From the Department of Genetics, Osaka University Medical School, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caspase-activated DNase (CAD) is responsible for
the DNA fragmentation that occurs during apoptosis. CAD is complexed
with an inhibitor of CAD (ICAD) in non-apoptotic, growing cells. Here, we report that mouse WR19L and human Jurkat T lymphoma cells express two alternative forms of ICAD, ICAD-L and ICAD-S, at similar levels. CAD was predominantly associated with ICAD-L in these cell lines. When
CAD was expressed alone in Sf9 cells, it was found in insoluble fractions. However, when CAD was co-expressed with ICAD-L and ICAD-S,
it was recovered as a soluble protein complexed predominantly with
ICAD-L. In vitro transcription and translation of CAD
cDNA did not produce a functional protein. Addition of ICAD-L but
not ICAD-S to the assay mixture resulted in the synthesis of functional CAD. These results indicated that ICAD-L but not ICAD-S works as a
specific chaperone for CAD, facilitating its correct folding during
synthesis. Recombinant CAD, as a complex with ICAD-L, was then produced
in Sf9 cells. The complex was treated with caspase 3, and CAD
was purified to homogeneity. The purified CAD had DNase activity with a
high specific activity.
Apoptosis is a process by which cells can be eliminated in
response to a wide range of stimuli, such as death factors (Fas ligand,
tumor necrosis factor, and TRAIL), anti-cancer drugs, Apoptosis is driven by members of the caspase family, which are
cysteine proteases (10). At least 14 caspase family members have been
identified, and they are divided into 3 subgroups based on their
substrate specificity (11, 12). Different apoptotic stimuli appear to
sequentially activate different sets of caspases, which ultimately
cleave various cellular substrates to cause the morphological changes
in cells and nuclei (6). The DNA fragmentation that occurs during
apoptosis also depends on caspase activation. We and others have
recently identified a caspase-activated DNase (CAD),1 which is also called
a caspase-activated nuclease or DNA fragmentation factor-40 (DFF-40)
(13-16). CAD is complexed with its inhibitor (ICAD), also called
DFF-45, in growing, non-apoptotic cells. Mouse CAD is a basic protein
consisting of 344 amino acids, while ICAD is an acidic protein with two
recognition sites for caspase 3. When caspase 3 is activated by
apoptotic stimuli, ICAD is cleaved, resulting in the release of CAD.
This released CAD appears to cause DNA fragmentation in nuclei.
Molecular cloning of mouse ICAD cDNA indicated that there are two
forms of ICAD, designated ICAD-L and ICAD-S for the long and short
forms, respectively. ICAD-L and ICAD-S are composed of 331 and 265 amino acids, respectively (13). They are translated from two different
mRNAs produced by alternative
splicing.2 Purification of
the latent form of CAD from human HeLa cells indicated that the ICAD
that is associated with CAD is ICAD-L (17). However, purification of
mouse ICAD by following its activity led us to identify ICAD-S (13).
Thus, it was not clear whether both ICAD-L and ICAD-S proteins were
expressed in mouse and human cells, and whether they had any functional differences.
In this study, we showed that mouse and human lymphoma cell lines
constitutively express both ICAD-L and ICAD-S at similar levels.
Fractionation of the cell lysates from human Jurkat cells, and from
Sf9 cells co-infected with a recombinant baculovirus containing
ICAD and CAD indicated that CAD is complexed predominantly with ICAD-L.
ICAD-L but not ICAD-S supported production of the functional CAD
protein in vitro. These results indicated that ICAD-L but
not ICAD-S has a chaperone-like function for CAD, and that it remains
complexed with CAD. Recombinant CAD was produced in insect cells as a
complex with ICAD-L, and was homogeneously purified after treatment
with caspase 3. The purified CAD showed high DNase activity.
Cells, Antibodies, and Recombinant Proteins
Mouse T-cell lymphoma WR19L and human Jurkat cells were
maintained at 37 °C in RPMI medium containing 10% fetal calf serum (Life Technologies, Inc., Gaithersburg, MD). Spodoptera
frugiperda (Sf9) insect cells (Invitrogen, Carlsbad, CA)
were cultured at 27 °C in TNM-FH medium (Invitrogen) supplemented
with 10% fetal calf serum.
Anti-human ICAD/DFF-45 (clone 6B8) and anti-FLAG (clone M2) monoclonal
antibodies were purchased from MBL (Nagoya, Japan) and Kodak
(Rochester, NY), respectively. Affinity purified, anti-murine ICAD
polyclonal antibody was custom prepared at MBL by immunizing rabbits
with recombinant murine ICAD-L. Anti-murine CAD antiserum was custom
prepared at the Peptide Institute (Osaka, Japan) by immunizing rabbits
with a C-terminal peptide (RRKQPARKKRPARKR, amino acids 330-344). The
antiserum was affinity purified using AF-amino Toyopearl beads (Tosoh
Co., Tokyo, Japan) to which the peptide had been attached using
m-maleimidbenzol-N-hydroxysuccinimide ester
(Pierce, Rockford, IL). The horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from DAKO (Denmark) and Transduction Laboratories (Lexington, KY), respectively. The recombinant ICAD fused with glutathione S-transferase (GST),
and the histidine-tagged recombinant human caspase 3 was prepared in
Escherichia coli as described previously (18).
Assay for CAD and ICAD
The activities of CAD and ICAD were determined with mouse liver
nuclei or plasmid DNA as described previously (13). For the ICAD assay,
the S-100 fraction from Fas-activated mouse W4 cells (13) or purified
recombinant CAD produced by Sf9 cells (see below) was used as
active CAD.
Western Blotting and in Vitro Transcription/Translation
Western blotting was performed as described previously (19). In
brief, proteins were separated by electrophoresis in a 12.5 or 10-20%
gradient polyacrylamide gel (Dai-ichi Chemical, Tokyo, Japan), and
electrophoretically transferred to a polyvinylidene difluoride membrane
(Hybond P, Amersham-Pharmacia, Arlington Heights, IL) at 100 V for
1 h. After treatment with 25% Block Ace (Dai-Nihon Pharmaceutical, Osaka Japan) or 5% skim milk, the filters were incubated with primary antibodies against human ICAD, mouse ICAD, mouse
CAD, or FLAG-epitope. Horseradish peroxidase-conjugated anti-mouse IgG
or anti-rabbit IgG was used as the secondary antibody, and proteins
were visualized using the enhanced chemiluminescence system
(Renaissance; NEN Life Science Products Inc., Boston, MA). The in
vitro transcription/translation was carried out using a kit from
Promega (Madison, WI) as described previously (13).
Production and Purification of ICAD and CAD in Sf9
Cells
Production of ICAD and the ICAD-CAD Complex in Sf9
Cells--
DNA fragments coding for FLAG-tagged mouse ICAD-L and
ICAD-S (F-ICAD-L and F-ICAD-S) were described previously (18). These fragments and a fragment coding for mouse CAD were inserted into the
pBlueBac III vector (Invitrogen), and high titer recombinant baculoviruses were generated using the MaxBac baculovirus expression system according to the manufacturer's instructions. To produce F-ICAD-L, F-ICAD-S, and the F-ICAD-L·CAD complex, Sf9 cells
were infected at a multiplicity of infection (m.o.i.) of 5.0 with the recombinant baculovirus. The cells were incubated at 27 °C for 3 days before harvest.
Preparation of Cell Lysates--
Cell lysates from the infected
Sf9 cells were prepared essentially as described previously
(13). In brief, cells were disrupted by three cycles of freezing and
thawing in buffer A (10 mM HEPES-KOH, pH 7.2, 2 mM MgCl2, 5 mM EGTA, 50 mM NaCl, and 1 mM dithiothreitol) supplemented
with protease inhibitors (1 mM p-APMSF
(p-amino-phenylmethanesulfonyl fluoride hydrochloride), 3 µg/ml aprotinin, 3 µg/ml leupeptin, and 3 µg/ml pepstatin A). The
lysates were spun for 30 min at 30,000 × g, and the
supernatant fractions were used as S-30 fractions. The S-30 fractions
were spun for 2 h at 100,000 × g to obtain the
S-100 fraction. Glycerol was added to the S-30 and S-100 fractions at a
final concentration of 10% (v/v).
Purification of the ICAD·CAD Complex--
To purify the
F-ICAD-L·CAD complex, the S-100 fraction from Sf9 cells
co-infected with the F-ICAD-L and CAD baculoviruses was applied to a
DEAE-Sepharose FF column equilibrated with buffer B (10 mM
HEPES-KOH, pH 7.2, 2 mM MgCl2, 5 mM
EGTA, 10% glycerol, 0.1 mM dithiothreitol, and 0.1 mM p-APMSF) containing 50 mM NaCl, and eluted with a 50-350 mM linear NaCl gradient. The
active fractions were applied to an SP-Sepharose FF column, and eluted
with a 50-600 mM linear NaCl gradient. The ICAD·CAD
complex, which eluted at 350-500 mM NaCl, was then applied
to a HiTrap Heparin column, and eluted with a 150-700 mM
linear NaCl gradient. About 3 mg of F-ICAD-L·CAD complex was obtained
from 1000 ml of Sf9 culture.
Preparation of Active CAD--
About 2.5 mg of purified
F-ICAD-L·CAD complex was treated at 25 °C for 36 h with 25 µg of recombinant caspase 3 in buffer B supplemented with 2 mM dithiothreitol. The sample was applied to a HiTrap
Heparin column (5 ml) equilibrated with buffer D (buffer B without
glycerol) containing 300 mM NaCl, and eluted with a 300-1000 mM linear NaCl gradient. About 0.2 mg of active
CAD was obtained.
Miscellaneous--
All purification steps were carried out using
an fast protein liquid chromatography system (Amersham-Pharmacia) at
4 °C. Protein concentration was determined by the bicinchoninic acid
method using a kit from Pierce. Quantification of homogeneous
preparations of recombinant proteins was carried out by measuring
A280 and using extinction coefficients
calculated as described previously (20).
Two Forms of ICAD and Predominant Association of CAD with
ICAD-L--
We previously identified two forms of mouse ICAD cDNAs
coding for ICAD-L and ICAD-S (13). To examine whether ICAD-L and ICAD-S
proteins are indeed expressed, cell lysates were prepared from mouse
WR19L and human Jurkat cells, and analyzed by Western blotting. As
shown in Fig. 1A, the
polyclonal antibody against mouse ICAD detected two bands at apparent
Mr of 45,000 and 34,000. These two bands were
not detected by rabbit non-immune serum. Sizes of the two proteins
agreed with those detected in the lysates from Sf9 cells
infected with baculovirus carrying ICAD-L and ICAD-S, respectively.
When the cell lysates were treated with caspase 3, both bands
disappeared. These results indicated that the 45,000 and 34,000 bands
represented ICAD-L and ICAD-S, respectively. ICAD-L appeared to be more
abundant than ICAD-S in WR19L cells. The cell lysates from human Jurkat
cells also showed two bands at Mr of 45,000 and
34,000 in Western blotting using an anti-human ICAD/DFF-45 monoclonal
antibody as the probe (Fig. 1B). Both bands were cleaved by
caspase 3. In Jurkat cells, ICAD-S appeared to be more abundant than
ICAD-L.
Mouse WR19L and human Jurkat cells carry a latent form of CAD (proCAD)
that exists as a complex with ICAD, and can be activated by caspase 3 (13, 16, 18). When the S-100 fraction from Jurkat cells was loaded onto
an SP-Sepharose column in buffer containing 50 mM NaCl,
proCAD was retained in the column, and eluted with 600 mM
NaCl (Fig. 2A). The S-100
fraction also contained the ICAD activity, which was recovered in the
flow-through fractions (Fig. 2B). To determine which form of
ICAD, ICAD-L or ICAD-S, is associated with CAD, the flow-through and
bound fractions were analyzed by Western blotting with the anti-ICAD
antibody. As shown in Fig. 2C, the ICAD in the flow-through
fraction was predominantly ICAD-S, while ICAD-L was predominant in the
bound fractions. Re-chromatography of the bound fractions on
SP-Sepharose showed co-elution of ICAD-L with the proCAD activity in a
linear NaCl gradient. The ICAD activity in the flow-through fraction of
SP-Sepharose was bound to a DEAE-Sepharose column, and co-eluted with
ICAD-S in a linear NaCl gradient (data not shown). These results
indicated that CAD is predominantly associated with ICAD-L. ICAD-S
exists mainly in a free form. Similar data were obtained using the
S-100 fraction from mouse WR19L cells (data not shown).
Predominant Incorporation of ICAD-L into the ICAD·CAD
Complex--
To confirm the predominant association of CAD with
ICAD-L, recombinant baculoviruses containing F-ICAD-L, F-ICAD-S, and
CAD were constructed, and used to infect Sf9 cells. After
culturing for 24 h, the cell lysates were spun at 30,000 × g for 30 min, and the supernatant and precipitate fractions
were designated as S-30 and P-30, respectively. As shown in Fig.
3A, the S-30 fraction from the
parental Sf9 cells or the cells infected with F-ICAD-L,
F-ICAD-S, or CAD baculovirus alone did not show CAD activity. However,
when the cells were co-infected with baculoviruses containing CAD and
F-ICADs, the CAD activity was detected in the S-30 fraction (Fig.
3A). The CAD activity in the S-30 fraction increased when
the m.o.i. of the CAD baculovirus was increased. Proteins in the S-30
and P-30 fractions were then analyzed by Western blotting using the
anti-FLAG antibody recognizing F-ICAD. As shown in Fig. 3B,
two bands of an equal intensity at Mr of 45,000 and 34,000 were exclusively detected in the S-30 fraction, suggesting
that both ICAD-L and ICAD-S are soluble proteins. In contrast, when
Sf9 cells were infected with the CAD baculovirus alone, the CAD
protein with Mr of 40,000 was exclusively found in the P-30 fraction, suggesting that CAD formed aggregates. However, co-expression of CAD with ICADs in Sf9 cells resulted in soluble CAD (Fig. 3C). At lower expression levels of CAD, all of the
CAD was found in the S-30 fraction. When CAD was overexpressed, some of
it was detected in the P-30 fraction. The expression levels of ICAD-S
and ICAD-L, and their solubility, were not affected by the expression
of CAD. These results suggest that the soluble and functional proCAD
could be produced only in the presence of ICAD.
To determine whether ICAD-L, ICAD-S, or both form a complex with CAD,
the cell lysates from Sf9 cells infected with various dosages of
CAD and ICAD baculovirus were loaded onto SP-Sepharose in 50 mM NaCl. The proCAD activity was retained on the column, and eluted with 600 mM NaCl (Fig.
4B). ICAD-L and ICAD-S in the cell lysates from Sf9 cells infected with ICAD-L and ICAD-S
baculovirus were found in the flow-through fractions (Fig.
4A). When CAD was co-expressed with ICAD-L and ICAD-S,
ICAD-L but not ICAD-S bound to the column, and co-eluted with CAD. At
the highest concentration of CAD, all of the ICAD-L protein was found
in the bound fractions, while only a small portion of the ICAD-S bound
to the column. These results indicated that CAD predominantly
associates and forms a complex with ICAD-L during its synthesis in
Sf9 cells.
ICAD-L Supports Synthesis of the Functional CAD in
Vitro--
Predominant association of ICAD-L with CAD during CAD
synthesis suggested that ICAD-L, but not ICAD-S works as a chaperone to
facilitate the correct folding of CAD during its synthesis. To examine
this possibility, ICAD-L and ICAD-S were prepared in Escherichia
coli as GST fusion proteins. At first, the inhibitory activity of
ICAD was determined with the purified CAD (see below). As shown in Fig.
5A, both ICAD-L and ICAD-S
efficiently inhibited CAD's DNase activity at a molar ratio of 5-10
for ICAD to CAD, suggesting that ICAD-L and ICAD-S have comparable
ability to bind intact CAD. When CAD cDNA was transcribed and
translated in vitro, no functional CAD was produced (Fig.
5B), although CAD protein was synthesized (data not shown).
The presence of ICAD-L in the reaction mixture stimulated synthesis of
functional CAD in a dose-dependent manner. In contrast,
almost no functional CAD was produced in the presence of ICAD-S.
Production of Recombinant CAD--
To produce CAD in a large
quantity, Sf9 cells were infected with baculoviruses of CAD and
ICAD-L at m.o.i. of 5.0, and the ICAD-L·CAD complex was purified from
the cell lysates by successive column chromatographies of
DEAE-Sepharose, SP-Sepharose, and HiTrap Heparin column. As shown in
Fig. 6A, the purified
ICAD·CAD complex showed bands of Mr of 45,000 and 40,000 on SDS-polyacrylamide gel electrophoresis, which correspond
to ICAD-L and CAD, respectively. Treatment of the ICAD·CAD complex
specifically cleaved ICAD into fragments of Mr
18,000, 12,000, and 12,000. Although the cleaved ICAD-L fragments had
some affinity to CAD, removal of glycerol from buffer reduced its
affinity to CAD. Therefore, the ICAD-L·CAD complex after treatment
with caspase 3 was loaded onto a heparin column, and the column was
developed with a liner NaCl gradient in a buffer without glycerol.
Under these conditions, CAD was eluted from the column later than the
cleaved ICAD fragments. CAD, thus purified, was homogeneous (Fig.
6A), and it digested naked DNA in a
dose-dependent manner (Fig. 6B). One nanogram of CAD efficiently digested 1 µg of DNA within 2 h in our assay
conditions.
Apoptosis induced by various stimuli is mediated by caspases,
which cleave various cellular substrates to promote the apoptotic process (10). One of these substrates is ICAD, also known as DFF-45
(13, 17, 18). Once ICAD/DFF-45 is cleaved by caspase-3, CAD (also known
as caspase-activated nuclease or DFF-40) is released, is activated, and
causes the characteristic fragmentation of chromosomal DNA into
nucleosomal units. Characterization of mouse ICAD cDNA indicated
that there are two different ICAD mRNAs (ICAD-L and ICAD-S), which
can be generated by alternative splicing. Although both forms of ICAD
are expressed at similar levels, CAD was predominantly associated with
ICAD-L in mouse and human cells. This was confirmed by expressing CAD
and ICAD in Sf9 cells, from which CAD was recovered as a soluble
protein preferentially complexed with ICAD-L. These results agree with
previous reports that DFF (proCAD) is purified from human HeLa cells as
a complex with DFF-45 (ICAD-L) (17). The expression of CAD alone in
Sf9 cells caused aggregation of the protein, and ICAD-L but not
ICAD-S supported production of functional CAD in a cell-free system,
suggesting a chaperone-like activity for ICAD-L but not ICAD-S. It is
likely that during synthesis of CAD on the ribosome, ICAD-L binds to
the nascent chain of CAD and facilitates its correct folding. Mice
deficient in the ICAD gene express CAD, but it does not cleave
chromosomal DNA during apoptosis (21), which agrees with our results
indicating an absolute requirement for ICAD to produce functional
CAD.
ICAD-L and -S are generated from a single gene with alternative
splicing.2 Alternative splicing often gives rise to
products with different functions. It has been reported that
alternative splicing can also affect the folding property of a
chaperone-like protein (22). Previously, we have expressed CAD in COS cells and in reticulocyte
lysates, and suggested that it has a DNase activity (13). However,
since the preparation contained many other proteins, the possibility
that CAD needs other co-factors to show its DNase activity was not
formally ruled out. Here, we showed that the recombinant homogeneous
CAD has the DNase activity with a high specific activity. If the
functional CAD containing a nuclear localization signal is produced in
cells, it will digest chromosomal DNA, and kills the cells. Cells have
developed a device to avoid such a deleterious situation. That is, the
correctly folded CAD is synthesized only in the presence of its
inhibitor, ICAD-L. ICAD-L remains as a complex with CAD in the cells
until it is cleaved by action of caspase 3. There is a precedent for
such a heteromeric structure for a chaperone and its target protein. Prolyl 4-hydroxylase, which catalyzes a post-translational modification of collagen, consists of two distinct polypeptides forming an ICAD-L but not ICAD-S was necessary to produce functional CAD, and
ICAD-L can inhibit the CAD's DNase. However, we found that both ICAD-L
and -S are expressed in various human and mouse cell lines. Why is
ICAD-S expressed in living cells? The analyses of the CAD and ICAD gene
promoters indicated that their expression is controlled by constitutive
promoter as found in many housekeeping genes.4 Since the expression
level of CAD can be controlled by ICAD-L, it is possible that splicing
for ICAD-L is prevalent in the tissues undergoing apoptosis at high
frequency, to generate more CAD. Another possibility is that ICAD-S
works as a dominant-negative form for the chaperone-like function of
ICAD-L. Death signaling molecules such as caspases, Ced-4, and Bcl-x
(29-32) have alternatively spliced forms with opposite functions.
Similarly, if the amount of ICAD-S is increased, the complex formation
between ICAD-L and CAD may be down-regulated. This may provide a
fine-tuning mechanism for apoptotic DNA fragmentation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or UV
radiation, and factor deprivation (1-4). This process is characterized
morphologically by cell shrinkage accompanied by blebbing of the plasma
membranes, and condensation and fragmentation of the nuclei (5-7). It
is also characterized biochemically by internucleosomal degradation of
chromosomal DNA (8, 9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Two forms of ICAD in mouse and human
cells. The S-100 fraction was prepared from mouse WR19L
(A) and human Jurkat cells (B) as described
previously (13). Twenty micrograms of protein before (lane
1) and after (lane 2) treatment with 0.2 µg of
caspase 3 at 30 °C for 2 h were analyzed by Western blotting
with rabbit anti-mouse ICAD antibody (A) or anti-human
ICAD/DFF-45 monoclonal antibody (B). As a control for
anti-mouse ICAD antibody, the S-100 fraction from mouse WR19L cells was
also analyzed by Western blotting with rabbit non-immune serum
(lane 3). The sizes of molecular marker proteins are shown
in kilodaltons on the left, and the bands for ICAD-L
(L) and ICAD-S (S) are indicated by
arrows on the right.
View larger version (28K):
[in a new window]
Fig. 2.
Separation of proCAD and ICAD by SP-Sepharose
column. The S-100 fraction (5 mg of protein in 1 ml) from human
Jurkat cells was loaded onto an SP-Sepharose column (1-ml bed volume)
equilibrated with buffer B containing 50 mM NaCl, and
proteins were eluted with 1 ml of buffer B containing 600 mM NaCl. A, CAD activity. The CAD activity in
the S-100 fraction (lane 1, 40 µg of protein), the
flow-through fraction (lane 2, 10-µl aliquots), and the
bound fraction (lane 3, 10-µl aliquots) was determined
with mouse liver nuclei as the substrate. Liver nuclei subjected to the
assay procedure without the enzyme sample are shown in lane
C. B, ICAD activity. The ICAD activity in the S-100
fraction (lane 1, 40 µg of protein), the flow-through
fraction (lane 2, 10-µl aliquots), and the bound fraction
(lane 3, 10-µl aliquots) was determined with the Fas-activated W4
cell lysate providing the activated CAD (13). The liver nuclei, treated
with Fas-activated W4 cell lysate alone, are shown in lane
C. C, Western blotting for ICAD. The S-100 fraction
(lane 1, 20 µg of protein), the flow-through fraction
(lane 2, 5-µl aliquots), and bound fraction (lane
3, 5-µl aliquots) were analyzed by Western blotting with
anti-human ICAD/DFF-45 antibody. The molecular mass of standard
proteins are shown in kilodaltons on the left. The bands
corresponding to ICAD-L (L) and ICAD-S (S) are
indicated by arrows on the right.
View larger version (36K):
[in a new window]
Fig. 3.
Production of CAD and ICAD in Sf9
cells. Sf9 cells were infected with recombinant
baculoviruses (ICAD-L, ICAD-S, and CAD) at m.o.i. as indicated above
each lane. After culturing the cells for 24 h, S-30 and P-30
fractions were prepared. A, the CAD activity in the S-30
fraction. The S-30 fraction (1 µg) from baculovirus-infected
Sf9 cells was treated (upper panel) or untreated
(lower panel) with caspase 3, and the CAD activity was
determined with plasmid DNA as the substrate. B and
C, distribution of ICAD and CAD. Proteins in the S-30
(upper panel) and P-30 (lower panel) fractions
from the baculovirus-infected Sf9 cells (corresponding to 5 × 104 cells per lane) were analyzed by Western blotting
with anti-FLAG antibody (B) or anti-CAD antibody
(C). The molecular masses of the standard proteins are shown
in kilodaltons on the left. The bands corresponding to
ICAD-L (L), ICAD-S (S), and CAD are indicated by
arrows on the right.
View larger version (50K):
[in a new window]
Fig. 4.
Co-fractionation of CAD with ICAD-L.
Sf9 cells were infected with recombinant baculoviruses (ICAD-L,
ICAD-S, and CAD) at m.o.i. as indicated above each lane, and S-30
fractions were prepared after culturing the cells for 24 h. The
S-30 fractions (100 µg of protein) in 100 µl of buffer B containing
150 mM NaCl were incubated with 20 µl (bed volume) of
SP-Sepharose at 4 °C for 1 h. The proteins that did not bind to
the column were recovered as the flow-through fractions (A),
while the proteins bound to the Sepharose beads were eluted with 100 µl of buffer B containing 600 mM NaCl (B).
Top panels, aliquots (1 µl) of the flow-through and bound
fractions were treated with caspase 3, and the CAD activity was
determined with plasmid DNA as the substrate. Middle and
bottom panels, proteins (4-µl aliquots) in the
flow-through and bound fractions were separated by electrophoresis on a
12.5% polyacrylamide gel, and analyzed by Western blotting using
anti-FLAG (middle panels) or anti-CAD (bottom
panels) antibodies. Sizes of the standard proteins are shown in
kilodaltons on the left. The bands corresponding to ICAD-L
(L), ICAD-S (S), and CAD are indicated by
arrows on the right.
View larger version (57K):
[in a new window]
Fig. 5.
Chaperone-like activity of ICAD-L for CAD
synthesis. A, CAD-inhibitory activity of ICAD. Pure CAD
(70 fmol) was incubated at 25 °C for 10 min without (lane
1) or with GST (lane 2, 1,500 fmol), GST-ICAD-L
(lane 3, 75 fmol; lane 4, 375 fmol; lane
5, 1,500 fmol), or GST-ICAD-S (lane 6, 75 fmol;
lane 7, 375 fmol; lane 8, 1,500 fmol). The CAD
activities were then determined with plasmid DNA as the substrate.
B, synthesis of functional CAD in a cell-free system. CAD
cDNA was subjected to the in vitro transcription and
translation coupled system in the presence of increasing amounts of GST
(lanes 2-5), GST-ICAD-L (lanes 6-9), and
GST-ICAD-S (lanes 10-13). Using 3-µl aliquots of the
reaction mixtures, the CAD activity with plasmid DNA as a substrate was
determined in the presence (upper panels) or absence
(lower panels) of caspase-3. The amount of proteins added to
the assay mixture were: lane 1, 0 pmol; lanes 2, 6, and 10, 0.75 pmol; lanes 3, 7, and
11, 1.5 pmol; lanes 4, 8, and 12, 3.75 pmol; and lanes 5, 9, and 13, 7.5 pmol.
View larger version (55K):
[in a new window]
Fig. 6.
Purification of the recombinant ICAD,
ICAD·CAD complex, and CAD. A, analysis of the
purified proteins by SDS-polyacrylamide gel electrophoresis. About 1.5 µg of the purified recombinant F-ICAD-L·CAD complex (lane
1) and CAD (lane 2) were fractionated by
electrophoresis on a 10-20% polyacrylamide gel, and stained with
Coomassie Brilliant Blue. B, DNase activity of the purified
CAD. Plasmid DNA (1 µg) was incubated at 30 °C for 2 h with
the purified CAD, and analyzed by electrophoresis on a 1.5% agarose
gel. The amounts of CAD used were: lane 1, 0 fmol;
lane 2, 2.8 fmol; lane 3, 7.0 fmol; lane
4, 14 fmol; lane 5, 28 fmol; lane 6, 70 fmol; lane 7, 140 fmol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Crystallin is a member of the small
heat shock protein family, and has a chaperone-like activity in
suppressing nonspecific aggregation of denatured proteins in
vitro (23). One of the subunits of
-crystallin (
A-subunits)
has a minor product (
Ains-crystallin) that carries an
insertion of 23 amino acids, which is produced by alternative splicing.
Both
A- and
Ains-crystallin display similar
heat-resistance, self-renaturation, and oligomerization properties, but
Ains-crystallin has less chaperone-like activity than
A-crystallin. Similarly, both ICAD-L and ICAD-S are resistant to
heat and easily renaturate
(13).3 Although they
interacted with intact CAD with similar efficiency, only ICAD-L was
associated with CAD in the cells, and supported production of
functional CAD in vitro. ICAD-L is 66 amino acids longer
than ICAD-S at the C terminus. This extra region of ICAD-L is probably
responsible for its interaction with the nascent chain of CAD. In
addition to facilitating folding of nascent polypeptides, chaperone-like proteins can enhance renaturation of their denatured target proteins (24, 25). Whether or not ICAD-L has such a property
remains to be studied.
2
2 heterotetramer (26). The
-subunit
has protein disulfide isomerase activity. The
-subunit forms highly
insoluble aggregates without
-subunits (27, 28). However, in the
presence of
-subunits/protein disulfide isomerase, the
-subunit
is correctly folded with intramolecular disulfide bonds, and becomes a
soluble protein to form an
2
2 heterotetramer. CAD is also rich in cysteine residues (14 residues in
344 amino acids), and correctly folded only in the presence of ICAD-L.
To examine how ICAD-L promotes the correct folding of CAD, it would be
necessary to analyze whether the cysteine residues of CAD are
disulfide-bonded, and thus whether any free thiol is involved in the
folding of the protein.
![]() |
ACKNOWLEDGEMENT |
---|
We thank S. Kumagai for secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture in Japan and the Technology Agency of the Japanese Government.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.
Supported by research fellowships of the Japan Society for the
Promotion of Science.
§ Present address: Neurogenetics Unit, Imperial College School of Medicine at St. Mary's, Norfolk Place, London W2 1PG, United Kingdom.
¶ To whom correspondence should be addressed: Dept. of Genetics, Osaka University Medical School, B-3, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6879-3310; Fax: 81-6879-3319; E-mail: nagata{at}genetic.med.osaka-u.ac.jp.
2 K. Kawane, H. Fukuyama, H. Sakahira, and S. Nagata, unpublished results.
3 H. Sakahira and S. Nagata, unpublished results.
4 K. Kawane and S. Nagata, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CAD, caspase-activated DNase; ICAD, inhibitor of CAD; DFF, DNA fragmentation factor; GST, glutathione S-transferase; p-APMSF, p-amino-phenylmethanesulfonyl fluoride hydrochloride; m.o.i., multiplicity of infection.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|