Caspase-3-mediated Processing of Poly(ADP-ribose) Glycohydrolase during Apoptosis

Abstract

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 Fasinduced 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 2 V and the unconventional site MDVD 307 2 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.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 b...

Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines

“…Because PARP is cleaved by activated caspase-3 and caspase-7 (Affar et al, 2001;Germain et al, 1999;Lazebnik et al, 1994;Tewari et al, 1995) and our results showed that PARP is not cleaved during cell death induced by V. parahaemolyticus (Fig. 7), this indicated that caspase-3 and caspase-7 are not activated during the process of cell death.…”

“…PARP is a 116 kDa enzyme that is cleaved into an 89 kDa inactive form to conserve the energy required for apoptosis (Affar et al, 2001); thus, detection of the cleaved form of PARP is a hallmark of apoptotic cell death. In contrast, for the other three mechanisms of cell death (autophagy, oncosis and pyroptosis), PARP is maintained as a full-length protein and the active form depletes its substrate NAD and ATP, leading to cell death.…”

“…The membrane integrity of the cells infected with wild-type and T3SS2 mutant strains was disrupted, while there was no evidence of similar changes in the cells infected with T3SS1 and T3SS1-T3SS2 double mutant strains. Changes typical of apoptosis, such as chromatin condensation, membrane blebbing and apoptotic bodies, were not observed in U937 and HeLa cells infected with either wild-type or mutant strains of V. parahaemolyticus.PARP is a 116 kDa enzyme that is cleaved into an 89 kDa inactive form to conserve the energy required for apoptosis (Affar et al, 2001); thus, detection of the cleaved form of PARP is a hallmark of apoptotic cell death. In contrast, for the other three mechanisms of cell death (autophagy, oncosis and pyroptosis), PARP is maintained as a full-length protein and the active form depletes its substrate NAD and ATP, leading to cell death.…”

BubR1 is involved in regulation of DNA damage responses

“…Although PARP-1 cleavage is mediated by caspase 3 (Affar et al, 2001), the molecular entity responsible for degradation of BubR1 after genotoxic stresses remains unknown. As inhibition of proteasome by MG132 can partially block the degradation of BubR1, especially the phosphorylated form ( Figure 5), we speculate that the proteasomal pathway is involved in regulating the stability of BubR1 after DNA damage.…”

3-[2-[4-(3-Chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole Dihydro-chloride 3.5 Hydrate (DY-9760e) Is Neuroprotective in Rat Microsphere Embolism: Role of the Cross-Talk between Calpain and Caspase-3 through Calpastatin

“…In addition to calpain activation, caspase-3 plays pivotal roles in apoptotic cell death by inducing Bid and poly(ADPribose) polymerase activation (Affar et al, 2001;Degli Esposti et al, 2003). Inappropriate imbalances between calpain and calpastatin activation may play critical roles in the delayed neuronal death including apoptosis (Rami et al, 2003).…”