Guy G. Poirier

Yama/CPP32β, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase

Abstract 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 CPP32β. 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.

PARP inhibition: PARP1 and beyond

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.

Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks

There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.

Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death

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.

Poly(ADP-ribose) (PAR) polymer is a death signal

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.

ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACG pentapeptide, rather than the QACG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

PARP-1, a determinant of cell survival in response to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a primary role in the process of poly(ADP-ribosyl)ation. This posttranslational modification of nuclear proteins is activated in response to DNA damage. Having been studied for more than 30 years, PARP-1 is now known to be implicated in several crucial cellular processes: DNA replication, transcription, DNA repair, apoptosis, and genome stability. In this review, we focus on recent findings suggesting that PARP-1 participates in DNA damage signaling in cell death. Of clinical relevance is its role in cancer therapy, irradiation, and chemotherapy, all of which may cause DNA damage and overactivate PARP-1, resulting in inflammation caused by necrosis. Therefore, we will discuss how inhibition of PARP-1 may enhance the efficiency of cancer therapy.

PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Molecular and biochemical features of poly (ADP-ribose) metabolism

In the past five years, poly(ADP-ribosyl)ation has developed greatly with the help of molecular biology and the improvement of biochemical techniques. In this article, we describe the physico-chemical properties of the enzymes responsible for the synthesis and degradation of poly(ADP-ribose), respectively poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase. We then discuss the possible roles of this polymer in DNA repair and replication as well as in cellular differentiation and transformation. Finally, we put forward various hypotheses in order to better define the function of this polymer found only in eucaryotes. (Mol Cell Biochem122: 171–193, 1993)