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INTRODUCTION |
Poly(ADP-ribose) polymerase
(PARP-1)1 synthesizes
poly(ADP-ribose) (pADPr) in response to DNA strand breaks. This nuclear
enzyme, present in most eukaryotic cells, is involved in the
maintenance of the DNA integrity (1, 2). Recently, other pADPr
synthesizing enzymes were identified, suggesting the presence within
mammalian cells of a PARP-1-like enzyme family. A protein named
tankyrase with homology to ankyrins and to the catalytic domain of
PARP-1 was isolated from human tissue and shown to be associated with telomeres (3). Other proteins homologous to the catalytic domain of
PARP-1 have also been reported (4-8).
Cells display a low basal level of pADPr, which can increase
dramatically in response to DNA damaging agents (9-11). This increase
in pADPr synthesis is transient and is followed by a rapid degradation
by poly(ADP-ribose) glycohydrolase (PARG) (10, 12, 13). Two forms of
PARG (74 and 59 kDa) have previously been purified from various tissues
(14-19). However, the PARG cDNA recently isolated encodes an
active protein of 111 kDa (20). Furthermore, we have recently reported
the presence of only the 111-kDa form of PARG, which is localized
mostly in the cytoplasm of the cells (21, 22). These findings raise
questions about the cellular mechanism of pADPr catabolism and the
physiological significance of the 59- and 74-kDa forms of PARG.
Programmed cell death, or apoptosis, is an essential mechanism for
appropriate embryogenesis, normal cell turnover, and the selection of
lymphocytes (23, 24). Apoptosis is characterized by the activation of a
cascade of cysteine proteases, named caspases, which trigger the
biochemical and morphological changes occurring during cell
dismantling. This includes nuclear condensation, DNA fragmentation, and
the formation of apoptotic bodies (25, 26). Caspases cleave a specific
set of cellular proteins like the lamins (27) and the inhibitor of
caspase-activated deoxyribonuclease (28) that results in their
inactivation. Caspase cleavage can also result in a constitutive
activation of proteins such as protein kinase C
(29, 30) as well as
their own precursors, procaspases (31).
Cells undergoing apoptosis show a transient synthesis of pADPr (32,
33). We have previously reported that PARP-1 is cleaved by caspases
during apoptosis (34-36). This cleavage separates its DNA-binding
domain from the catalytic domain, resulting in the inactivation of the
enzyme. Furthermore, we have recently shown that during etoposide
(VP-16)-induced apoptosis, a transient pADPr synthesis is concomitant
with the cleavage and inactivation of PARP-1 (37). This suggests a
regulation of pADPr metabolism during apoptosis, which could also
involve a modulation of PARG activity. In fact, PARG contains potential
caspase cleavage sites, raising the possibility that this enzyme could
also be cleaved during apoptosis. In this report we show that PARG is
cleaved by caspase-3 during apoptosis. This cleavage releases
enzymatically active C-terminal fragments of 85 and 74 kDa in human cells.
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EXPERIMENTAL PROCEDURES |
Materials--
[32P]NAD (30 Ci/mmol) and
[35S]methionine (1175 Ci/mmol) were purchased from
PerkinElmer Life Sciences. VP-16, staurosporine, activated calf thymus
DNA, 1,5-isoquinolinediol, and 1-methyl-3-nitro-1-nitroguanidine (MNNG)
were obtained from Sigma-Aldrich.
-NAD and the protease inhibitor
mixture tablets were from Roche Molecular Biochemicals. The
tetrapeptide caspase inhibitors, acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) and acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), and the
colorimetric substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA)
were purchased from Biomol Research Laboratories.
z-Acetyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk) was from
Calbiochem-Novabiochem. Anti-Fas antibody was supplied by PanVera Inc.
Calf thymus PARP-1 was purified as described previously (38).
Recombinant human caspase-3, -6, and-7 were purified to homogeneity as
previously reported (39).
Cell Culture and Treatments--
Human leukemia HL-60 and Jurkat
cells, breast carcinoma MCF-7, bovine kidney MDBK, mouse embryo
C3H10T1/2, and monkey kidney Cos-7 cells were grown according to
American Type Culture Collection instructions. The cells were treated
with the apoptotic inducers in a complete growth medium at the
indicated concentrations for different lengths of time. The cell
permeable irreversible caspases inhibitor z-DEVD-fmk (3 µM) was added to the culture medium 30 min before treatment.
Western Blots and Preparation of Apoptotic Cell
Extracts--
After drug treatment, cells were washed with ice-cold
HEPES-saline buffer A (10 mM HEPES, pH 7.4, 140 mM NaCl, 7 mM KCl, 6 mM glucose)
and lysed in the ice-cold hypotonic buffer B (25 mM HEPES,
pH 7.5, 1 mM EGTA, 5 mM MgCl2,
0.1% Triton X-100, 250 µM PMSF, 2 mM
dithiothreitol, antiprotease mixture tablet (1 tablet/10 ml)). Aliquots
were taken for protein determination (40). The remaining cell extract
was completed with reducing loading buffer or homogenized in buffer B
by 20 strokes in a Dounce homogenizer and centrifuged at 15,000 × g for 10 min at 4 °C to obtain the apoptotic cell
extract. DEVDase activity was routinely assayed in the apoptotic cell
extract using the substrate DEVD-pNA to verify caspase activation (41).
For fractionation experiments, staurosporine (400 nM)
treated and control Jurkat cells were homogenized in buffer B without
Triton X-100. Following incubation at 4 °C for 15 min and addition
of 140 mM KCl, the homogenates were centrifuged at
1,000 × g for 3 min. The supernatant was further
centrifuged at 100,000 × g for 60 min at 4 °C to
obtain S-100 cytosol and pellet. The 1,000 × g and
100,000 × g pellets were resuspended in the same
buffer and mixed to form the organelles fraction. S-100 cytosol and
organelles fractions were used to determine PARG distribution along
with the markers for the different cellular organelles. SDS-PAGE and
blotting were done according to Duriez et al. (42). PARG was
detected using the affinity purified polyclonal G-03 antibody directed
against the peptide (849AYCGFLRPGVSSEN862) in
the conserved C-terminal domain of the protein (22). After the
peroxidase-based chemiluminescence revelation, the membranes were
stripped in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol at 65 °C for 30 min to remove the
antibodies (43) and reprobed with the monoclonal CII10
antibody, which recognizes full-length PARP-1 and its 89-kDa fragment
(35). Calnexin, a specific marker of endoplasmic reticulum, was
detected with an anti-calnexin polyclonal antibody (StressGen
Biotechnologies). Cytochrome oxidase (subunit II), a specific marker of
mitochondria, was detected with an anti-cytochrome oxidase monoclonal
antibody (Molecular Probes). Lactate dehydrogenase and 5'-nucleotidase
were used as specific markers for cytoplasm and plasma membrane,
respectively. Both enzyme activities were determined with kits from Sigma.
Protease Cleavage Assay--
A bovine PARG cDNA lacking the
region coding for the N-terminal 74 amino acids (21) was used to
translate PARG in vitro with the transcription/translation
TNT kit (Promega) in the presence of [35S]methionine. The
translation mixture (4 µl) was incubated in a total volume of 12.5 µl for 3 h at 37 °C, either with purified caspases
resuspended in buffer C (50 mM HEPES, pH 7.4, 100 mM NaCl, 10% sucrose, 10 mM EDTA, and 1.6 mM CHAPS) or with the apoptotic cell extract diluted in
buffer B. The reaction was stopped by addition of reducing loading
buffer, and the digestion products were analyzed by SDS-PAGE and
autoradiography. The amount of radioactivitiy in the bands was
electronically quantified on the Instant Image Analyzer (Packard
Instrument Company). For the cleavage inhibition assays, the
tetrapeptide inhibitor DEVD-CHO or YVAD-CHO was added to the apoptotic
cell extract at the indicated concentrations.
Mapping of the Caspase Cleavage Sites in PARG by
Mutagenesis--
Site-directed mutagenesis was achieved using the
QuikChange kit (Stratagene). The human DEID sequence in positions
253-256 was introduced into the corresponding region (positions
254-257) of the bovine protein (replacing the bovine EEVD site) using
the primer 5'-CAGGGTGTCAGCAGGACGAGATAGACGTGGTGTCCG-3'. The aspartic acid in position 308 of bovine PARG was mutated to alanine using the
primer 5'-GAGTCACCAATGGATGTAGCTAATTCCAAAAATAGTTGTCAGG-3'. The mutations were confirmed by DNA sequencing (44).
Analysis of pADPr Levels--
pADPr determination was done using
the immunodot-blot technique as described previously (45). Briefly,
dihydroxyboronyl Bio-Rex purified pADPr was diluted in 0.4 M NaOH containing 10 mM EDTA and loaded on
Hybond N+ membrane using a dot-blot manifold system (Life Technologies,
Inc.). The presence of pADPr was detected by the anti-pADPr LP96-10
antibody followed by peroxidase-conjugated anti-rabbit IgG and
chemiluminescence reaction. Quantification was performed using a cooled
CCD camera equipped with a Chemi Imager 4000, and the data were
analyzed with the Digital Imaging and Analysis Systems (Alpha Innotech
Inc.).
Partial Purification of PARG and Caspase-3 Cleavage
Assay--
Jurkat cells (5 × 109) were sedimented at
1,900 × g for 5 min at 4 °C and washed with buffer
A. The cell pellet was resuspended in 10 ml of ice-cold buffer (20 mM KPO4, pH 7.4, 1 mM EGTA, 5 mM MgCl2, 5 mM
-mercaptoethanol,
0.5 mM PMSF, antiprotease mixture) and homogenized with 20 strokes using a Dounce homogenizer. Cell homogenates were centrifuged
at 100,000 × g for 60 min at 4 °C. The supernatant
was completed with 10 ml of buffer (20 mM KPO4, pH 7.4, 800 mM KCl, 2 mM EDTA, 20% glycerol,
0.2% Triton X-100, 5 mM
-mercaptoethanol, 0.5 mM PMSF, antiprotease mixture) and mixed with 5 ml of
Red-Agarose preswollen resin (Amicon Corp.) equilibrated with buffer D
(20 mM KPO4, pH 7.4, 400 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 5 mM
-mercaptoethanol, 0.5 mM PMSF,
antiprotease mixture). After 1 h of gentle shaking at 4 °C, the
mixture was washed with buffer D and with the same buffer containing
600 mM of KCl, and the enzyme was eluted with buffer D
containing 1-1.2 M KCl. The active fraction was desalted with four columns of Sephadex G-25 resin (Amersham Pharmacia Biotech) packed in a 60-ml syringe (3 × 7-cm) using buffer E (50 mM Tris-HCl, pH 9.0, 150 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 5 mM
-mercaptoethanol, 0.5 mM PMSF,
antiprotease mixture) and applied on 5 ml of prepacked Heparin
Sepharose (HiTrap Heparin; Amersham Pharmacia Biotech) equilibrated
with the same buffer. The elution was done with buffer E containing
200-300 mM KCl. The active fraction was adjusted to pH 7.4 and applied on a 1 × 3-cm phosphocellulose column (Whatman)
equilibrated with buffer F (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 5 mM
-mercaptoethanol, 0.5 mM PMSF,
antiprotease mixture). After washing with buffer F containing 100-300
mM KCl, the elution was done with the same buffer
containing 400-450 mM of KCl. The active fraction was used
for DHB-Sepharose-polymer affinity chromatography as described
previously (17) except that the pADPr used for the purification was
synthesized without the formic acid resuspension and trichloroacetic
acid reprecipitation step, and the elution was done at pH 7.4. Partially purified PARG (550 ng) was cleaved by caspase-3 (200 or 600 ng) in a total volume of 600 µl of buffer C without NaCl. After
3 h at 37 °C, the reaction was stopped by 100 nM of
Ac-DEVD-CHO and used for PARG activity determination. Controls
consisting of PARG with Ac-DEVD-CHO or PARG alone were also included
for incubation and activity analysis.
PARG Activity Determination Assays--
PARG activity was
measured essentially as described by Jonsson et al. (12).
The cells were washed with ice-cold buffer A and homogenized in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM sucrose, 1 mM
-mercaptoethanol, 0.1%
Nonidet P-40 using a Dounce homogenizer. The reaction mixture for the
assay contained 50 mM KPO4, pH 7.5, 50 mM KCl, 100 µg/ml bovine serum albumin, 10 mM
-mercaptoethanol, 0.1 mM PMSF, 10 µM
[32P]pADPr. The reaction was started by the addition of
the cell extracts or the purified enzyme and stopped after 10 min by
the addition of SDS at a 0.1% final concentration. An aliquot of
10,000 cpm was applied on polyethyleneimine-cellulose TLC plate.
The plate was first developed at room temperature in methanol, dried, and then developed in 0.3 M LiCl, 0.9 N acetic
acid. The TLC plate was electronically autoradiographed on the Instant
Image Analyzer as described above. The radioactivity in the ADP-ribose
spot, and the origin was used to calculate the activity. Full-length or
caspase-3-digested purified PARG was used for the determination of the
kinetic parameters (Km and
Vmax), using various concentrations of
[32P]pADPr (0.1, 0.3, 1, 3, 10, and 30 µM)
to establish Lineweaver-Burk reciprocal plots.
One-dimensional PARG Activity Zymogram--
An activity zymogram
consisting of an SDS-PAGE of PARG in a gel containing
32P-automodified PARP-1 was done according to Brochu
et al. (46). After renaturation, the presence of PARG was
determined by the disappearance of radioactivity in the bands.
Fluorescence Microscopy Analysis--
Fusion protein between GFP
in frame with the N-terminal of 103-kDa PARG (GFP-PARG) or its 74-kDa
apoptotic fragment (GFP-74) was generated to determine their cellular
distribution. GFP-PARG was obtained by insertion of a 2.68-kilobase
XhoI-EcoRI polymerase chain reaction fragment
amplified from pCMV-PARG plasmid (21) with primers
5'-CTGCACTCGAGGTATGTGTCAGGATTCAGAAGCAG-3' and
5'-GACGTGAATTCTCGGCCCCCGTCCTTTGT-3' into a pEGFP-C1 expression vector
(CLONTECH). GFP-74 was obtained by insertion of a
2-kilobase XhoI-EcoRI polymerase chain reaction fragment amplified from pCMV-PARG with primers
5'-CTGCACTCGAGGTATGGACACTAAAGGAATCAAGAC-3' and
5'-GACGTGAATTCTCGGCCCCCGTCCTTTGT-3' into the GFP expression vector. The
resulting plasmids were transiently transfected in Cos-7 cells by a
lipofection method (Life Technologies, Inc.). The expression of the
fusion proteins was verified 24 h after the transfection by
Western blotting on total cell extract with a polyclonal anti-GFP
antibody (CLONTECH). The distribution of PARG in
normal cells or in cells undergoing apoptosis following treatment with
1 µM staurosporine was directly monitored with an
Axioskop microscope (Zeiss). Images were captured with a CCD camera
(DAGE-MTI) coupled to the Metamorph Imaging System (Universal Imaging Corporation).
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RESULTS |
Cleavage of Endogenous PARG during Apoptosis--
Human Jurkat
leukemia cells were treated with the topoisomerase II inhibitor VP-16
or the PKC inhibitor staurosporine and analyzed by Western blot for
PARG cleavage (Fig. 1, A and
B). In untreated cells, PARG was present in two bands of
~111 kDa corresponding possibly to post-translational modifications
or different translation origins. In cells undergoing apoptosis, these
bands were cleaved into ~ 85- and 74-kDa fragments at
approximately the same time as PARP-1 cleavage. The two apoptotic
fragments are in the C-terminal region of the protein because they are
recognized by the anti-peptide antibody. PARG cleavage could be
inhibited by the cell permeable caspases inhibitor z-DEVD-fmk,
suggesting the involvement of caspases in PARG cleavage. PARG was also
cleaved into the ~85- and 74-kDa fragments in HL-60 cells treated
with VP-16 (Fig. 1C) and Jurkat cells treated with anti-Fas
antibody (Fig. 1D) or the ligand Trail (data not shown).
These results suggest that PARG processing is a general feature of
apoptosis.

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Fig. 1.
Cleavage of PARG during apoptosis.
Jurkat and HL-60 cells were treated with apoptotic inducers for the
indicated times. Following treatments, cell lysates (40 µg of
protein) were resolved using 8% SDS-PAGE and subsequently analyzed by
immunoblotting using the polyclonal anti-PARG C-terminal G-03 antibody
and the monoclonal anti-PARP-1 CII10 antibody. Jurkat cells
were treated with 120 µM VP-16 (A) or 300 nM staurosporine (B). The irreversible caspases
inhibitor z-DEVD-fmk (3 µM) was added to the culture
medium 30 min before treatment. C, HL-60 cells treated with
68 µM VP-16. D, Jurkat cells treated with 1 µg/ml of anti-Fas antibody. The results are representative of three
independent treatments. DMSO, dimethyl sulfoxide;
MW, molecular mass.
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Because pADPr synthesis occurs in apoptotic cells (32, 33, 37), we
asked whether PARG cleavage is specific to apoptosis or whether it
could also occur during the early pADPr metabolism in response to DNA
strand breaks. HL-60 cells were treated with MNNG, and the pADPr level
was determined by the immunodot-blot method (Fig.
2A). MNNG treatment induced a
transient synthesis of pADPr, which was abolished by the PARP inhibitor
1,5-isoquinolinediol. Western blot analysis of PARG showed that the
protein remained unaffected during MNNG treatment (Fig. 2B),
unlike following VP-16 treatment. This indicates that, in living cells,
pADPr catabolism does not require PARG cleavage.

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Fig. 2.
Absence of PARG cleavage during pADPr
metabolism. A, HL-60 cells were treated with 50 µM MNNG and pADPr was detected by immunodot-blot using
LP96-10 antibody (closed circles). As control, cells were
pretreated with 1 mM 1,5-isoquinolinediol for 5 min before
MNNG treatment (open circles). B, HL-60 cells
were treated with 50 µM MNNG or 68 µM
VP-16, and cell lysates (40 µg of protein) were immunoblotted for
PARG using G-03 antibody. The results are representative of four
independent experiments. DMSO, dimethyl sulfoxide;
MW, molecular mass.
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Cleavage of PARG by Caspase-3 during Apoptosis--
To determine
the nature of the protease responsible for PARG cleavage during
apoptosis, in vitro translated bovine PARG was incubated
with different amounts of apoptotic HL-60 cell extract (Fig.
3A). Two PARG translation
products, corresponding to 103 and 91 kDa were observed in the control.
These bands probably correspond to two translation start points from
the partial PARG cDNA. Following the addition of the apoptotic cell
extract, PARG was cleaved to a 74-kDa fragment in a
dose-dependent manner, suggesting that an apoptotic
protease is responsible for this cleavage. The 85-kDa fragment produced
in human cells (Fig. 1) was not observed with bovine PARG. Similar
results were obtained with cytosolic extracts from Jurkat cells treated
with staurosporine or anti-Fas antibody (data not shown). The small
N-terminal fragments derived from the cleavage of the 103- and 91-kDa
products were hardly detectable because of their low radioactivity
(data not shown). To study the involvement of caspases in PARG
cleavage, the specific tetrapeptide caspase inhibitors DEVD-CHO and
YVAD-CHO were used (Fig. 3B). The cleavage of PARG was
completely inhibited by 50 nM of DEVD-CHO but only
partially inhibited by 500 nM of YVAD-CHO, suggesting that
a caspase-3-like activity present in apoptotic HL-60 cells is involved
in PARG processing. Similar patterns of inhibition were obtained with
cytosolic extracts from Jurkat cells treated with staurosporine or
anti-Fas antibody (data not shown). To further determine which caspase
is responsible for PARG cleavage, in vitro translated bovine
PARG was incubated with caspase-3, -6, and -7. Using 4 ng of purified
caspases per assay (~7 nM), a weak cleavage of PARG was
observed with caspase-3. Caspase-6 and -7 did not produce detectable
cleavage at this concentration. At 20 ng/assay (~ 35 nM),
PARG was cleaved to a significant degree by caspase-3 and marginally by
caspase-7 (Fig. 3C). These results suggest that caspase-3
could be responsible for PARG cleavage in vivo. To test this
hypothesis, caspase-3-deficient MCF-7 cells were used to determine
whether PARG could be cleaved in the absence of this protease following
apoptotic treatment (Fig. 3D). In this cell line, PARG was
not cleaved in response to staurosporine treatment, whereas PARP-1 was
almost totally cleaved.

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Fig. 3.
Cleavage of PARG by caspase-3. In
vitro translated bovine [35S]PARG was incubated with
apoptotic cytosols or purified caspases for 3 h at 37 °C. The
reaction was then stopped by the addition of reducing loading buffer,
the proteins were resolved using 8% SDS-PAGE and analyzed by
autoradiography. A, in vitro translated PARG was
cleaved with different amounts of cell extract from HL-60 cells treated
with VP-16. The lower panel shows the counts/min obtained
from the bands following the cleavage assay. The circles and
squares represent the 103- and 91-kDa bands, respectively.
The triangles represent the 74-kDa band. B,
bovine PARG was incubated with HL-60 apoptotic cytosol (20 µg of
protein) in the presence of the indicated concentrations of the
tetrapeptide caspases inhibitors DEVD-CHO and YVAD-CHO. C,
bovine PARG was incubated with 4 ng (~7 nM) and 20 ng
(~35 nM) of purified caspases. D, MCF-7 cells
were treated for 16 h with 1 µM staurosporine after
which cell extract (40 µg of protein) was used for SDS-PAGE and
immunobloting with anti-PARG or anti-PARP-1 antibodies. The results are
representative of three independent experiments. DMSO,
dimethyl sulfoxide; MW, molecular mass.
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Identification of PARG Cleavage Sites--
In vitro
translated bovine PARG was cleaved only into a 74-kDa fragment, whereas
human PARG was cleaved into 85- and 74-kDa fragments (Figs. 1 and 3).
Analysis of the PARG sequence (Fig. 4A) revealed the presence of a
potential DEID motif, present only in human PARG, that could generate
the 85-kDa fragment. To determine whether the 85-kDa fragment is
produced in other mammalian cell types, cell extracts from MDBK
(bovine), C3H10T1/2 (mouse), and Jurkat (human) cells were treated with
caspase-3 and assayed for PARG cleavage with anti-PARG antibody (Fig.
4B). PARG was cleaved to its 74-kDa fragment in all cell
types, but only human and mouse PARG showed the presence of additional
fragments (of 85 and 66 kDa, respectively). In MDBK cells, three faint
bands migrating below 66 kDa are present in the control as well as the
treated extract and are thus unrelated to caspase cleavage. To
determine whether the human DEID sequence corresponds to the caspase
cleavage site that generates the 85-kDa fragment in human cells, we
introduced this sequence in the corresponding region of bovine PARG.
The resulting PARG mutant construct was translated in vitro
and subjected to a cleavage assay using recombinant caspase-3 or the
apoptotic cell extract (Fig. 4C). Cleavage of the mutant
PARG produced the 85-kDa fragment in addition to the 74-kDa fragment.
This cleavage was also obtained with the apoptotic cell extract and was
completely inhibited by 50 nM of DEVD-CHO but not by 500 nM of YVAD-CHO. To determine the caspase-3 cleavage site
that produces the 74-kDa fragment, several aspartic acid residues were
mutated to alanine. Mutation at amino acid position 308 of bovine PARG
abolished the cleavage of PARG by caspase-3 (Fig. 4D). This
cleavage site, MDVD, is conserved in mouse, bovine, and human PARG
(Fig. 4A). Mutations of other aspartic acid residues near
this site to alanine did not affect the efficiency of the cleavage
(data not shown).

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Fig. 4.
Mapping of caspase cleavage sites on
PARG. A, schematic representation of PARG structure and
the putative cleavage sites. B, cytosolic extracts (40 µg
of protein) prepared from MDBK (bovine), C3H10T1/2 (mouse), and Jurkat
(human) cells were treated with 40 ng of caspase-3 for 3 h at
37 °C and immunoblotted for PARG with G-03 antibody. C,
the sequence DEID, present only in human PARG, was introduced into the
corresponding region of bovine PARG. The resulting PARG mutant
construct was translated in vitro and subjected to cleavage
by recombinant caspase-3 (20 ng) or apoptotic cytosolic extract (20 µg of protein) prepared from HL-60 cells treated with 68 µM VP-16. The inhibitors DEVD-CHO and YVAD-CHO were used
at 50 and 500 nM, respectively. D, the aspartic
acid residue at amino acid position 308 of bovine PARG was mutated to
alanine. The resulting PARG mutant construct was translated in
vitro and subjected to cleavage by 20 ng of caspase-3. The results
are representative of three independent experiments. MW,
molecular mass; NLS, nuclear localization signal.
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PARG Is Cleaved without Loss of Glycohydrolase Activity--
To
determine whether PARG cleavage results in a change in its catalytic
function, enzymatic activity was measured in total extracts of HL-60
cells at various times after VP-16 treatment. PARG activity was not
altered for up to 360 min of treatment (Fig. 5A). A PARG activity gel was
therefore used to determine whether the proteolytic products of PARG
were active. The 85- and 74-kDa fragments still possess the capacity to
hydrolyze the pADPr like the full-length enzyme (Fig. 5B).
Similar results were obtained in Jurkat cells treated with
staurosporine or anti-Fas antibody (Fig. 5C). To further
characterize the kinetic parameters of PARG and its apoptotic
fragments, we purified 848-fold full-length PARG from a cytosolic
fraction of Jurkat cells (Fig.
6A). The purified enzyme was
used for in vitro cleavage by caspase-3 followed by PARG
activity assay. A representative blot shown in Fig. 6B indicates that almost all the full-length PARG is cleaved to generate the 85- and 74-kDa fragments. Kinetic analysis showed similar Km and Vmax for PARG and a
mixture of the apoptotic fragments, 85 and 74 kDa (Fig. 6C).
The complete cleavage of PARG to the 74-kDa fragment did not result in
a significant change of the Km and
Vmax (Fig. 6, B and C).
The DEVD-CHO added to stop the caspase-3 reaction did not interfere
with the PARG assay, because its addition to the reaction mixture did
not inhibit the enzyme activity (data not shown). These results
corroborate those obtained with PARG activity determination in total
cell extract and by activity gel.

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Fig. 5.
PARG activity during apoptosis.
A, PARG activity in a total cell extract from HL-60 cells
treated with 68 µM VP-16 (closed triangle) or
dimethyl sulfoxide (open circle). Data are the means ± S.D. of three experiments. B, activity zymogram consisting
of SDS-PAGE followed by the renaturation of proteins using a gel
containing PARP-1-bound [32P]pADPr. Total cell extracts
(20 µg of protein) from HL-60 cells treated with 68 µM
VP-16 were prepared at the indicated time as for Western blot.
C, activity zymogram of the total cell extract (20 µg of
protein) from Jurkat cells treated with 1 µM
staurosporine or 1 µg/ml of anti-Fas antibody for 3 h. The
results are representative of three independent experiments.
DMSO, dimethyl sulfoxide; MW, molecular
mass.
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Fig. 6.
Purification of PARG and enzymatic properties
of its apoptotic fragments. A, purification of
full-length PARG from Jurkat cells cytosol. B, purified PARG
(550 ng) was cleaved by caspase-3 (200 or 600 ng) for 3 h at
37 °C. The reaction was stopped by 100 nM of Ac-DEVD-CHO
and used for PARG immunoblotting and activity determination.
C, full-length and caspase-3-digested PARG were used for the
determination of the kinetic parameters using various concentrations of
[32P]pADPr. The enzymatic activities were used to
establish the Lineweaver-Burk reciprocal plots to deduce the
Km and Vmax. The
numbers in parentheses represent the S.D.
obtained from three experiments. MW, molecular mass.
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Cellular Localization of PARG and of Its 74-kDa Apoptotic
Fragment--
Because the cleavage of PARG does not alter its
catalytic activity, we examined whether it could result in a change of
its cellular localization. Homogenates from control and apoptotic Jurkat cells fractionated into S-100 cytosol and pellet containing nuclei and the other organelles were analyzed for the presence of PARG
by immunoblotting (Fig. 7A).
In control and apoptotic Jurkat cells, PARG was mostly associated to
the cytosolic fraction, which contains most of the lactate
dehydrogenase activity. The organelles fraction containing nuclei,
mitochondria, endoplasmic reticulum and plasma membranes did not
contain a significant amount of PARG or its apoptotic fragments. To
further directly monitor the cellular localization of PARG during
apoptosis, GFP was fused to the N terminus of both PARG, and the 74-kDa
apoptotic fragment and the resulting recombinant proteins were
transiently expressed in Cos-7 cells (Fig. 7B). In the
absence of treatment, both GFP-PARG and GFP-74 showed predominant
cytoplasmic localization (Fig. 7C), whereas GFP was
localized in the nucleus and in the cytoplasm as expected. The
localization of GFP-PARG is consistent with the previously reported
distribution for PARG (22). Cos-7 cells treated with staurosporine for
7 h underwent apoptosis as determined by the typical apoptotic
morphological changes and about 50% of PARP-1 cleavage (data not
shown). Only the cells with an apoptotic morphology were analyzed for
PARG distribution by fluorescence microscopy. Following staurosporine
treatment, most of the fluorescence remained in the cytoplasm, and no
detectable redistribution of PARG or its 74-kDa fragment to the nucleus
was observed (Fig. 7C). These results suggest that the
active 74-kDa fragment of PARG may play a role in the cytoplasm of
apoptotic cells.

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Fig. 7.
Cellular localization of PARG during
apoptosis. A, Jurkat cells, treated with staurosporine
(400 nM) for 3 h were homogenized and centrifuged to
obtain S-100 cytosol and organelles as described under "Experimental
Procedures." Both fractions (40 µg of protein) were resolved by
SDS-PAGE and immunoblotted for PARG, PARP, calnexin, and cytochrome
c oxidase. Lactate dehydrogenase (LDH) and
5'-nucleotidase activities were determined in both fractions and
expressed at percentage of total cellular activity for each enzyme.
B, expression of fusion proteins GFP-PARG and GFP-74
apoptotic fragment in Cos-7 cells after 24 h of transient
transfection. The Western blotting on total cell extract was done using
a polyclonal anti-GFP antibody. C, the cellular distribution
of GFP-PARG and GFP-74 in untreated cells and cells undergoing
apoptosis following treatment with 1 µM staurosporine was
directly monitored using a Zeiss axioskop microscope with the
appropriate filter. The nuclei are indicated with arrows.
The results are representative of three independent experiments.
MW, molecular mass.
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DISCUSSION |
Two forms of PARG, 59 and 74 kDa, have been previously purified
from mammalian cells. However, bacterial expression of the bovine PARG
cDNA recently isolated, produced a protein of 111 kDa (20). We
recently showed that under normal growth conditions, PARG is present
only as a doublet at 111 kDa with a cytoplasmic localization (21).
These findings raised questions about the biological significance and
the roles of the 59- and the 74-kDa forms of PARG. In this study we
show that, during apoptosis, PARG is specifically cleaved by caspase-3
to produce a catalytically active 74-kDa fragment in different
mammalian species.
We established here a new purification procedure that allowed us to
isolate for the first time the full-length PARG from mammalian cells
(Fig. 6). Purified PARG under these conditions did not contain its
59-kDa form previously reported (14, 17, 20). This C-terminal 59-kDa
fragment of PARG was not observed during apoptosis or normal pADPr
metabolism (Figs. 1 and 2). By using previously reported methods (17)
to purify PARG from Jurkat cells, we noticed that the 59-kDa fragment
was produced despite the use of an antiprotease mixture. During later
steps of purification, the 111-kDa PARG was totally cleaved to 59 kDa
(data not shown), indicating that the enzyme contains a site that is
very sensitive to proteases. However, whether this fragment of PARG
could be produced in vivo in particular circumstances
remains to be determined.
Human PARG is cleaved during Fas receptor-, staurosporine- and
VP-16-induced apoptosis to generate fragments of 85 and 74 kDa,
suggesting that this cleavage is a common feature of apoptosis. The
involvement of caspase-3 in PARG cleavage was evidenced using an
in vitro cleavage assay in the presence of apoptotic cell
extract, specific caspases inhibitors, and recombinant caspases. PARG
cleavage by caspase-7 was negligible, despite the fact that caspase-3
and caspase-7 are execution caspases with similar specificity and Km for the tetrapeptide DEVD-pNA (47). PARG was not cleaved in caspase-3-deficient MCF-7 cells during staurosporine-induced apoptosis, during which caspase-7 is known to be activated (37).
PARG was cleaved by caspase-3 to produce the 74-kDa fragment at the
unconventional site MDVD that lacks an aspartic acid at the P4
position. However, previous inhibition studies using synthetic peptides
have shown that this P4 aspartic acid is required for the cleavage by
caspase-3 (48, 49). In contrast, our results support a recent report
showing a noncanonical EEED site for caspase-3 in topoisomerase I (50).
These results suggest that other parameters such as the tertiary
structure and post-translational modification of proteins could also be
involved in the specificity of cleavage by caspases. For instance, the
sequence DEAD in the retinoblastoma protein is cleaved by caspase-7 but
not by caspase-3 (51). In addition, we have recently observed that
poly(ADP-ribosyl)ation of PARP-1 stimulates its cleavage by caspase-7
but not by caspase-3 (37). The DEID site that produces the 85-kDa
fragment in human cells is quite similar to the typical sequence
P4DXXDP1
for caspase-3 found in
other proteins cleaved by this protease (48). However, this fragment is
produced only in human cells, indicating that its production is not
essential in other mammalian species. Further cleavage of this fragment
by caspase-3 to a 74-kDa fragment could occur during apoptosis. On the
other hand, we cannot exclude the possibility that this fragment could
have a specific role to play during apoptosis in human cells.
During apoptosis, many different enzymes are cleaved by caspases to
dissociate their regulatory domain from their catalytic domain. This
results in the inactivation or the constitutive activation of these
proteins (31). Our results indicate that the C-terminal domain of PARG
produced during apoptosis contains the active site of the enzyme and is
able to hydrolyze pADPr in vitro (Figs. 5 and 6). This is
consistent with previous results indicating that the PARG C-terminal
59-kDa fragment possesses glycohydrolase activity (20). The N-terminal
domain is likely to be responsible for the regulation of PARG function
in vivo. Because the glycohydrolase assay in our study was
done with free pADPr, we could not rule out the possibility for
distinct manners of action of PARG and its apoptotic fragments on
poly(ADP-ribosyl)ated proteins in vivo. Indeed, some
specific interactions between the N-terminal domain of full-length PARG
and other proteins may occur that obviously are impossible for the
apoptotic fragments. If the cleavage of PARG in mammalian apoptotic
cells is to circumvent certain regulation of the enzyme function by the
N-terminal domain, we could expect that the 74-kDa fragment would
continuously catabolyze pADPr and possibly obliterate its cellular
functions (e.g. signaling of cellular stress). Consistent
with this hypothesis, PARP-1 is cleaved during apoptosis resulting in
the suppression of its pADPr synthesis function. The fact that the
74-kDa fragment remains mostly in the cytoplasm may suggest a role in
relation with the other PARP-1-like proteins. In fact, recent studies
have reported the presence of a new PARP (vPARP) associated with the
ribonucleoproteins, vault particles in the cytoplasm (7). A cytoplasmic
distribution was also reported for the telomeres associated PARP
(tankyrase) (52, 53). On the other hand, we have previously shown that the nuclear accumulation of pADPr following MNNG treatment is abolished
in PARG overexpressing Cos-7 cells in comparison with the control
vector transfected cells (22). However, overexpression of PARG results
in its cytoplasmic localization without any detectable redistribution
of the enzyme following MNNG treatment (22). This suggests that a very
small but sufficient amount of PARG (determined indirectly by the rapid
polymer degradation) is translocated to the nucleus and catabolyze
pADPr. The same model could be applied to the 74 kDa fragment because
it still possesses the putative nuclear localization signal (20).
Further studies are necessary to understand the role of PARG cleavage
during apoptosis. The generation of PARG-deficient mice and cells will
be a useful model to study this role (e.g. reintroduction of
an uncleavable PARG mutant into the enzyme-deficient cells).