Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis.

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N:-methyl-N:'-nitro-N:-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N:-methyl-N:'-nitro-N:-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to gamma-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.

Characterization of sPARP-1. An alternative product of PARP-1 gene with poly(ADP-ribose) polymerase activity independent of DNA strand breaks.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that catalyzes the synthesis of poly(ADP-ribose) (pADPr) from its substrate NAD(+) upon binding to DNA strand breaks. Poly(ADP-ribosyl)ation has been implicated in many cellular processes including replication, transcription, and the maintenance of genomic stability. However, studies with mice and cells lacking PARP-1 reveal a critical role for the enzyme in the maintenance of genomic integrity only. Recently, a significant level of poly(ADP-ribose) polymerase activity has been detected in fibroblasts derived from mice lacking PARP-1 following treatment with genotoxic agents (Shieh, W. M., Amé, J-C., Wilson, M. V., Wang, Z-Q., Koh, D. W., Jacobson, M. K., and Jacobson, E. L. (1998) J. Biol. Chem. 273, 30069-30072). We have isolated a cDNA that originates from PARP-1 (-/-) fibroblasts and encodes a polypeptide of 493 amino acid residues bearing poly(ADP-ribose) polymerase activity. This protein, that we named sPARP-1 for short poly(ADP-ribose) polymerase-1, has a calculated mass of 55.3 kDa and is identical in deduced amino acid sequence to the catalytic domain of PARP-1. Radiation hybrid analysis assigned the sPARP-1 gene to the chromosome 1H5-H6 in an immediate proximity to the known location of PARP-1 gene, indicating that sPARP-1 and PARP-1 are most probably products of the same gene. Active sPARP-1 is present in both PARP-1 (+/+) and PARP-1 (-/-) cells as demonstrated by activity-Western blotting and immunostaining using a specific antibody developed against sPARP-1. Like PARP-1, sPARP-1 is localized in the cell nucleus, uses NAD(+) as a substrate and is inhibited by nicotinamide analogues. sPARP-1 produces pADPr of similar length and structure to that of PARP-1. However, contrary to PARP-1, sPARP-1 does not require DNA strand breaks for its activation, although it is stimulated following genotoxic treatments.

Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.

The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.

Rapid detection of poly(ADP-ribose) polymerase by enzyme-linked immunosorbent assay during its purification and improvement of its purification.

We report a new detection method for the purification of poly(ADP-ribose) polymerase (PARP). PARP purification generates many fractions in which PARP is usually detected by a time consuming activity assay. The development of a new method was also needed in order to decrease the utilization of radioactivity. This new method, based on an enzyme-linked immunosorbent assay (ELISA), is very rapid, sensitive, and avoids most radioactivity. Moreover, to illustrate this method, a new matrix was used, the Heparin Sepharose. This matrix was chosen for its affinity for the DNA binding proteins and because it allows the separation of whole PARP from its proteolytic fragments.

Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase.

Intracellular cysteine proteases are important mediators of apoptosis. Indeed, some nuclear proteins and enzymes are cleaved during apoptosis, in particular poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand interruptions and is involved in DNA repair. PARP is cleaved into two fragments of 29 and 85 kDa (apparent molecular mass) in human promyelomonocytic leukemia cells, HL-60, treated with etoposide to induce apoptosis. These cells possess protease activities, caspases, that share many features with the ICE/CED-3 family. The cleavage occurs between Asp-214 and Gly-215, a site that is conserved in human, bovine, and chicken PARP. This cleavage has been shown to be an early marker of apoptosis. To monitor apoptosis, to understand the role of PARP cleavage by caspases, and to study the role of the two fragments in DNA repair, members of our laboratory have developed two polyclonal antipeptide antibodies directed against the two human PARP sequences: [196-214] for LP96-22 and [215-228] for LP96-24. Moreover, these antibodies will be useful to map the necrotic cleavage of PARP, which generates fragments different from those obtained during apoptosis, and thus to discriminate between apoptotic and necrotic cell death.