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J Biol Chem, Vol. 274, Issue 30, 20759-20762, July 23, 1999
,,From the Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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DNA fragmentation factor (DFF) functions
downstream of caspase-3 and directly triggers DNA fragmentation during
apoptosis. Here we described the identification and characterization of
DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional
assays have shown that only DFF45, not DFF35, can assist in the
synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a
chaperone and assist in the synthesis of active DFF40. Whereas the
amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40,
the amino acid residues 23-100, which is homologous between DFF35/45
and DFF40, may function to inhibit the activity of DFF40. In contrast
to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40
more strongly than DFF45 and can inhibit its nuclease activity. These
findings suggest that DFF35 may function in vivo as an
important alternative mechanism to inhibit the activity of DFF40 and
further, that the inhibitory effects of both DFF35 and DFF45 on DFF40
can put the death machinery under strict control.
Apoptosis is fundamentally important in a variety of physiological
and pathological processes. Apoptotic cells undergo an orchestrated
cascade of events characterized by distinct morphological changes
including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1, 2).
Activation of the caspase cascade is a key molecular event in the
process of apoptosis (3, 4). Apoptotic signals, including growth factor
and interleukin deprivation, activation of Fas, ionizing radiation, and
a series of chemicals acting as upstream signals, can convert the
precursors of caspases into the active enzymes (2). Several important
downstream substrates of caspase, such as gelsolin (5), p21-activated
kinase-2 (PAK-2) (6), and DNA fragmentation factor 45 (DFF45)1 (7), whose cleavages
clearly induce specific well characterized steps in apoptosis, have
been recently identified. The cleavage of chromatin into the
nucleosomal fragments, which distinguishes apoptosis from oncosis and
necrosis, is a key element in the cell death process and is believed to
be mediated by Mg2+/Ca2+ required and
Zn2+-sensitive nuclease (8-12).
We have previously identified a triplet of nuclease proteins named
NP42-50 that causes DNA degradation in vitro
when cells undergo apoptosis (13). The similarity in molecular weight
and biochemical characteristics between NP42-50 and the
recently identified DFF40 led us to further investigate these
molecules. DFF is a heterodimeric protein composed of DFF45 and DFF40
subunits. DFF45 has been found to be the substrate of caspase-3, and
DFF40 has also been cloned and found to be a DNA fragmentation nuclease
(7, 14, 15). Cleavage of the DFF45 by caspase-3 during apoptosis
releases DFF40 that degrades chromosomal DNA into nucleosomal
fragments. Similar findings have also been described recently in the
mouse. The mouse DFF is composed of three molecules: one
caspase-activated DNase (CAD) and two forms of CAD inhibitors (ICAD-L
and ICAD-S) (16, 17). Mouse CAD and ICAD-L are apparently the
counterpart of human DFF40 (CPAN) and DFF45, respectively, whereas the
human counterpart of mouse ICAD-S has not been identified.
In the present study we described the cloning of human DFF35, the short
isoform of DFF45 and mapped the function domains of DFF35/45.
Functional analysis has shown that the intact structure/conformation of
DFF45 is essential for it to function as a chaperone and assist in the
synthesis of active DFF40. In contrast, DFF35, although it can bind to
DFF40 and inhibit its nuclease activity, cannot assist in the synthesis
of active DFF40. While DFF35 binds to DFF40 more strongly than does of
DFF45, suggesting that DFF35 may function in vivo as an
additional inhibitor of DFF40, augmenting the regulatory control of
DFF45. Taken together, our studies suggest that the inhibitory effects
of both DFF35 and DFF45 on DFF40 place this important component of the
cell death machinery under strict control.
cDNA Cloning, Expression, Mutation of
DFF35/DFF45--
Outer encoding region primers
5'-GCTTCCTTGGCATTCCCGCTGCTGC-3' (for both DFF35 and DFF45),
5'-GCAGAGGTGAACAAAAGGGCCCACACACC-3' (for DFF35), and
5'-GGTGGGCAGGTCCCACCTTGTGGAGG-3' (for DFF45) were designed for
amplifying both DFF35 and DFF45 from Jurkat cell total RNA and human
fetal liver cDNA library by PCR. The PCR-amplified fragments were
cloned into pCR2.1 (Invitrogen) for sequencing (ABI 200 sequencer).
DFF35 and DFF45 were further amplified by
5'-GCTCTAGAGATGGAGGTGACCGGGGACG-3' (for both DFF35 and DFF45), 5'-CCCAAGCTTGGGTCAGTGACCCTGGTTTCC-3' (for DFF35), and
5'-CCCAAGCTTGGGCTATGTGGGATCCTGTCT-3' (for DFF45) and cloned into
pGEX-KG (Amersham Pharmacia Biotech) for GST fusion protein. D117E and
D224E double mutants of DFF35 were generated by two-step PCR using
20-nucleotide primers carrying the mutated nucleotides. Deletion
mutants of DFF45 were also generated by two-step PCR. All the mutations
were confirmed by DNA sequencing. GST fusion proteins were expressed in
Escherichia coli and adsorbed by glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) and then eluted by either reduced
glutathione (GSH) containing buffer (50 mM Tris, 20 mM GSH, pH 9.6) or thrombin (Sigma) according to the instructions of the manufacturer. The Flag-tagged DFF45 and DFF35 were
generated by cloning into pCDNA3.0-Flag vector (Eastman Kodak Co.).
In Vitro Cell-free DNA Fragmentation Assay--
Jurkat cells
were incubated with anti-Fas monoclonal Ab 7C11 (1 µg/ml) at 37 °C
for 2 h, the crude cytoplasm fraction containing DNA fragmentation
nuclease was extracted as described previously (7). Jurkat nuclei (5 µl, 6.5 × 105 total), were incubated with 100 ng of
nuclease fraction at 37 °C for 2 h. DNA fragmentation was
determined by extraction of genomic DNA and analyzed by 2% agarose gel
electrophoresis. Indicated GST fusion proteins or thrombin-cleaved
proteins treated or untreated with 100 ng/ml caspase-3 at 30 °C for
2 h and then inactivated with 20 nM CHO-DEVD
(Calbiochem) at 4 °C for 2 h were included in the above
in vitro system to test their inhibitory effects on DNA fragmentation.
Western Blot Analysis--
A polyclonal Ab against the
N-terminal peptide of both DFF35 and DFF45 (EVTGDAGVPESGEIRTLPKC) was
from Upstate Biotechnology. A polyclonal Ab against intermediate
peptide of both DFF35 and DFF45 (KQEESKAAFGEEVDAVD) and a polyclonal Ab
against C-terminal peptide of DFF45 only (KASPPGDLQNPKRARQDPT) were
from Santa Cruz Biotechnology. A monoclonal Ab against epitope Flag
(M5) was purchased from Sigma. 293T cells were transfected with
Flag-tagged plasmids by calcium phosphate method. Jurkat cells were
incubated with staurosporine (Sigma) at the final concentration of 1 µM for the indicated time. Immunoblot was performed with
the horseradish peroxidase-conjugated goat anti-rabbit (N-terminal) or
donkey anti-goat (internal and C-terminal) Ab using ECL system
(Amersham Pharmacia Biotech).
cDNA Cloning and Transcription and Translation of DFF40 in
Vitro--
The primers 5'-GCGAGGACGATCTGAGCAGCTTGGGCAG-3' and
5'-AAATGATGCCCACGTCAGGCCTCAAACA-3' were used to amplify DFF40 from
human lymphocyte cDNA library and directly for sequencing. To
express DFF40 in a cell-free system, the encoding cDNA of DFF40 was
placed under T7 promoter of pCDNA 3.1 (Invitrogen), and the coupled
transcription and translation of DFF40 was performed by using TNT
in vitro transcription/translation kit (Promega) according
to the instructions of the manufacturer. pCDNA-DFF40 (1 µg) was
incubated at 30 °C for 2 h with 40 µl of TNT reagents and 20 µCi [35S]methionine (NEN Life Science Products) in the
absence or presence 200 ng of indicated recombinant proteins.
Assay for DFF40 Nuclease Activity and Binding Ability--
To
test DFF40 nuclease activity, a 5-µl aliquot of synthesized DFF40,
after being treated with caspase-3 and inactivated with CHO-DEVD, was
incubated with Jurkat nuclei (5 µl, 6.5 × 105
total) to test its ability to induce DNA fragmentation or DNA digestion
in assay buffer (10 mM HEPES, pH 8.0, 4 mM
MgCl2, 4 mM dithiothreitol, 5 mM
EGTA, 75 mM NaCl, and 16.7 µg/ml histone HI).
Caspase-3-treated or -untreated recombinant DFF-35, DFF45, or its
mutant proteins were included in the system to test their effects on
DFF40 activity. To test binding ability, a 10-µl aliquot of
synthesized DFF40, after treatment with caspase-3 and inactivation by
CHO-DEVD, was diluted to 500 µl, and 20 µg of GST-DFF35, GST-DFF45, or its GST mutant protein-Sepharose 4B beads were added and incubated at 4 °C for 2 h. Binding complex was eluted by 30 µl of
GST-containing buffer. A 15-µl aliquot was used for SDS-PAGE
analysis, and the gel was treated with autoradiography enhancer (NEW
Life Science Products), dried, and then visualized by exposing to Kodak
X-AR film.
In an effort to search for the short form of DFF45 and to better
understand the molecular details of DNA fragmentation, an expressed
sequence tag (EST) clone was found of approximately 1.5 kilobases which
contained an open reading frame encoding a novel protein of 268 amino
acid residues. A homology search in GenBankTM data bank
indicated that this protein was identical to DFF45 (consisting of 331 amino acids) up to amino acid position 261, after which the sequences
diverged (Fig. 1A), suggesting
that the mRNAs encoding these two proteins were generated through
alternative splicing. The two potential caspase-3 cleavage sites within
the protein are exactly the same as those of DFF45. More importantly, the C-terminal region of this protein is highly homologous with the
mouse short isoform of ICAD, ICAD-S (16), and has three additional
amino acids, QGH, at the C-terminal when compared with mouse ICAD-S
(Fig. 1B). These results clearly suggest that this protein
is the human counterpart of mouse ICAD-S. We therefore designated this
protein as DFF35 because it appears to be the short form of DFF45 with
a molecular weight of 35 kDa.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Comparison of amino acid sequences between
DFF35 and DFF45 (A) or mouse ICAD-S
(B). Two putative cleavage sites for caspase-3
are indicated by double underlines. Three peptides (N, I,
and C) used to generate polyclonal antibodies are indicated by
single underlines. Identical amino acids between DFF35 and
mouse ICAD-S are in bold face.
To further confirm whether the RNA transcript and the encoded protein
exist in vivo, Jurkat cell total RNA and fetal liver cDNA were amplified by PCR using DFF45 or DFF35 outer encoding region primer sets (5' primer is the same for both DFF35 and DFF45, but
3' primers are different) and cloned (Fig.
2A). Sequences of the PCR
products showed an exact match with the sequences of DFF45 and EST
clone, respectively (data not shown). Moreover, polyclonal Ab specific
for an N-terminal or intermediate peptide of DFF35/45 recognized two
bands corresponding to DFF45 and DFF35 by Western blot analysis of the
Jurkat cell lysate, whereas a polyclonal Ab specific for a C-terminal
peptide of DFF45 recognized only one band corresponding to DFF45, but
not DFF35 in the same blot (Fig. 2B). To further exclude the
possibility that DFF35 is because of the degradation of DFF45,
flag-tagged DFF35, DFF45, or an irrelevant control plasmid p53 was
transiently transfected into 293T cells. Although the blot with
anti-DFF35/45 indicated the existence of two endogenous bands, only the
one exogenous band was observed on the blot with either anti-Flag or
anti-DFF35/45 corresponding to the indicated transfected plasmid (Fig.
2C). These results clearly demonstrated that the novel
35-kDa isoform does exist in vivo at both mRNA and
protein levels.
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A modified in vitro apoptosis system that mimics the later
stages of the apoptotic cascade under cell-free conditions was used to
analyze the function of DFF35. Addition of cytosolic extracts prepared
from apoptotic cells to Jurkat nuclei induced the formation of
internucleosomal DNA fragments. GST-DFF35 fusion protein could completely block the DNA cleavage induced by apoptotic cytosolic extract but not GST-DFF35 cleaved by caspase-3. However, GST-DFF35 double mutant (at caspase-3 cleavage sites D117E and D224E) could block
DNA degradation either in the absence or presence of caspase-3 (Fig.
3A). Cleavage of both DFF45
and DFF35 and subsequently generated DNA fragmentation in
vivo were also observed in Jurkat cells killed by staurosporine
(Fig. 3, B and C). These results indicate that the caspase-3 cleavage sites are required for dismantling the inhibitory ability of DFF35, as well as DFF45, and releasing the nuclease responsible for DNA cleavage. Such a nuclease seems to be
DFF40.
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DFF40 was then cloned by reverse transcriptase-PCR from Jurkat cells
and ligated into pCDNA3.1 downstream of the T7 promoter and
subjected to coupled in vitro transcription and translation in the absence or presence of recombinant DFF35 or DFF45. DFF40 was
synthesized in vitro only in the presence of
pCDNA3.1-DFF40 and bound to DFF45 or DFF35 in the subsequent GST
pull-down experiment (Fig.
4A). Functional assay showed
that the DFF40 synthesized in the presence of DFF45, but not DFF35 or
vector only, could induce DNA fragmentation in the cell-free system
assay, suggesting that DFF45 may act as a chaperone to assist DFF40
folding into a functional conformation with high DNase activity (Fig.
4B). However, both DFF35 and DFF45 could inhibit fully
functional synthesized DFF40 in vitro and such an inhibition
can be dismantled by caspase-3 cleavage (Fig. 4C). These
results show that the excess carboxyl terminus of DFF45 is
indispensable to achieve the correct folding of DFF40. To our surprise,
an equal amount of GST-DFF35 can pull down much more in
vitro synthesized DFF40 than DFF45 (Fig. 4A), indicating DFF35 has a much stronger ability to bind to DFF40 than
DFF45 does.
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By comparing the protein sequences of DFF35/45 with DFF40, we could
delineate a homologous region at the N-terminal between the nuclease
and its inhibitors (the amino acids 23-100 of DFF35/45 and the amino
acids 10-87 of DFF40). Further comparison found that this region was
also conserved in the mouse CAD and ICAD-S/L (44%) (Fig.
5B). We hypothesize that this
region may be involved in mediating the binding or inhibitory effect of
DFF35/45 on DFF40 or ICAD-S/L on CAD. We therefore constructed four
deletion mutants of DFF45, which deleted the region of DFF45 ranging
from 23-100 (DFF45D1), 101-261 (DFF45D2), 101-180 (DFF45D3), and
181-260 amino acids (DFF45D4), respectively. As shown in Fig.
5C, DFF40 was synthesized in the presence of all four
deletion mutants at levels comparable with that of wild type DFF45, but
only wild type DFF45, DFF45D1, and DFF45D4, but not DFF45D2 and
DFF45D3, bound to DFF40 (Fig. 5C). It suggests that DFF40
binding domain is within 101-180 amino acids of DFF35/45. Functional
analysis shows that only DFF40 synthesized in the presence of DFF45 but
not in the presence of any mutant has nuclease activity (Fig.
5D). Although both DFF45D1 and DFF45D4, as well as DFF45,
can bind with DFF40, only DFF45 and DFF45D4, but not DFF45D1, can
inhibit the nuclease activity of DFF40 (Fig. 5E). It
suggests that the homologous region (the amino acid 23-100 of
DFF35/45) may function as the inhibitory domain of DFF35/45 to inhibit
the nuclease activity of DFF40. It is reasonable to hypothesize that
the physical interaction of DFF35 or DFF45 with DFF40 is a prerequisite
for their inhibition and/or folding function. Thus, DFF45D2 and DFF45D3
cannot inhibit the activity of DFF40 because of their failure to bind
to DFF40 (Fig. 5E). DFF35 and the deletion mutants of DFF45
cannot assist in the synthesis of active DFF40, suggesting that an
intact structure/conformation of DFF45 is essential for its function as
a chaperone involved in the folding DFF40 into an active enzyme.
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Several enzymes including cyclophins, DNase I, and DNase II have been
shown to be partially involved in DNA degradation during apoptosis
(18-20). Both DFF45 and DFF35 can completely block nuclease activity
of DFF40 but have no inhibitory effect on DNase I or II (data not
shown), suggesting that the inhibitory effect of both DFF45 and DFF35
is DFF40-specific. Further, our findings suggest that DFF45 can both
bind DFF40 and assist in the folding of nascent DFF40 into a functional
nuclease with high activity and inhibit its activity as well. DFF40 is
a highly active nuclease that is harmful or even lethal to normal
cells. Thus, it is not surprising that DFF35 with its strong binding
ability can also serve as an important safety net to ensure a complete
control of DFF40 activity in cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Zhimin Yuan and Dr. Prasad Kanteti for helpful discussion and critically reading this manuscript.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF087573.
These authors contributed equally to the work.
§ To whom correspondence and requests for reprints should be addressed: Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Fax: 617-632-2690; E-mail: stuart_schlossman@dfci.harvard.edu.
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ABBREVIATIONS |
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The abbreviations used are: DFF, DNA fragmentation factor; CPAN, caspase-activated nuclease; CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; GST, glutathione S-transferase; EST, expressed sequence tag; PCR, polymerase chain reaction; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; CHO-DEVD, cell-permeable caspase-3 inhibitor.
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TOP
ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
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 D. Lechardeur, L. Drzymala, M. Sharma, D. Zylka, R. Kinach, J. Pacia, C. Hicks, N. Usmani, J. M. Rommens, and G. L. Lukacs Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (ICAD/CAD) J. Cell Biol., July 24, 2000; 150(2): 321 - 334. [Abstract] [Full Text] [PDF]
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 J.-M. Kim, L. Luo, and B. R. Zirkin Caspase-3 Activation Is Required for Leydig Cell Apoptosis Induced by Ethane Dimethanesulfonate Endocrinology, May 1, 2000; 141(5): 1846 - 1853. [Abstract] [Full Text] [PDF]
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H. Sakahira, A. Iwamatsu, and S. Nagata Specific Chaperone-like Activity of Inhibitor of Caspase-activated DNase for Caspase-activated DNase J. Biol. Chem., March 10, 2000; 275(11): 8091 - 8096. [Abstract] [Full Text] [PDF]
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P. Widlak, P. Li, X. Wang, and W. T. Garrard Cleavage Preferences of the Apoptotic Endonuclease DFF40 (Caspase-activated DNase or Nuclease) on Naked DNA and Chromatin Substrates J. Biol. Chem., March 10, 2000; 275(11): 8226 - 8232. [Abstract] [Full Text] [PDF]
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C. L. Perkins, G. Fang, C. N. Kim, and K. N. Bhalla The Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide- or Paclitaxel-induced Mitochondrial Events during Apoptosis Cancer Res., March 1, 2000; 60(6): 1645 - 1653. [Abstract] [Full Text]
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D. McIlroy, M. Tanaka, H. Sakahira, H. Fukuyama, M. Suzuki, K.-i. Yamamura, Y. Ohsawa, Y. Uchiyama, and S. Nagata An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes Genes & Dev., March 1, 2000; 14(5): 549 - 558. [Abstract] [Full Text]
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D. Chen, R. A. Stetler, G. Cao, W. Pei, C. O'Horo, X.-M. Yin, and J. Chen Characterization of the Rat DNA Fragmentation Factor 35/Inhibitor of Caspase-activated DNase (Short Form). THE ENDOGENOUS INHIBITOR OF CASPASE-DEPENDENT DNA FRAGMENTATION IN NEURONAL APOPTOSIS J. Biol. Chem., December 1, 2000; 275(49): 38508 - 38517. [Abstract] [Full Text] [PDF]
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