(Received for publication, May 15, 1995; and in revised form, June 12, 1995)
From the
The proteolytic cleavage of poly(ADP-ribose) polymerase (PARP)
is an early biochemical event, which occurs during apoptosis. A recent
study suggested that PARP cleavage can be mediated by a novel cytosolic
protease (prICE) that resembles interleukin-1 converting enzyme
(ICE), but cannot be mediated by ICE itself (Lazebnik, Y. A.,
Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W.
C.(1994) Nature 371, 346-347). We have used a COS cell
co-transfection assay to investigate if ICE or any known ICE-like
protease is active in PARP cleavage within the cell. Here we report
that co-expression of human PARP with human ICE, or the ICE homologs TX
and Nedd-2, resulted in a cleavage of PARP identical to that observed
in apoptotic cells. Experiments with purified recombinant human ICE
indicated that PARP polypeptide can be specifically cleaved in
vitro by ICE in a time- and enzyme concentration-dependent manner.
PARP cleavage, however, requires a 50-100-fold higher ICE
concentration than does processing of the interleukin-1
precursor
at an equivalent substrate concentration. The abilities of ICE, TX, and
Nedd-2, when expressed at high intracellular concentrations, to cleave
PARP are consistent with their induction of apoptosis in transfected
cells.
Programmed cell death (apoptosis) is a morphologically and
biochemically defined form of active cell death, distinct from
necrosis, that occurs in many cell types and organisms. It is
characterized by a set of cellular events such as nuclear condensation
and DNA fragmentation. Recently, it has become clear that apoptosis
plays an important role in early development, homeostasis, and in
diseases such as neurodegenerative disorders and cancer (for review,
see Ellis et al.(1991). Apoptosis of cells can occur in
response to many stimuli, such as glucocorticoid, ionizing radiation,
growth factor deprivation, and the activation of Fas antigen by the Fas
ligand or anti-Fas antibodies. Apoptosis is mediated through multiple
pathways that involve a complex array of biochemical regulators and
molecular interactions. It has been best characterized genetically in
the worm Caenorhabditis elegans, in which 131 cells undergo
apoptosis during development. Among the 14 genes identified that
function in different steps of apoptosis in the worm, ced-3 and ced-4 genes are indispensable for cell death to occur
(Ellis et al., 1991; Ellis and Horvitz, 1986). The Ced-3
protein has been found to be 28% identical to the mammalian
interleukin-1 converting enzyme (ICE) (
)(Yuan et
al., 1993), a cysteine protease with a substrate cleavage
specificity for Asp-X (Howard et al., 1991; Thornberry et
al., 1992). ICE processes the inactive interleukin-1
precursor (pre-IL-1
) to the proinflammatory cytokine (Thornberry et al., 1992). Overexpression of ICE in transfected cells
induces apoptosis, which can be inhibited by the co-expression of
Bcl-2, a general suppressor of apoptosis (see references in Hengartner
and Horvitz(1994)) or a viral protein CrmA, a potent serpin-like
inhibitor of ICE protease activity (Ray et al., 1992; Miura et al., 1993), suggesting that ICE protease activity plays an
important role in apoptosis. Furthermore, we have reported that
thymocytes derived from ICE-deficient mice are resistant to apoptosis
induced by an anti-Fas antibody, suggesting a physiological role for
ICE in Fas-mediated apoptosis of normal thymocytes (Kuida et
al., 1995).
An early biochemical event that accompanies
apoptosis in many cell types is the proteolytic cleavage of
poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA
repair (Cherney et al., 1987). During apoptosis, nucleosomal
DNA fragmentation and nuclear condensation are accompanied by a rapid
and quantitative cleavage of PARP from a 116-kDa polypeptide to an
approximately 31-kDa fragment containing the N-terminal DNA binding
domain and an approximately 85-kDa polypeptide containing the
automodification domain and the NAD-binding domain (Kaufmann et
al., 1993 (see also Fig.1)). Using a cell-free system,
Lazebnik et al.(1994) identified a protease activity in the
apoptotic cytosolic extract that cleaved bovine PARP at an Asp residue
at position 216, a site that is conserved in PARP of many species. This
cytosolic protease activity had properties resembling ICE, as it
required an Asp at the substrate P position and was
inhibited by a tetrapeptide aldehyde inhibitor of ICE (Lazebnik et
al., 1994). These results suggested that a protease resembling ICE
(prICE), may be involved in apoptosis of mammalian cells and that prICE
may function as a mammalian equivalent of ced-3. This
conclusion has prompted intensive investigations to identify an ICE
homolog that would be singularly responsible for cleaving PARP during
apoptosis.
Figure 1:
PARP is cleaved by ICE and ICE homologs
in transfected COS cells. A, schematic drawing of the two
human PARP cDNA clones used in the experiments. The full-length cDNA
clone encodes a polypeptide of 1014 amino acid residues with the
functional domains indicated (Kaufmann et al., 1993). The
truncated form contains only the first 337 amino acid residues that
spans through most of the DNA-binding domain of PARP and the identified
prICE cleavage site (Asp-Gly
) as
indicated. Both clones contain a T7 epitope tag fused to their N
termini allowing for the detection of these proteins by immunoblotting. B, cleavage of PARP by ICE, TX, and Nedd-2 in COS cells. COS
cells were transfected with PARP cDNA alone or in combination with the
T7-tagged p30 version of ICE, TX, or Nedd-2 cDNA as indicated.
Twenty-four hours later, cells were harvested, and the expressed
proteins were analyzed by Western blot with an anti-T7 antibody.
PARP(F) denotes full-length PARP, PARP(T) truncated PARP, and PARP* the
tagged N-terminal fragment of cleaved PARP proteins. Numbers on the left indicate molecular mass standards in
kilodaltons. C, COS cells were transfected with a fixed
amount (3 µg) of truncated PARP cDNA in combination with increasing
amounts (0-3 µg) of T7-p30 ICE (top) or
T7-p30 TX cDNA (bottom). The expressed proteins were
analyzed by Western blot with an anti-T7 antibody as described in B. Mobilities of the p30 ICE or TX proteins and their
corresponding autoprocessing products, ICE p20 and TX p20, are
indicated, respectively, along with PARP(T) and PARP*. Molecular mass
markers on the left are in
kilodaltons.
Recently, several mammalian ICE homologs with conserved active site residues have been cloned and sequenced. These include Nedd-2/Ich-1L, TX, and CPP32. Overexpression of each of these ICE homologs induces apoptosis in transfected cells (Faucheu et al., 1995; Wang et al., 1994; Kumar et al., 1994; Fernandes-Alnemri et al., 1994). Among these, TX encodes a cysteine protease that has the highest resemblance to ICE with a more than 50% sequence identity, while Nedd-2/Ich-1L has a 30% overall protein sequence identity to ICE. In addition, an alternately spliced Ich-1 mRNA encodes a C-terminally truncated protein ICH-1S that appears to function in an anti-apoptotic manner (Wang et al., 1994). We have investigated the cleavage of PARP by ICE, TX, and Nedd-2 in transfected cells and in vitro. In this report, we demonstrate that ICE, as well as TX and Nedd-2, are capable of cleaving PARP, consistent with their ability to induce apoptosis in transfected cells.
T7-tagged full-length human
PARP cDNA (Cherney et al., 1987) was isolated from human
placenta and leukocyte cDNA libraries (Clonetech) as two overlapping
fragments of approximately 1 and 2 kb by PCR as follows. A 5` end 1-kb
fragment was amplified by PCR from a human placenta cDNA library using
primers 1 and 2. This fragment was re-amplified with primers 3 and 4 to
provide a T7-tag at the N terminus. The amplified DNA was then digested
with XbaI and EcoRI and joined with an EcoRI-digested 2-kb 3` end fragment amplified from a human
leukocyte cDNA library with primers 5 and 6 and re-amplified by nested
PCR using primers 7 and 8. T7-tagged C-terminally truncated PARP
encoding the first 337 amino acids was obtained by PCR using primers 2
and 3. Both the full-length and the truncated forms of PARP cDNAs were
cloned into the pcDLSR vector as XbaI and EcoRI
fragments. The PCR primers are as follows: primer 1,
5`-GCGCTCTAGAGCTCCATGGCGGAGTCTTCGGATAAGCTCTATCGAGTC-3`; primer 2,
5`-GATTTCTCGGAATTCTTACTTTGGGGTTACCCACTCCTTCCGGTTGGG-3`; primer 3,
5`-GCGCTCTAGAGCTCCATGGCTAGCATGACTGGTGGACAGCAAATGGGTGCGGAGTCTTCGGATAAGCTCTATCGAGTCGAGTAC-3`;
primer 4, 5`-GGCGCGGAATTCCTTTGGGGTTACCCACTCCTTCCGGTT-3`; primer 5,
5`-TACAGAGGATAAAGAAGCCCTGAAGAAGCA-3`; primer 6,
5`-TAGGACTAGTCTATGCAACAGAATCTCTCT-3`; primer 7,
5`-TGCCTATTACTGCACTGGGGACGTCACTGC-3`; primer 8,
5`-AAGCGCTTCGGGTGAATTCATACCACAGCC-3`.
Transient transfection of COS cells with the DEAE-dextran method was carried out as described (Gu et al., 1995). Briefly, COS cells in 6-well culture plates were transfected with 3 µg each of plasmid DNA. The cells were harvested 24 h after the transfection, and the expressed proteins were analyzed by immunoblotting as described (Gu et al., 1995).
We first co-transfected a plasmid encoding PARP(F) or PARP(T) with a plasmid encoding either an N-terminally truncated p30 version of ICE or a similarly truncated ICE homolog into COS cells by transient transfection. Expression and cleavage of PARP proteins as well as the expression and autoprocessing of ICE or ICE homologs were investigated by Western blots. As we have reported previously (Gu et al., 1995), T7-tagged p30 ICE or TX autoprocesses into p20 forms. We observed a similar processing of T7-tagged p30 Nedd-2 (Fig.1B). Surprisingly, when co-expressed with any of these proteases in COS cells, either the full-length (116 kDa) or the truncated form of PARP (43 kDa) was cleaved into a T7-tagged 31-kDa fragment, consistent with processing at the previously identified cleavage site (Fig.1B). Similarly, cleavage of PARP was observed in co-transfection experiments with full-length (p45) ICE or TX (data not shown). The cleavage was specific since active site mutations in ICE or TX that abolish their autoprocessing (Gu et al., 1995) also abolish PARP cleavage (data not shown). In addition, co-expression of the viral protein CrmA inhibited the cleavage. These results were consistent with either a direct intracellular processing of PARP by ICE, TX, or Nedd-2, or alternatively, these proteases were activating an as yet unidentified COS cell protease that in turn cleaves PARP.
One line of evidence
that suggested that ICE or TX was cleaving PARP directly came from
co-transfection experiments in which COS cells were transfected with a
fixed amount of PARP plasmid plus increasing amounts of ICE or TX
plasmids (Fig.1C). We observed that PARP cleavage was
directly proportional to the expression level of ICE or TX in the
cells. We also observed that ICE was slightly more potent than TX in
cleaving PARP, consistent with their relative abilities in inducing
apoptosis in transfected cells. ()The direct correlation
between the ICE/TX protease levels and PARP cleavage suggested that ICE
or TX was cleaving PARP directly rather than indirectly by activating
another protease.
Figure 2:
Cleavage of pre-IL-1 and PARP by ICE in vitro.
S-Labeled pre-IL-1
or the
truncated PARP(T) protein prepared by the in vitro transcription-translation was incubated at an equivalent substrate
concentration, as determined by
S counts (12,000 and
11,000 cpm/reaction for pre-IL1-
and PARP(T), respectively), with
the indicated concentrations of purified, active human ICE for 1 h at
37 °C, as described under ``Experimental Procedures.''
The reactions were then stopped and the cleavage products analyzed by
SDS-PAGE and fluorography. Top, cleavage of pre-IL-1
.
Mobilities of pre-IL-1
and mature IL-1
are indicated. Openarrowheads indicate an alternate cleavage
product by ICE and the propeptide. Bottom, cleavage of PARP.
PARP(T) denotes the truncated form of PARP protein, and PARP* indicates
the primary cleavage products (31 and 12 kDa). At the highest
concentrations of active ICE, the 31-kDa protein was apparently further
cleaved into two smaller fragments (openarrowheads).
Molecular mass standards on the left are in
kilodaltons.
We confirmed the specificity of in vitro PARP cleavage by ICE by the inclusion of specific ICE
inhibitors in the reactions (Fig.3). Two ICE inhibitors were
used in the experiments: Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) and
benzyloxycarbonyl-Val-Ala-Asp-[(2,6-dichlorobenzoyl)oxy]methyl
ketone (Cbz-VAD-CH-DCB). Both compounds are potent
inhibitors of ICE in vitro (Thornberry et al., 1992;
Dolle et al., 1994). Under the conditions of the assay (75
nM active ICE), Cbz-VAD-CH
-DCB, an irreversible
inhibitor, completely inhibited the cleavage activity at an
approximately equimolar concentration to the enzyme (78 nM).
Inhibition by Ac-YVAD-CHO, a reversible inhibitor, was
concentration-dependent with complete inhibition at 260 nM.
Similar experiments with partially purified TX protease also showed
cleavage of PARP (data not shown). Thus, these results demonstrate that
PARP is a substrate for ICE, TX, and Nedd-2, consistent with
observations that all these proteins can induce apoptosis when
overexpressed in transfected cells.
Figure 3:
Inhibition of in vitro PARP
cleavage by specific ICE inhibitors. S-Labeled PARP(T) was
incubated with purified ICE (75 nM) for 1 h at 37 °C in
the presence of various concentrations of ICE inhibitor Ac-YVAD-CHO or
Cbz-VAD-CH
-DCB as indicated. The reactions were then
stopped and analyzed as described in the legend of Fig.2.
Our results are in
contradistinction to those reported by Lazebnik et al.(1994),
who observed no cleavage of bovine PARP by purified human ICE in
vitro. Although we cannot formally rule out the possibility that
human ICE does not recognize bovine PARP as a substrate, we would
suggest that this apparent discrepancy most likely arises from our
observation that it requires 50-100-fold higher ICE
concentrations to cleave PARP than to cleave pre-IL-1. Similarly,
by comparing apoptosis induction by ICE cDNA constructs under the
control of different strength promoters, we have observed that high
expression levels of ICE or TX are required to induce apoptosis in
transfected COS cells.
The requirement for an increased
amount of enzyme may indicate that PARP is not a substrate for ICE in
normal cells under physiological conditions and that apoptosis induced
by ICE or ICE-like proteases in transfected cells may be due to
promiscuous hydrolysis by these proteases. Induction of apoptosis by
overexpression of ICE homologs, therefore, is insufficient evidence for
claiming a role for a specific protease in apoptosis of normal cells.
There is increasing evidence, however, that ICE homologs may
function selectively within the cell. We observed that TX and Nedd-2
have no detectable activity toward pre-IL-1 even when they are
overexpressed at very high levels in COS cells. Furthermore, thymocytes
from ICE-deficient mice fail to respond to the Fas-mediated apoptosis
(Kuida et al., 1995), indicating that ICE may play a
physiological role in cell death in normal cells. We propose that
apoptotic signals can lead to an increase in the intracellular ICE-like
protease activity, either by increased expression or activation.
Indeed, Los et al.(1995) have observed an increase in ICE-like
protease activity in apoptotic cells triggered by an anti-Fas antibody.
These results suggest that there may be an intracellular control
mechanism to protect normal cells expressing ICE-like proteases from
being driven into apoptosis under normal conditions. In this regard, it
is interesting to note that intact IL-1
-secreting peripheral blood
mononuclear cells, as well as the monocytic THP.1 cells, contain a
large amount of ICE protein in the inactive precursor form. Upon cell
lysis, these precursor proteins are readily processed to the active
p20/p10 form (Ayala et al., 1994). This apparent inhibition of
ICE autoprocessing indicates that ICE activity in these monocytes may
be tightly regulated by posttranslational mechanisms.
PARP is one of
the few polypeptides yet identified that is processed during apoptosis
and is the only one for which a cleavage sequence has been reported
(Lazebnik et al., 1994). Although no causal link has been
established between PARP cleavage and apoptosis, it is interesting that
PARP is cleaved next to an Asp residue at a site identical to one of
the two ICE cleavage sites in pre-IL-1 and that it is cleaved by a
protease with properties similar to ICE. Outside of the
ICE/ced-3 family of cysteine proteases, only one
other eukaryotic protease (granzyme B, a serine protease involved in
cytotoxic T-cell-induced apoptosis) is known to have a similar
substrate cleavage specificity (Odake et al., 1991; Heusel et al., 1994). The discovery that PARP can be cleaved directly
by ICE, TX, and Nedd-2, all of which are able to induce apoptosis in
overexpressing cells, further strengthens the correlation between PARP
cleavage and apoptosis.