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.

The yeast Xrs2 complex functions in S phase checkpoint regulation.

The Nbs1 complex is an evolutionarily conserved multisubunit nuclease composed of the Mre11, Rad50, and Nbs1 proteins. Hypomorphic mutations in the NBS1 or MRE11 genes in humans result in conditions characterized by DNA damage sensitivity, cell cycle checkpoint deficiency, and high cancer incidence. The equivalent complex in the yeast Saccharomyces cerevisiae (Xrs2p complex) has been implicated in DNA double-strand break repair and in telomere length regulation. Here, we find that xrs2Delta, mre11Delta, and rad50Delta mutants are markedly defective in the initiation of the intra-S phase checkpoint in response to DNA damage. Furthermore, the absence of a functional Xrs2p complex leads to sensitivity to deoxynucleotide depletion and to an inability to efficiently slow down cell cycle progression in response to hydroxyurea. The checkpoint appears to require the nuclease activity of Mre11p and its defect is associated with the abrogation of the Tel1p/Mec1p signaling pathway. Notably, DNA damage induces phosphorylation of both Xrs2p and Mre11p in a Tel1p-dependent manner. These results indicate that the Tel1p/ATM signaling pathway is conserved from yeast to humans and suggest that the Xrs2p/Nbs1 complexes act as signal modifiers.

Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding.

Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm-zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm-ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.

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.

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.

Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions. DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively. In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions. PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends. The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.

Proteolysis of poly(ADP-ribose) polymerase by caspase 3: kinetics of cleavage of mono(ADP-ribosyl)ated and DNA-bound substrates.

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents and during DNA base excision repair. After DNA damage, the enzyme binds to nicks in DNA through its N-terminal zinc fingers and catalyzes the formation of poly(ADP-ribose) on various nuclear acceptors including itself. When DNA damage is extensive, cells induce their own demise by activating the proteases that induce apoptosis (caspases) which cleave PARP and other death substrates. Here we report the development of a new approach to investigate the sensitivity of mono(ADP-ribosyl)ated and DNA-bound PARP to cleavage during apoptosis. The development of a stoichiometric labeling procedure of the enzyme has allowed us to evaluate the catalytic properties of caspase 3 toward mono(ADP-ribosyl)ated PARP at various enzyme:substrate molar ratios. We show that low levels of automodification (< or = 3 U of ADP-ribose per chain) do not inhibit the proteolysis of the substrate. In addition, we demonstrate that binding of unmodified PARP to DNA influences the kinetics of its cleavage by caspase 3.

Interaction of DNA-dependent protein kinase and poly(ADP-ribose) polymerase with radiation-induced DNA strand breaks.

Two of the enzymes involved in the response of mammalian cells to ionizing radiation are the DNA-dependent protein kinase and poly(ADP-ribose) polymerase. These enzymes are known to be activated by binding to DNA strand breaks, but previous studies designed to look at strand break specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase, we compared enzyme activation by a series of DNA substrates. Irradiated plasmid DNA activated DNA-dependent protein kinase in a dose-dependent manner. When calculated in terms of the molar concentration of double-strand breaks, the enzyme activation by irradiated DNA was comparable to that by restriction enzyme-cleaved DNA. Linear DNA purified after plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comparison of the enzyme activation by plasmid molecules with different 3'- and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence the response of the DNA-dependent protein kinase. Similar experiments with poly(ADP-ribose) polymerase demonstrated that single- and double-strand breaks activate this enzyme with almost equal efficiency, but because of their greater number, single-strand breaks dominate the response of poly(ADP-ribose) polymerase to irradiated DNA.

Granzyme B/perforin-mediated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptotic fragment and less abundant 64-kDa fragment.

Cytotoxic lymphocytes utilize granule associated serine proteases (granzymes) and perforin to induce apoptosis. Although the importance of granzyme B has been established by gene ablation experiments, biochemical events initiated by the granzyme remain enigmatic. We show here that exposure of Jurkat cells to granzyme B and perforin results in cleavage of poly(ADP-ribose) polymerase to an apoptotic 89 kDa fragment and to lesser amounts of a 64 kDa fragment. The 64 kDa fragment is produced directly by granzyme B while the 89 kDa fragment is presumably generated by activated ICE/Ced-3 proteases. Establishing the intracellular function of GrB in the apoptotic response, these results indicate that granzyme B enters perforin treated targets activating the ICE/Ced-3 family proteases which then cleave poly(ADP-ribose) polymerase to its apoptotic fragment. Intracellular granzyme B appears to be translocated to the nucleus where the protease directly cleaves poly(ADP-ribose) polymerase.

[The usefulness of an exercise test on a treadmill shortly after a myocardial infarction]. / Utilité de l'épreuve d'effort sur tapis roulant peu après un infarctus du myocarde.

A program of reconditioning through walking was prescribed for 130 patients following an exercise test on a treadmill 3 weeks after a myocardial infarction. At 8 and at 12 weeks the patients again underwent an exercise test. The protocol is safe and permits the detection of angina, arrhythmias and dyspnea during the exercise, thus avoiding delays in treatment. The heart rate and the systolic blood pressure were measured at the end of each stage of the test and after 3 minutes of recuperation. About 75% of the patients attained the target energy output of the two submaximal tests (4 and 7 mets at 3 and 8 weeks respectively); an output of 7 mets permits a patient to resume his or her usual daily activities. The results of the tests at 3 and 12 weeks (the latter a maximal test) showed that the probability of an aerobic capacity of 7 mets or greater at 12 weeks is 86% if the 3-week test is completed. Clinical observations alone did not have the same prognostic value 3 weeks after the infarction.