Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Inhibition of homodimerization of poly(ADP-ribose) polymerase by its C-terminal cleavage products produced during apoptosis

The biochemical role of the C-terminal fragment of poly(ADP-ribose) polymerase (PARP) was investigated in HeLa cells undergoing UV-mediated apoptosis. During the course of apoptosis, the C-terminal cleavage product of PARP interacted with intact PARP and down-regulated PARP activity by blocking the homodimerization of PARP. The basic leucine zipper motif in the auto-modification domain of the C-terminal fragment of PARP represented the site of association, and Leu(405) was critical to the ability of the basic leucine zipper motif to associate with intact PARP. The expression of the C-terminal fragment of PARP stimulated UV-mediated apoptosis. These results suggest that the C-terminal cleavage product of PARP produced during apoptosis blocks the homodimerization of PARP and inhibits the cellular PARP activity. The inhibition of the cellular PARP activity might prevent cellular NAD(+) depletion and stimulate apoptosis by maintaining the basal cellular energy level required for the completion of apoptosis.     

Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7.

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.     

Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.     

PARP degradation in apoptotic Syrian hamster embryo (SHE) cells compared to HL60 cell line

In this study, we attempted to identify apoptotic Syrian hamster embryo (SHE) cells by detecting the specific cleavage of poly(ADP-ribose)polymerase (PARP). Apoptosis was unequivocally identified in serum-deprived SHE cells. After protein electrophoresis and transfer, the anti-PARP antibody (C-2-10) was applied in order to visualize PARP degradation and the anti-polymer antibody (LP96-10) was used to identify PARP and its expected 89-kDa fragment on the membrane after renaturation and NAD+ addition. Results showed that PARP rapidly disappeared during apoptosis in SHE cells, but the resulting fragment remained undetectable with the anti-PARP antibody and no stable polymerase activity of this fragment was measured using anti-polymer antibody. Serum-starved SHE cells were compared to the etoposide-treated HL60 cell line as a control for typical apoptosis-related PARP cleavage. These results underline the fact that while PARP degradation is a criterion for apoptosis, the diagnosis of apoptosis can not rely exclusively on the appearance of its 89-kDa fragment as this signal may fail to appear in some cell systems.     

Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.     

Liver poly(ADP-ribose)polymerase is resistant to cleavage by caspases

In hepatocytes the DNA repair enzyme poly(ADP-ribose)polymerase (PARP) is not proteolytically cleaved during apoptosis. The reason for this was investigated using a cell-free system that consisted of isolated nuclei from hepatocytes or thymocytes and cytosolic extracts from hepatocytes or thymocytes undergoing apoptosis. It was found that liver PARP is resistant to proteolytic cleavage by the caspases present in the cytosolic extracts. Furthermore, liver PARP was not cleaved by recombinant human caspase-3. It is concluded that PARP proteolysis cannot be used as a marker for hepatocyte apoptosis.     

Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells.

An early transient burst of poly(ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines (Simbulan-Rosenthal, C., Rosenthal, D., Iyer, S., Boulares, H., and Smulson, M. (1998) J. Biol. Chem. 273, 13703-13712) followed by cleavage of poly(ADP-ribose) polymerase (PARP), catalyzed by caspase-3. This inactivation of PARP has been proposed to prevent depletion of NAD (a PARP substrate) and ATP, which are thought to be required for later events in apoptosis. The role of PARP cleavage in apoptosis has now been investigated in human osteosarcoma cells and PARP -/- fibroblasts stably transfected with a vector encoding a caspase-3-resistant PARP mutant. Expression of this mutant PARP increased the rate of staurosporine and tumor necrosis factor-alpha-induced apoptosis, at least in part by reducing the time interval required for the onset of caspase-3 activation and internucleosomal DNA fragmentation, as well as the generation of 50-kilobase pair DNA breaks, thought to be associated with early chromatin unfolding. Overexpression of wild-type PARP in osteosarcoma cells also accelerated the apoptotic process, although not to the same extent as that apparent in cells expressing the mutant PARP. These effects of the mutant and wild-type enzymes might be due to the early and transient poly(ADP-ribose) synthesis in response to DNA breaks, and the accompanying depletion of NAD apparent in the transfected cells. The accelerated NAD depletion did not seem to interfere with the later stages of apoptosis. These results indicate that PARP activation and subsequent cleavage have active and complex roles in apoptosis.     

Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism.

Poly(ADP-ribosyl)ation is a posttranslational modification that alters the functions of the acceptor proteins and is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Following DNA damage, activated poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the elongation and branching of poly(ADP-ribose) (pADPr) covalently attached to nuclear target proteins. Although the biological role of poly(ADP-ribosyl)ation has not yet been defined, it has been implicated in many important cellular processes such as DNA repair and replication, modulation of chromatin structure, and apoptosis. The transient nature and modulation of poly(ADP-ribosyl)ation depend on the activity of a unique cytoplasmic enzyme called poly(ADP-ribose) glycohydrolase which hydrolyzes pADPr bound to acceptor proteins in free ADP-ribose residues. While the PARP homologues have been recently reviewed, there are relatively scarce data about PARG in the literature. Here we summarize the latest advances in the PARG field, addressing the question of its putative nucleo-cytoplasmic shuttling that could enable the tight regulation of pADPr metabolism. This would contribute to the elucidation of the biological significance of poly(ADP-ribosyl)ation.     

Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases

The poly(ADP-ribose) polymerase (PARP-1), a 113 kDa nuclear enzyme, is cleaved in fragments of 89 and 24 kDa during apoptosis. This cleavage has become a useful hallmark of apoptosis and has been shown to be done by DEVD-ase caspases, a family of proteases activated during apoptosis. Interestingly, PARP-1 is also processed during necrosis but a major fragment of 50 kDa is observed. This event is not inhibited by zVAD-fmk, a broad spectrum caspase inhibitor, suggesting that these proteases are not implicated in the necrotic cleavage of PARP-1. Since lysosomes release their content into the cytosol during necrosis, the proteases liberated could produce the cleavage of PARP-1. We therefore isolated lysosomal rich-fractions from Jurkat T cells. Our results reveal that the in vitro lysosomal proteolytic cleavage of affinity purified bovine PARP-1 is composed of fragments corresponding, in apparent molecular weight and function, to those found in Jurkat T cells treated with necrotic inducers like 0.1% H2O2, 10% EtOH or 100 microM HgCl2. Moreover, we used purified lysosomal proteases (cathepsins B, D and G) in an in vitro cleavage assay and found that cathepsins B and G cleaved PARP-1 in fragments also found with the lysosomal rich-fractions. These findings suggest that the necrotic cleavage of PARP-1 is caused in part or in totality by lysosomal proteases released during necrosis.     

Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.     

Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the auto-modification site does not show any immunoreactivity with the antibodies.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

In vivo activated caspase-3 cleaves PARP-1 in rat liver after administration of the hepatocarcinogen N-nitrosomorpholine (NNM) generating the 85 kDa fragment

We reported previously that treatment of rats with the hepatocarcinogen N-nitrosomorpholine (NNM) caused severe hepatotoxicity associated with apoptosis of hepatocytes beginning 12 h after administration of NNM. We observed that poly(ADP-ribose) polymerase 1 (PARP-1), one of the major nuclear targets for caspases, was proteolytically degraded generating primarily 64 and 54 kDa fragments. Interestingly, at 20, 30, and 40 h post-treatment a 85 kDa cleavage product of PARP-1 resembling that generated by caspase-3 appeared additionally in hepatocytes. More detailed analysis performed in the present study revealed that the 85 kDa fragment of PARP-1 was generated in the liver in 10 of 17 (60%) animals examined between 20 and 40 h after NNM administration. The caspase-3 generated 85 kDa fragment was detected solely in hepatocytes undergoing apoptosis as evidenced by immunostaining performed with the antibody recognizing exclusively PARP-1 cleaved at position 214/215. The appearance of the 85 kDa fragment of PARP-1 in the liver nuclei coincided temporally with an significant increase of caspase-3 activity in hepatocytes. In contrast, in testis samples obtained from the same animals, no changes characteristic for apoptosis such as induction of caspases activity or degradation of nuclear PARP-1 could be detected. Our results evidence unequivocally that PARP-1 in liver is not resistant to caspases and can be processed in vivo by activated caspase-3 producing the p85 kDa fragment. Moreover, the caspase-3 induced PARP-1 fragmentation coinciding with the increase of caspase-3 activity was detected solely in the target organ and exclusively in hepatocytes undergoing apoptosis. Considering the fact that the caspase-3 mediated PARP-1 cleavage occurred only in 60% of animals tested between 20 and 40 h, it becomes obvious that the cellular response in vivo to the same trigger(s) strongly varies and may depend on a variety of intrinsic factors. It remains to elucidate which additional factors may be involved in the modulation of cellular response to the strong insults thereby activating different pathways and generating distinct outcomes.     

Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells

The cleavage of poly(ADP-ribose) polymerase (PARP) by caspase (casp)-3 is an essential link in the apoptotic pathway in animal cells. In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis. Using a heat shock (HS)-induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS. An activation of casp-3-like protease was also observed. The results suggest that apoptosis in plants and animals may share common mechanisms. On the other hand, when cells were preincubated with 4 mM 3-aminobenzamide or 2-8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly. Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp-related events.