©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage of Poly(ADP-ribose) Polymerase by Interleukin-1 Converting Enzyme and Its Homologs TX and Nedd-2 (*)

(Received for publication, May 15, 1995; and in revised form, June 12, 1995)

Yong Gu Charlyn Sarnecki Robert A. Aldape David J. Livingston Michael S.-S. Su (§)

From theFrom Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-1beta 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-1beta 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.


INTRODUCTION

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-1beta converting enzyme (ICE) (^1)(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-1beta precursor (pre-IL-1beta) 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(1) 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.


EXPERIMENTAL PROCEDURES

Plasmids and COS Cell Transfection

Plasmids for the expression of full-length or the T7-tagged p30 form of human ICE or TX proteins were described previously (Gu et al., 1995). The full-length murine Nedd-2 cDNA was obtained from Dr. Makoto Noda (Kyoto University, Japan). An N-terminally T7-tagged p30 version of Nedd-2 cDNA lacking the first 136 amino acid residues was generated by polymerase chain reaction (PCR) with the following primers: 5`-TCATCTAGAGCTCCATGGCTAGCATGACTGGTGGACAGCAAATGGGTACAAGTCTCCCTTTCTCGGTG-3` and 5`-TCAAGTTCTAGATTATCACGTGGGTGGGTAGCC-3` (Kumar et al., 1994). Amplified DNA was digested with XbaI and subcloned into the pcDLSRalpha vector under the control of the SRalpha promoter as described (Gu et al., 1995).

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 pcDLSRalpha 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).

In Vitro Cleavage of pre-IL-1beta and PARP by ICE

S-Labeled pre-IL-1beta and PARP(T) proteins, which contain 12 and 11 methionine residues, respectively, were prepared by in vitro transcription-translation (IVTT) using the TNT T7 coupled reticulocyte lysate system (Promega) and [S]methionine (1000 Ci/mmol, Amersham Corp.). S-Labeled substrate was incubated with purified recombinant human ICE (Wilson et al., 1994) in a total volume of 22 µl for 1 h at 37 °C in a buffer containing 20 mM HEPES, pH 7.4, 2 mM dithiothreitol, 0.1% Triton X-100, 10 µg/ml leupeptin, and 0.4 mM phenylmethylsulfonyl fluoride. The reactions were stopped by the addition of SDS-PAGE sample buffer. Cleavage products were analyzed by SDS-PAGE followed by fluorography and densitometry.


RESULTS AND DISCUSSION

Cleavage of PARP by ICE and ICE-like Proteases in Transfected COS Cells

The prICE activity that cleaves PARP during apoptosis represents a possible candidate for the mammalian equivalent of C. elegans cell death gene ced-3. We developed a co-transfection assay in COS cells to test if any of the known ICE-like proteases would cleave PARP in vivo. We isolated human PARP cDNA from human cDNA libraries by polymerase chain reaction. Two PARP clones were used in these experiments: a full-length clone (PARP(F)) encoding a polypeptide of 1014 amino acids and a truncated form (PARP(T)) containing the N-terminal 337 amino acid residues that spans most of the DNA-binding domain and contains the identified prICE cleavage site (Asp-Gly). Both the full-length and the truncated forms of PARP contain a short T7 epitope tag fused to their N termini, allowing for the detection of these proteins and their cleavage products on Western blots by an anti-T7 antibody (Fig.1A) (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. (^2)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.

In Vitro Cleavage of PARP by ICE

To confirm that ICE can cleave PARP directly, we set up an in vitro cleavage assay using highly purified recombinant human ICE (Wilson et al., 1994) as the source of enzyme and [S]methionine-labeled PARP(T) prepared by IVTT as the substrate. We observed that incubation of IVTT PARP with purified ICE resulted in the cleavage of the 43-kDa PARP(T) into two fragments (approximately 31 and 12 kDa) similar to that observed in the COS cell co-transfection assay (Fig.2). At high ICE concentrations, the 31-kDa fragment was apparently cleaved further into two smaller fragments of approximately 21 and 10 kDa. This in vitro cleavage of PARP was time- and enzyme concentration-dependent. PARP cleavage could be observed within 10 min after the addition of purified ICE, and it increases linearly for the first 30 min (data not shown). When we compared the enzyme concentration dependence of PARP cleavage with that of pre-IL-1beta (also prepared by IVTT) at an equivalent substrate concentration, we found that a 50-100-fold higher concentration of ICE was required to cleave PARP than to process pre-IL-1beta (Fig.2).


Figure 2: Cleavage of pre-IL-1beta and PARP by ICE in vitro.S-Labeled pre-IL-1beta 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-beta 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-1beta. Mobilities of pre-IL-1beta and mature IL-1beta 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(2)-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(2)-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(2)-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-1beta. 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.^2 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-1beta 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-1beta-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-1beta 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vertex Pharmaceuticals Incorporated, 40 Allston St., Cambridge, MA 02139-4211. Tel.: 617-576-3111; Fax: 617-499-7315; su{at}vpharm.com.

^1
The abbreviations used are: ICE, interleukin-1beta converting enzyme; PARP, poly(ADP-ribose) polymerase; PARP(F), T7-epitope-tagged, full-length poly(ADP-ribose) polymerase; PARP(T), T7-epitope-tagged, C-terminally truncated polypeptide of poly(ADP-ribose) polymerase; pre-IL-1beta, interleukin-1beta precursor; prICE, protease resembling ICE; PCR, polymerase chain reaction; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; IVTT, in vitro transcription-translation; Ac-YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde; Cbz-VAD-CH(2)-DCB, benzyloxycarbonyl-Val-Ala-Asp-[(2,6-dichlorobenzoyl)oxy]methyl ketone.

^2
Y. Gu, unpublished results.


ACKNOWLEDGEMENTS

We thank H. Katz for oligonucleotide synthesis and DNA sequencing, J. Thomson and T. Fox for providing purified recombinant human ICE, and N. Margolin and S. Raybuck for recombinant TX protease. We thank Dr. Makoto Noda (Kyoto University, Japan) for kindly providing the murine Nedd-2 cDNA clone. We are grateful to Drs. V. Sato, J. Boger, S. Raybuck, and P. McCaffrey for their comments on the manuscript.


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