Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE-related gene, designated Yama, that encodes a protein identical to CPP32 beta. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.
Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.
Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death.
Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents.Current efforts to develop poly(ADP-ribose) polymerase (PARP) inhibitors as anticancer drugs represent the culmination of over 40 years of research. After Paul Mandel's research group first described a nuclear enzymatic activity that synthesizes an adenine-containing RNA-like polymer 1 , independent studies by French and Japanese teams demonstrated that this polymer, designated poly(ADP-ribose) (pADPr), is composed of two ribose moieties and two phosphates per unit polymer [2][3][4][5] . The purification of an enzyme that could generate large amounts of pADPr, PARP1 (REFS 6,7 ), led to the discovery that PARP1 is activated by DNA strand breaks [8][9][10] . Seminal work by Sydney Shall's group showed that PARP1 is involved in DNA repair and also suggested the potential use of PARP inhibitors to enhance the cytotoxic effects Correspondence to G.G.P. guy.poirier@crchul.ulaval.ca. Competing interests statementThe authors declare no competing financial interests. DATABASES NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of alkylating agents 10 . Examination of knockout mouse models 11 strengthened the hypothesis that PARP1 participates in DNA repair and simultaneously provided the first evidence for the existence of PARP2 (REF. 12 ). A parallel set of experiments demonstrated that PARP1 hyperactivation leads to nicotinamide adenine dinucleotide (NAD + ) and ATP depletion after various types of DNA damage 13,14 (BOX 1), potentially contributing to a unique form of metabolic cell death, which is now termed parthanatos 15 . PARP was thrust into the limelight by the discovery that PARP inhibition is particularly toxic in cancer cell lines 16,17 and human tumours 18 that lack BRCA1 or BRCA2. Despite this progress, there is still much that we do not understand about the biology of the PARP family and pADPr, as detailed below. Box 1 PARP1 hyperactivation and cell deathNicotinamide adenine dinucleotide (NAD + ) is the source of ADP-ribose used by poly(ADPribose) polymerases (PARPs) to produce poly(ADP-ribose) (pADPr). Because hyperactivation of PARP1 consumes the cytosolic and nuclear pools of NAD + to generate pADPr, pADPr synthesis translates DNA damage intensity into changes in cellular energy. Low to moderate DNA damage triggers pADPr-dependent DNA repair. In the context of excessive DNA damage, however, PARP1 hyperactivation leads to extended pADPr synthesis and extensive NAD + consumption 8,13,14 . Depending on the cellular context, this intense pADPr synthesis can induce cell death through several mechanisms. Long and branched pADPr (60mers and longer) can directly trigg...
Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation. How PARP-1 activation leads to AIF release is not known. Here we identify PAR polymer as a cell death signal that induces release of AIF. PAR polymer induces mitochondrial AIF release and translocation to the nucleus. PAR glycohydrolase, which degrades PAR polymer, prevents PARP-1-dependent AIF release. Cells with reduced levels of AIF are resistant to PARP-1-dependent cell death and PAR polymer cytotoxicity. These results reveal PAR polymer as an AIF-releasing factor that plays important roles in PARP-1-dependent cell death.excitotoxicity ͉ poly(ADP-ribose) polymerase
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