From the Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 10081, Taiwan, Republic of China
Received for publication, January 22, 2001, and in revised form, March 28, 2001
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ABSTRACT |
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Abrin A-chain (ABRA) inhibits protein
synthesis by its N-glycosidase activity as well as
induces apoptosis, but the molecular mechanism of ABRA-induced cell
death has been obscure. Using an ABRA mutant that lacks
N-glycosidase activity as bait in a yeast two-hybrid
system, a 30-kDa antioxidant protein-1 (AOP-1) was found to be an
ABRA(E164Q)-interacting protein. The interaction was further confirmed
in vitro by a glutathione S-transferase pull-down assay. The colocalization of endogenous AOP-1 and exogenous ABR proteins in the cell was demonstrated by confocal
immunofluorescence. We also demonstrated that ABRA attenuates AOP-1
antioxidant activity in a dose-dependent manner and the
intracellular level of reactive oxygen species (ROS) increases in
ABR-treated cells. Moreover, ROS scavengers
N-acetylcysteine and
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl delayed programmed cell
death. This indicates that ROS are important mediators of
ABR-induced apoptosis. When ectopically expressed, AOP-1 blocked
the release of cytochrome c and prevented
apoptosis in ABR-treated cells. These findings suggest that
the binding of ABRA to AOP-1 promotes apoptosis by inhibiting the
mitochondrial antioxidant protein AOP-1, resulting in the increase of
intracellular ROS and the release of cytochrome c from the
mitochondria to the cytosol, which activates caspase-9 and
caspase-3.
Abrin (ABR)1 and ricin
belong to the type II ribosome inactivating proteins which are
heterodimeric glycoproteins that contain a toxophoric A-chain with
protein synthesis inhibitory activity and a lectin B-chain that binds
to D-galactose moieties on the cell membrane (1). The
A-chain is transferred across the plasma membrane by the B-chain via
endocytotic vesicles into cells (2). In addition to their ability to
inhibit protein synthesis, recent studies have shown that these toxins
are able to induce apoptosis (3-5). Baluna and colleagues (6, 7) have
suggested that inhibition of protein synthesis and apoptosis may be
mediated by different segments of the ricin A-chain molecule. Besides
the inhibition of protein synthesis, it is an attractive hypothesis that ABRA adopts alternative molecular mechanisms to trigger apoptotic programs.
Apoptosis is a form of cell death that leads to elimination of excess
or damaged cells. Apoptosis contributes to tissue homeostasis and
embryonic development (8). A range of stimuli including DNA damage,
growth factor withdrawal, anticancer drugs, and members of the tumor
necrosis factor receptor family of death receptors can induce apoptotic
signals (9-11). The apoptotic cascade in mammalian cells is a
multistep process (12). In most cases, the apoptotic cascade is
initiated by loss of integrity of the outer mitochondrial membrane
accompanied by release of cytochrome c from the
intermembrane space of mitochondria to cytosol. The cytosolic
cytochrome c serves as a cofactor with the apoptotic protease activating factor (Apaf-I) to activate pro-caspase 9 (13-15).
Caspase-9 then activates other caspases, which undermine the structural
integrity of the cells by cleaving a key structural protein substrate
(16).
The signaling events that affect the particular apoptotic mediators are
currently a focus of intense study. Reactive oxygen species (ROS)
participate in a wide variety of cellular functions, including cell
proliferation, differentiation, and apoptosis (17, 18). Addition of ROS
or depletion of cellular antioxidants induces apoptosis (17, 19, 20),
and ROS are likely to act as signaling intermediates that are involved
in the signal transduction mechanism for apoptosis (17, 21-23).
Elucidation of the mechanism that is involved in ABR-triggered
apoptosis is important for the development of cancer chemotherapeutic agents. In order to identify ABRA-interacting proteins, the yeast two-hybrid system was used. Since ABRA inhibits the growth of yeast
cells by its N-glycosidase activity, ABRA(E164Q) with a mutation of its N-glycosidase catalytic site was employed.
In this paper, we report on an ABRA(E164Q)-interacting protein, AOP-1,
which is an antioxidant protein with two catalytic conserved cysteine
residues and has 93.3% identity to SP-22 (24, 36), a mitochondria
antioxidant protein. We show that ABRA can directly inhibit the
antioxidant activity of AOP-1 in vitro and two antioxidants
N-acetylcysteine (NAC) and
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-OH-tempo) can delay
ABR-induced apoptosis, suggesting that ABR may deplete cellular
antioxidants. Moreover, the ectopic expression of AOP-1 prevents both
mitochondrial cytochrome c release and phenotypic apoptosis
in response to ABR. Taken together, our results support AOP-1 as one of
the target molecules of ABRA-induced apoptosis.
Materials--
A Matchmaker Two-Hybrid System 2 kit was
purchased from CLONTECH (Palo Alto, CA).
Restriction endonucleases were obtained from New England Biolabs, Inc.
(Beverly, MA). Chemicals for nucleotide autosequence analysis were
purchased from PE Applied Biosystems (Foster, CA). Taq DNA
polymerase, large scale RNA production system-T7, and rabbit
reticulocyte lysate translation system, and the CaspACETM assay system
were purchased from Promega (Madison, WI).
[35S]Methionine was obtained from PerkinElmer Life
Sciences. DCFH-DA, Hoechst 33258, N-acetylcysteine,
and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl were purchased from
Sigma. Glutathione-Sepharose 4B gel and pGEX-2T were obtained from
Amersham Pharmacia Biotech (Uppsala, Sweden). Abrin-a was purified from
Abrus precatorius as described (1). An in
situ cell death detection kit was purchased from Roche Molecular Biochemicals GmbH (Mannheim, Germany). The protease inhibitor Cbz-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) was purchased from
Kamiya Biomedical Co. (Seattle, WA). The fluorescent tetrapeptide protease substrate, Ac-LEHD-AFC, was obtained from Calbiochem (San
Diego, CA). Anti-cytochrome c antibodies (7H8.2C12 and
6H2.B4) were from PharMingen (San Diego, CA).
Anti- Cell Culture--
HeLa cells and 293 T cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies,
Inc.) under 5% CO2 at 37 °C.
Yeast Two-hybrid System--
Since wild type ABRA is detrimental
to yeast, mutant ABRA(E164Q) cDNA was fused to pAS2-1 vector to
screen a Jurkat cDNA library cloned in the pGAD10 vector. A total
of 1.2 × 106 clones were analyzed, and the
transformed yeast was selected on synthetic dropout agar plates lacking
leucine, tryptophan, and histidine in the presence of 50 mM
3-amino-1,2,4-triazole (Sigma). Twenty-four colonies that developed
color on filter paper were restreaked on selective plates to allow
plasmid segregation and tested again for In Vitro Binding Assay--
GST-ABRA(E164Q) fusion protein was
expressed in Escherichia coli and purified by affinity
chromatography on glutathione-agarose as described (25). In
vitro translated 35S-labeled AOP-1 was prepared by
using pBluescript-AOP-1 as a template with an RNA production system-T7
and rabbit reticulocyte lysate translation system as described by the
supplier with modifications. The translation reaction contained 2 mM GTP and 2 mM MgCl2 in addition
to the original formula. For the binding assay, BSA-washed glutathione-agarose beads were bound to GST-ABRA(E164Q) or GST in
buffer containing 10 mM Na2HPO4,
150 mM NaCl, 2.7 mM KCl, 1.8 mM
KH2PO4, 10 mM DTT, and 1% Triton
X-100 for 1 h at 4 °C and then washed six times with the same
buffer. After the GST- or GST-ABRA(E164Q)-bound beads were incubated
with 35S-labeled AOP-1 (50 µl of in vitro
translated product) for 2 h at 4 °C, the reaction products were
extensively washed with the same buffer and extracted with SDS sample
buffer. The extracts were analyzed by SDS-PAGE and exposed to Kodak
x-ray film.
Transfection of HeLa Cells with AOP-1 cDNA--
A 768-base
pair cDNA containing the entire coding region of AOP-1 and a flag
tag at its C terminus was subcloned into a mammalian expression vector,
and the plasmid purification was prepared using a Qiagen Maxi Kit
(Groningen, Netherlands). HeLa cells were transfected with the
mammalian expression vector pcDNA3-AOP-1 using LipofectAMINE reagent according to the manufacturer's instructions. The
LipofectAMINE-DNA complexes were left on the cells for 5 h, and
then the transfection mixtures were removed and replaced with normal
growth medium to grow for 24 h.
Preparation of Subcellular Fractions and
Immunoblotting--
Cells were washed twice with PBS, and the pellet
was suspended in 0.5 ml of buffer containing 20 mM Hepes,
pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
protease inhibitors (Complete Mixture, Roche Molecular Biochemicals),
and 250 mM sucrose. The cells were homogenized by 40 strokes in a Dounce homogenizer. The homogenates were centrifuged at
500 × g for 10 min at 4 °C to remove nuclei and
unbroken cells, and the supernatant was re-centrifuged at 8,000 × g for 10 min at 4 °C to pellet the crude mitochondria fraction. The supernatant was centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant was referred to
as the cytosolic fraction. Samples containing 40 µg of protein were
subjected to 12% SDS-PAGE and electroblotted on a polyvinylidene
difluoride membrane. The membrane was probed with anti-cytochrome
c monoclonal antibody 7H8.2C12 at a dilution of 1:1,000,
anti-flag M5 antibody at a dilution of 1:1,000, and anti- Immunocytochemistry and Confocal Microscopy--
HeLa cells were
grown on glass coverslips for 24 h in DMEM containing 10% FBS.
Cells were transfected with pcDNA3-AOP-1 as described previously.
Apoptosis was induced by ABR treatment. The reaction was terminated by
washing the culture cells three times with PBS, followed by fixation in
freshly prepared 4% paraformaldehyde in PBS for 12 min. The fixed
cells were washed three times in PBS, followed by permeabilization in
0.2% Triton X-100 in PBS for 2 min. The cells were then blocked for 30 min in blocking buffer (0.2% Triton X-100 and 10% FBS in PBS),
followed by a 1-h incubation with mouse monoclonal antibody against
cytochrome c (6H2.B4) at a dilution of 1:1,000 and chicken
polyclonal antibody against DYKDDDK(flag) at a dilution of 1:500. The
cells were washed four times at 10 min each in blocking buffer followed
by a 1-h incubation with a fluorescein-conjugated goat anti-mouse
antibody for cytochrome c and a rhodamine-conjugated rabbit
anti-chicken antibody for flag. After extensive washing, slides were
mounted and examined under a Zeiss Axipophot inverted microscope with a
100× oil objective lens.
To assess colocalization of AOP-1 with ABR in HeLa cells, uptake
experiments with ABR and immunofluorescence for detecting endogenous
AOP-1 were performed. Cells were incubated in serum-free medium
containing 10 µg/ml FITC-ABR for 30 min at 4 °C and then warmed at
37 °C for 45 min. The treated cells were placed back on ice, rinsed
with 0.1 M galactose to remove surface-bound FITC-ABR, fixed, permeabilized, and stained for AOP-1 using rabbit polyclonal antibody and a rhodamine-conjugated goat anti-rabbit antibody. Samples
were imaged on a confocal laser scanning microscope (Leica TCS SP2),
with a 100× oil objective lens.
Assessment of Apoptosis--
Apoptotic cell death was examined
by the terminal deoxynucleotidyl transferase-catalyzed deoxyuridine
triphosphate (dUTP)-nick end labeling (TUNEL) method as described by
the supplier (Roche Molecular Biochemicals). Samples (104
events) were analyzed with a Becton-Dickinson FACSCalibur, and the
distribution of cells was determined.
To assess DNA ladder formation, HeLa cells (1 × 106)
were treated with various concentrations of ABR for 24 h at
37 °C. The DNA were digested overnight at 37 °C in 0.5 mg/ml
proteinase K and 0.5% sarcosyl in PBS, treated with 10 µg of RNase A
for 1 h at 37 °C, then gently extracted with phenol and
chloroform, and analyzed on 1.5% agarose gel.
The viability of cells was assessed by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay. Cells were preincubated with NAC or 4-OH-tempo for 2 h
,followed by treatment with 5 ng/ml ABR for 15 h. The medium was
replaced by 500 µl of MTT solution (1 mg/ml in DMEM supplemented with
10% FBS) and further incubated for 1 h at 37 °C. The well was
then washed three times with PBS, and ethanol was added to the well.
The absorbance at 570 nm was measured by an enzyme-linked immunosorbent
assay reader.
Measurement of Intracellular ROS--
Levels of intracellular
ROS were measured as described (26) with minor modifications. Briefly,
HeLa cells (1 × 106/ml) were treated with 100 ng/ml
ABR for various periods of time and they were incubated with 10 µM DCFH-DA in the dark for 30 min before the cells were
harvested. The cells were washed with PBS and analyzed (excitation, 488 nm; emission, 515-540 nm) on a Becton-Dickinson FACSCalibur.
Assay of AOP-1 Antioxidant Activity--
The primers,
5'-GGGAATTCCATATGGCACCTGCTGTCACCCAGCATGCA-3' and
5'-CGGGATCCCTACTGATTTACCTTCTGAAA-3', were designed to PCR amplify a
585-base pair fragment from pGAD10-AOP-1. The amplified DNA fragment
encoding the amino acids 62-256 of AOP-1 was subcloned in-frame to
the NdeI/BamHI sites of the bacterial expression
vector pET-15b. The expression plasmid was transformed into bacteria BL21(DE3) pLysS, and the recombinant AOP-1 (re-AOP-1) was purified as
described by the supplier.
The thiol-specific antioxidant activity of AOP-1 was measured by the
method described by Kim et al. (27) with slight
modifications. Assays were performed in a 50-µl reaction mixture
containing 0.5 unit of glutamine synthetase, 10 mM DTT, 12 µM FeCl3, 100 mM Hepes (pH 7.4),
with re-AOP-1 (0.06 mg/ml) and various amounts of ABRA(E164Q) and were
incubated at 30 °C. At various time periods, aliquots (10 µl) were
removed to assay the residual glutamine synthetase activity.
Measurement of DEVDase and LEHDase Activities--
The
CaspACETM assay system was used according to the instructions of the
manufacturer. Briefly, cytosol extracts (50 µg) were incubated with
245 µl of reaction buffer containing 80 µl of caspase buffer, 5 µl of Me2SO, and 10 mM DTT for 30 min at
30 °C and then Ac-DEVD-AMC or Ac-LEHD-AFC was added at a final
concentration of 50 µM. The reaction mixtures were
incubated for 1 h at 30 °C. Cleavage of fluorogenic substrate
was quantitated by using a fluorescence spectrophotometer (F-4500,
Hitachi) at 360/460 nm or 400/505 nm.
Interaction of AOP-1 with ABRA(E164Q)--
To identify
proteins interacting with ABRA, a yeast two-hybrid system was employed.
Two clones that specifically interacted with ABRA(E164Q) were confirmed
by a colony-lift
To confirm that AOP-1 did indeed bind to ABRA(E164Q), human AOP-1 was
transcribed and translated in vitro. We used GST-ABRA(E164Q) linked to glutathione-Sepharose beads to examine the interactions between ABRA(E164Q) and AOP-1. After incubation with
35S-labeled AOP-1, these GST-ABRA(E164Q) beads (or the
control bait, GST beads) were pelleted and washed extensively. The
proteins which remained bound tightly to GST or to GST-ABRA(E164Q) were resolved by SDS-PAGE. The 35S-labeled AOP-1 was visualized
by autoradiography. The AOP-1 protein was bound specifically by the
GST-ABRA(E164Q) (Fig. 2). Taken together,
these data suggest that AOP-1 is an ABRA(E164Q)-interacting protein.
AOP-1 Localized in the Mitochondria--
To determine whether
AOP-1 localizes in the mitochondria as SP-22 does, we examined the
localization of AOP-1 within the cells and compared it with the
distribution of Colocalization of AOP-1 and ABR--
The ability of AOP-1 and ABR
to associate in vivo would indicate a possible role for this
interaction in ABR-induced apoptosis. We studied the intracellular
localization of AOP-1 and ABR by confocal microscopy of HeLa cells,
which were treated with FITC-conjugated ABR and stained for endogenous
AOP-1. In Fig. 4, green
fluorescence indicated the endocytotic FITC-ABR
(left panel) and red
fluorescence showed the mitochondrial punctate staining of
AOP-1 (center panel). As shown in the
right panel, there was colocalization of AOP-1 and ABR in yellow (white arrows). The
data suggest that ABR interacts with AOP-1 in vivo.
Induction of Apoptosis and Endogenous ROS by ABR--
The typical
laddering pattern of fragmented DNA associated with apoptosis appeared
after exposure of cells to 100 ng/ml ABR (Fig.
5A). The nuclei of HeLa cells
treated with ABR and then stained with Hoechst 33258 showed that
chromatin condensation was produced in apoptotic cells (data not
shown). These results are consistent with previous studies (34) and
show that abrin is able to trigger programmed cell death.
To determine whether the level of ROS in ABR-treated cells was
increased, we used FACS analysis with DCFH-DA, which is a nonpolar compound that readily diffuses into cells and is hydrolyzed to DCFH by
esterase. When DCFH is oxidized within the cell, it becomes a highly
fluorescent 2',7'-dichlorofluorescein. The fluorescence of untreated
HeLa cells represented the endogenous ROS, and 300 µM
H2O2 treatment was used as a positive control
(Fig. 5B). The intracellular ROS was increased 2-fold at
4 h after ABR treatment. The results suggest that the level of ROS
is increased at the early phase in ABR-treated cells.
Since increases of ROS generation in ABR-treated cells are the early
sign of the ABR toxicity, it is suggested that ROS are important
mediators of ABR-induced cell death. To test whether ROS levels play a
role in mediating the death signal of ABR, two antioxidants NAC and
4-OH-tempo, which is known to be localized into the mitochondria, were
used, and the results showed that the antioxidants inhibited
ABR-induced apoptosis in a dose-dependent manner (Fig.
5C). These data further demonstrate that the generation of
ROS plays an important role in the induction of apoptosis by ABR.
ABRA(E164Q) Inhibited the AOP-1 Antioxidant Activity in
Vitro--
To further examine whether ROS are important mediators of
ABR-induced cell death, we tested the effects of re-ABRA(E164Q) on the
antioxidant activity of recombinant AOP-1 in vitro. The antioxidant activity of re-AOP-1 was detected by monitoring the ability of the protein to inhibit the inactivation of glutamine synthetase using a DTT/Fe3+/O2 metal-catalyzed
oxidation system (27). Various amounts of ABRA(E164Q) were added to the
reaction mixture, and the protection value of AOP-1 (0.06 mg/ml) was
decreased from 81% protection (AOP-1 alone) to 32% (molar ratio:
AOP-1:ABRA(E164Q) = 1:2; Fig. 6).
The inhibitory effects of ABRA(E164Q) on AOP-1 antioxidant activity
were dose-dependent. The results indicate that ABRA(E164Q) not only specifically interacts with AOP-1, but also directly inhibits
the antioxidant activity of AOP-1, which may elevate the level of ROS,
followed by cell death.
ABR Induced the Translocation of Mitochondrial Cytochrome c into
the Cytosol and the Activation of Caspases--
To study whether the
release of cytochrome c from the mitochondria to the cytosol
is involved in ABR-triggered apoptosis, the amount of cytochrome
c in the cytosol was determined by immunoblot analysis.
Untreated cells did not contain any detectable amounts of cytochrome
c in the cytosol, whereas the level of cytosolic cytochrome
c was increased significantly at 4 h after ABR
treatment (Fig. 7A). The
results suggest that ABR induces apoptosis by releasing cytochrome
c from the mitochondria to the cytosol. The intracellular distribution of cytochrome c in HeLa cells was further
examined by immunofluorescence staining. Treatment of HeLa cells with
ABR for 4 h induced the loss of mitochondrial cytochrome
c staining (Fig. 7B). Arrows indicated
that cells with ABR treatment have more diffuse cytochrome c
immunostaining. These findings indicate that ABR-triggered apoptosis is
through the release of cytochrome c.
To assay the role of caspases in ABR-mediated apoptosis, zVAD-fmk, a
broad spectrum inhibitor of mammalian caspases, was preincubated with
HeLa cells and then the cells were treated with ABR for 24 h. FACS
analysis with TUNEL indicated that zVAD-fmk prevented cell death in a
dose-dependent manner, and 80 µM zVAD-fmk
inhibited about 50% of cell death caused by ABR treatment (Fig.
8A). The results indicate that
caspases are involved in ABR-triggered apoptosis.
To assess the involvement of caspase-9 and caspase-3, activation of
caspases was evaluated by the cleavage of the fluorogenic substrates,
Ac-LEHD-AFC and Ac-DEVD-AMC. After treatment of HeLa cells with ABR for
4 h, caspase-9 and caspase-3 activities were increased to 4- and
10-fold, respectively (Fig. 8, B and C).
Caspase-1 activity was not increased after ABR treatment (data not
shown). These results demonstrate that ABR-triggered apoptosis is
through the release of cytochrome c and the activation of
caspase-9, which in turn activates caspase-3.
AOP-1 Attenuates Apoptosis and Cytochrome c Release--
As
described above, the antioxidant activity of AOP-1 was inhibited by
ABRA in vitro and antioxidants such as NAC and 4-OH-tempo delayed ABR-induced cell death. We examined whether ectopic expressed AOP-1 could attenuate the apoptosis induced by ABR. The anti-apoptotic activity of AOP-1 was assayed by FACS analysis. When HeLa cells were
transiently transfected with 3 µg of AOP-1 plasmid before the
treatment with 100 ng/ml ABR for 24 h, cell death decreased from
79% to 41% (Fig. 9A). These
results suggest that the overexpression of AOP-1 can attenuate
ABR-induced apoptotic cell death.
To investigate whether the overexpression of AOP-1 is capable of
regulating the release of cytochrome c from the mitochondria in ABR-treated cells, we performed Western blot analysis. As shown in
Fig. 9B, HeLa cells transfected with various amounts of
AOP-1 plasmid were treated with ABR and the release of cytochrome
c from the mitochondria was decreased in a
dose-dependent manner. The results indicate that
overexpression of AOP-1 can inhibit the translocation of cytochrome
c from the mitochondria to the cytosol in ABR-treated cells.
To further confirm the inhibitory activity of AOP-1 on the
translocation of cytochrome c, immunofluorescence staining
of HeLa cells with anti-cytochrome c and anti-AOP-1-flag
antibodies was carried out. ABR induced the accumulation of cells with
diffuse cytochrome c staining, which was significantly
inhibited by AOP-1 (Fig. 9C). Arrows indicated
that non-transfected cells with ABR treatment possessed more diffuse
cytochrome c immunostaining. In contrast, AOP-1 transfected
cells with ABR treatment maintained a punctate cytochrome c
immunostaining pattern. These findings demonstrate that AOP-1 maintains
cytochrome c within the mitochondria and is an important
target molecule in ABR-induced apoptosis.
In this report, we have identified a novel ABR-interacting
protein, AOP-1, which was originally reported to be a thiol-specific antioxidant protein in vitro (35), but its physiological
function was unclear. Analysis of the amino acid sequence has revealed that the N-terminal 61 amino acids have characteristics of signals that
target translocation of proteins into mitochondria. By Western blot
analysis and immunocytochemistry (Fig. 3), we have shown that AOP-1 is
located in the mitochondria and not in the cytoplasm. AOP-1 is 93.3%
identical (97.4% similar) to SP-22, which has been reported to be one
of the antioxidant proteins located in the mitochondria (29). Both of
them conserve the two critical motifs found in all human peroxide
reductases (the two cysteine-containing segments
Cys47(FFYPLDFTFVCPTEI) and Cys168(HGEVCPA)).
These cysteine motifs have been suggested as being important for the
catalysis of peroxides (36, 37). Proteins imported into the
mitochondria are usually cleaved by proteases, thereby losing their
N-terminal signal sequence. The resulting recombinant AOP-1 protein
showed antioxidant activity (Fig. 6). These results suggest that AOP-1
can scavenge ROS as SP-22 does by cooperating with mitochondrial
thioredoxin and can protect mitochondrial components from the action of
superoxide anions or hydrogen peroxide (38, 39). It will be interesting
to determine whether there are other factors cooperating with AOP-1 to
function as an antioxidant complex in the mitochondria.
Mitochondria play a key role in the regulation of apoptosis. The
oxidant stress triggers mitochondrial permeability transition pore
opening by oxidizing thiol groups in the pore protein (40, 41). Thus,
the mitochondrial redox potential is important for regulating the
opening of mitochondrial permeability transition pore, which in turn
allows the release of cytochrome c and then induction of
caspase-dependent apoptosis (42-44). Interestingly, ABR
induces cells to generate ROS and the antioxidants NAC and 4-OH-tempo
can delay ABR-caused cell death. The overexpression of AOP-1 can
attenuate the apoptosis and block the release of cytochrome
c in ABR-treated cells (Fig. 9). These data indicate that
AOP-1 functions in the protection of cells from apoptosis and may be
implicated as an endogenous regulator of apoptosis.
We have shown that recombinant ABRA(E164Q) inactivates the antioxidant
activity of AOP-1 in vitro (Fig. 6). Hence, we speculate that ABRA interacts with AOP-1 and inactivates the antioxidant activity
of AOP-1, which may initiate apoptosis by a pathway distinct from
inhibition of protein synthesis. Notably, zVAD-fmk, a broad spectrum
caspase inhibitor, was unable to completely abrogate apoptosis caused
by ABR. One explanation for this is that the activation of caspase is
not the only pathway involved in apoptosis. The stress caused by the
inhibition of protein synthesis partially contributes to ABR-triggered
apoptosis. It has recently been demonstrated that the segment of the
ricin A-chain (SVTLALDVTNAY) linked to antibodies can induce the
apoptosis of human vein endothelial cells (7). The data suggested that
the ricin A-chain-mediated inhibition of protein synthesis and
apoptosis might be exerted by different motifs of the ricin A-chain molecule.
In summary, we propose the model shown in Fig.
10. The binding of ABRA(E164Q) to AOP-1
can shield the sulfhydryl groups and result in the inhibition of the
antioxidant activity. As a consequence of the loss of AOP-1 antioxidant
activity, the mitochondrial redox potential is shifted to a more
oxidized state and the mitochondria generate large amounts of ROS and
release cytochrome c, which in turn activates caspases to
trigger apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin antibody was purchased from Zymed
Laboratories Inc. (San Francisco, CA). Anti-flag M5 antibody was
purchased from Eastman Kodak Co. Chicken anti-DYKDDDK (flag) polyclonal
antibody, high fluorescent FITC-labeled goat anti-mouse antibody, and
rhodamine-conjugated rabbit anti chicken antibody were from Chemicon
International (Temecula, CA).
-galactosidase activity.
For quantitative data, the liquid culture assay using
o-nitrophenyl-
-D-galactopyranoside as a
substrate was performed, and the hydrolysis of the substrate was
measured at A420. Two clones were specific for
ABRA(E164Q). Nucleotide sequence analysis of cDNA inserts was
performed using a PE Applied Biosystems automated sequencer.
tubulin
antibody at a dilution of 1:2,000 in the buffer followed by horseradish
peroxidase-conjugated goat anti-mouse IgG. Antigen-antibody complexes
were visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter assay and were quantified by
a liquid culture assay (Table I).
Sequencing of these cDNA clones identified two major transcripts, which consisted of an open reading frame encoding 256 amino acids (Fig.
1). Computer analysis using NCBI/BLASTN
(28) revealed that the open reading frame was identical to human
protein AOP-1 (24). Data base searches revealed that AOP-1 shared
93.3% identity with SP-22, a bovine mitochondrial
thioredoxin-dependent peroxide reductase, and the
N-terminal sequence contained the basic features of mitochondrial
targeting signal (29) (Fig. 1). The mitochondria targeting signal was
composed of 20-60 amino acid residues with abundant positive charges,
no negative charges, and frequent hydroxylated residues, and they
were rich in hydrophobic residues (30-32). The sequence upstream of
the cleavage site was coincident with the specificity of two processing
peptidases for mitochondrial proteins. The amino acid sequences
RX
(F/L/I)XX(T/S/G)XXXX
were
cleaved sequentially in two steps by mitochondrial processing peptidase and mitochondrial intermediate peptidase (33). The data showed a high
probability that AOP-1 was localized in the mitochondria.
Two-hybrid interaction assay between ABRA and AOP-1 constructs
-galactosidase units were determined by the liquid culture
assay.
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Fig. 1.
Alignment of amino acid sequences of AOP-1
and SP-22. The sequence upstream of the cleavage sites
coincides with the specificity of two processing peptidases for
mitochondrial proteins (33). The vertical arrows
indicate the processing sites in mitochondria. Identical aligned
residues are shaded in black, and similar aligned
residues are shaded in gray. Squares
represent two catalytic cysteine residues (Cys47 and
Cys168) (24), and residue numbers are indicated on the
right.
View larger version (20K):
[in a new window]
Fig. 2.
Interaction of AOP-1 with ABRA(E164Q).
The input of lysate containing 1 µl of in vitro
translation (IVT) 35S-labeled AOP-1 is shown in
the left lane. 35S-Labeled AOP-1 was
incubated with GST-ABR or GST. GST fusion protein and
35S-labeled interacting protein were recovered on
glutathione-agarose and analyzed on 12% SDS-PAGE.
-tubulin. We constructed a plasmid clone
pcDNA3-AOP-1 that was designed to express AOP-1 with a flag tag at
the C-terminal end (AOP-1-flag). This plasmid DNA was transfected into
HeLa cells, and the distribution of AOP-1-flag was detected by
subcellular fractionation and Western blotting. As shown in Fig.
3A, the subcellular
localization of AOP-1-flag was found in the mitochondria, and this
indicated that AOP-1 was distinctly separated from
-tubulin. To
further confirm the mitochondrial localization of AOP-1,
immunofluorescence staining was carried out by using an anti-cytochrome
c antibody (a well characterized mitochondria marker). In
HeLa cells, the anti-cytochrome c antibody gave a punctate
staining pattern characteristic of mitochondrial localization and the
AOP-1-flag colocalized with cytochrome c (Fig.
3B). The same results were obtained by rabbit anti-AOP-1 polyclonal antibody to detect endogenous AOP-1 (data not shown). We
conclude that AOP-1 localizes in the mitochondria.
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Fig. 3.
Subcellular localization of AOP-1.
A, AOP-1 is localized in the mitochondria.
Immunoblotting was performed with anti-flag, and anti- -tubulin
antibodies and performed as described under "Experimental
procedures." B, confocal microscopy. The cells transfected
with pcDNA3-AOP-1-flag were stained for flag using an anti-flag
antibody and monoclonal anti-cytochrome c antibody with a
rhodamine-conjugated secondary antibody (red
fluorescence, left panel) and for
mitochondria using an anti-cytochrome c antibody with
FITC-labeled secondary antibody (green
fluorescence, center panel). The
overlay of both images is shown in the right
panel.
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Fig. 4.
Colocalization of AOP-1 with ABR by confocal
microscopy. HeLa cells were treated with FITC-ABR
(green fluorescence, left
panel) and stained for endogenous AOP-1 (red
fluorescence, right panel). The
overlay of both images is shown in the right
panel. Arrows indicate sites of colocalization of
AOP-1 and ABR. Inset shows a higher magnification of
colocalization sites (yellow color).
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Fig. 5.
ABR induces DNA fragmentation and the
generation of ROS. A, gel electrophoresis of DNA. HeLa
cells were treated with the indicated concentrations of ABR for 24 h before DNA electrophoresis on a 1.5% agarose gel as described under
"Experimental Procedures." B, FACS analysis of
ABR-induced cells incubated with DCFH-DA. Cells were treated with ABR
and then loaded with DCFH-DA as described under "Experimental
Procedures." Cells loaded with DCFH-DA were defined as having a
fluorescence of 24 arbitrary units. Data are representative of three
similar experiments. C, effects of antioxidants on cell
death induced by ABR. Cell death was determined by MTT assay. Cells
were treated with NAC or 4-OH-tempo for 1 h before ABR treatment.
The left bar represents the viability of cells
treated with only 5 ng/ml ABR. Data are expressed as the mean ± S.D. of three independent experiments.
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Fig. 6.
Attenuation of AOP-1 antioxidant activity by
ABRA(E164Q). Thiol-specific antioxidant activity of AOP-1 was
measured by the method described under "Experimental Procedures."
The reaction mixture was changed by deleting AOP-1 ( ) or by adding
the following: 1 mM EDTA (
), 0.06 mg/ml AOP-1 (
)
,0.06 mg/ml AOP-1 with 0.068 mg/ml ABRA(E164Q) (molar ratio:
AOP-1:ABRA(E164Q) = 2:1) (
); AOP-1:ABRA(E164Q) = 1:1
(
); or AOP-1:ABRA(E164Q) = 1:2 (
)). Data are the mean of
triplicate experiments. GS, glutamine
synthetase.
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Fig. 7.
The release of cytochrome c
from the mitochondria into the cytoplasm in ABR-treated HeLa
cells. A, immunoblot analysis. HeLa cells were treated
with 100 ng/ml ABR and cultured for different time periods. Cytosolic
fractions were prepared from ABR-treated cells, and Western blot
analysis was performed using anti-cytochrome c
(Cyt. c; 7H8.2C12) and anti- -tubulin
antibodies. The
-tubulin was indicated as a control of protein
loading. B, confocal microscopy. HeLa cells treated with 100 ng/ml ABR for 4 h were stained for cytochrome c using
an anti-cytochrome c antibody (6H2.B4) with an FITC-labeled
secondary antibody. The PBS-treated cells (upper
panels) show a punctate cytochrome c
immunostaining pattern. The cells treated with 100 ng/ml ABR for 4 h (lower panels) show more diffuse cytosolic
pattern. The cells with significant diffuse cytochrome c
immunostaining are indicated by arrows. The left
panels are images of Nomarski.
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Fig. 8.
Activation of caspases in ABR-treated
cells. A, influence of zVAD-fmk on the apoptosis of
ABR-treated HeLa cells. HeLa cells were pretreated with the indicated
concentration of zVAD-fmk for 5 h and treated with ABR (100 ng/ml)
for an additional 24 h. Details of staining, washing, and FACS
analysis are described under "Experimental Procedures."
B, caspase-9 was activated during ABR-triggered
apoptosis. The cytosolic extracts were prepared from HeLa cells
treated with 100 ng/ml ABR for the indicated periods of time at
37 °C. Ac-LEHD-AFC cleavage assay was performed as described under
"Experimental Procedures." C, activation of caspase-3
during ABR-triggered apoptosis. The cytosolic fractions were prepared
and Ac-DEVD-AMC cleavage was assayed as described under "Experimental
Procedures." These results are representative of three separate
experiments. The data are mean ± S.D. of three samples.
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Fig. 9.
Effects of AOP-1 on attenuation of apoptosis
and the release of cytochrome c from the mitochondria
into the cytoplasm in ABR-treated cells. A, FACS
analysis of ABR-triggered apoptosis in HeLa cells and HeLa/AOP-1 cells.
HeLa cells were transiently transfected with 3 µg of AOP-1 plasmids
and then treated with 100 ng/ml ABR for 24 h. Quantification of
the DNA fragmentation was performed by the TUNEL method, and the
details are described under "Experimental Procedures."
B, immunoblot analysis. HeLa cells transfected with various
amounts of AOP-1 plasmids were treated with 100 ng/ml ABR. Western blot
was performed as described previously. C, confocal
microscopy. HeLa cells transfected with AOP-1-flag and treated with 100 ng/ml ABR for 5 h were stained and experiments performed as
described previously. The red fluorescence
(left panel) represents AOP-1 transfected cells,
and the green fluorescence (center
panel) represents cytochrome c staining. The
overlay of both images is shown in the right
panel. The non-transfected cells with more diffuse cytosolic
cytochrome c immunostaining are indicated by
arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
The model of ABR-triggered apoptosis.
ABR-induced programmed cell death involves at least two pathways. One
is the inhibition of protein synthesis by its N-glycosidase
activity to influence unknown cellular factors, and the other is the
alteration of the function of mitochondria by specific interaction with
AOP-1. Overexpression of AOP-1 prevents cytochrome c
(Cyt. c) release and attenuates apoptosis in
ABR-treated cells. Once the antioxidant ability of AOP-1 is blocked by
ABRA, the redox balance is disrupted, followed by the generation of
ROS, the release of cytochrome c, and activation of the
caspase cascade. Finally, apoptosis occurs.
ABR or immunotoxins have a dose-limiting toxicity in vascular leak
syndrome in mammals and humans (45-50). Since the antioxidants NAC and
4-OH-tempo significantly delay the apoptosis induced by ABR, it is
suggested that antioxidants can be employed in reducing the toxicity of
immunotoxins and hence improve the efficiency of cancer chemotherapy.
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ACKNOWLEDGEMENTS |
---|
We thank Judy Siirila for careful reading of the manuscript and Yun-long Tseng for preparing the FITC-labeled ABR and technical support.
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FOOTNOTES |
---|
* This work was supported in part by Grant 89-B-FA01-1-4 from the Ministry of Education and Grant NSC-89-2316-B002-010 from the National Science Council, Republic of China.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.
Present address: Dept. of Medical Technology, Yuanpei Technical
College, Hsin-Chu 300, Taiwan, Republic of China.
§ To whom correspondence should be addressed. Tel.: 886-2-2312-3456 (ext. 8206); Fax: 886-2-2341-5334; E-mail: jylin@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M100571200
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ABBREVIATIONS |
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The abbreviations used are: ABR, abrin; ABRA, abrin A-chain; ABRA(E164Q), mutant form of abrin A-chain; ROS, reactive oxygen species; AOP-1, antioxidant protein-1; Apaf-I, apoptotic protease activating factor; SP-22, 22-kDa substrate protein; NAC, N-acetylcysteine; 4-OH-tempo, 4-hydroxy-2,2,6,6-tetramethylpiperi-dine-1-oxyl; z-VAD-fmk, Cbz-Val-Ala-Asp-(OMe)-fluoromethyl ketone; Ac-LEHD-AFC, Ac-Leu-Glu-His-Asp-AFC; Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-AMC; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; DTT, dithiothreitol; TUNEL, terminal deoxynucleotidyl transferase-catalyzed deoxyuridine triphosphate-nick end labeling; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphynyltetrazolium bromide; GST, glutathione S-transferase; DCFH, 2',7'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescein diacetate; DCF, 2',7'-dichlorofluorescein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorting.
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