250 µM), or
para-chloromercuriphenylsulfonic acid (PCMPS, 30 µM) (x ± S.E. of five
experiments).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
---|
We thank Dr. Rita Bernhartd (Universität des Saarlandes, Saarbrucken, Germany) for kindly providing adrenodoxin.
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FOOTNOTES |
---|
* This work was supported by a special grant from the Ligue Nationale contre le Cancer, as well as by grants from Agence Nationale de Recherches sur le Sida, Fondation pour la Recherche Medicale, and the European Union QLG1-1999-00739 (to G. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Recipient of a short term fellowship from Caja de Ahorros de la Inmaculada.
d Recipient of a postdoctoral fellowship from the Fondation pour la Recherche Medicale.
e Recipient of a Ph.D. fellowship from the French Ministry of Science & Technology.
h These authors contributed equally to this work and share senior co-authorship.
i To whom correspondence should be addressed: CNRS, UMR 1599, Inst. Gustave Roussy, Pavillon de Recherche I, 39, rue Camille-Desmoulins, F-94805 Villejuif, France. Tel.: 33-1-42-11-60-46; Fax: 33-1-42-11-60-47; E-mail: kroemer@igr.fr.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010498200
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ABBREVIATIONS |
---|
The abbreviations used are:
AIF, apoptosis-inducing factor;
m, mitochondrial
transmembrane potential;
DTNB, 5.5'-dithiobis-(2-nitrobenzoate-(Nbs2);
NBT, 2,2'-di-p-nitrophenyl-5-5'-diphenyl-3,3' [3-3'-dimetoxy-4-4'difenilen]tetrazolium
chloride;
SOD, superoxide dismutase;
DTT, dithiothreitol;
HPLC, high
performance liquid chromatography;
Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane;
PAGE, polyacrylamide gel electrophoresis;
DCPIP, 2,6-dichlorophenolindophenol.
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