Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi

Larrea, Salomé Catalina Vilchez; Alonso, Guillermo D.; Schlesinger, Mariana; Torres, Héctor N.; Flawiá, Mirtha M.; Villamil, Silvia Hebe Fernandez

doi:10.1016/j.ijpara.2010.11.008

Cited by 24 publications

(45 citation statements)

References 81 publications

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“…Exposure of T. cruzi epimastigotes to DNA-damaging agents shows a drastic increase in the levels of pADPr in the nucleus, thus confirming pADPr synthesis in vivo and suggesting a physiological role for PARP in the trypanosomatid DNA repair signaling process [13]. We have also demonstrated that inhibition of PARP reduces epimastigote growth in culture and affects cell infection by T.…”

Section: Introductionsupporting

confidence: 61%

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Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

et al. 2013

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Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.

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Section: Introductionsupporting

confidence: 61%

“…Anti-TcPARG antibodies were obtained as previously described [13]. Parasites were fixed with 3.8% (W/V) formaldehyde in PBS at 4 ° C, permeabilized with fresh PBS-0.1%Triton X-100 and blocked for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

et al. 2013

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“…The activity of TcPARP has previously been detected to increase in the presence of DNA strand breaks [7], [8]. The highest activity was achieved by using 20 µg/ml of the DNA in assay buffer.…”

Section: Resultsmentioning

confidence: 98%

Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

et al. 2012

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Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

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“…Proteins with long poly(ADP-ribose) chains (>100 ADP-ribose) cannot easily migrate into the SDS-PAGE because of the size and phosphate moieties in PAR (Figure S2E). Thus, we used dot blot to examine PAR binding in vivo , which is a better approach to recover the long PAR chains during the analysis (Affar et al, 1998; Fiorillo et al, 2005; Vilchez Larrea et al, 2011). Following DNA damage, PAR is quickly synthesized at the DNA damage sites (D'Amours et al, 1999; Kim et al, 2005).…”

Section: Resultsmentioning

confidence: 99%

Function of BRCA1 in the DNA Damage Response Is Mediated by ADP-Ribosylation

2013

Cancer Cell

273

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SUMMARY Carriers of BRCA1 germline mutations are predisposed to breast and ovarian cancers. Accumulated evidence shows that BRCA1 is quickly recruited to DNA lesions and plays an important role in the DNA damage response. However, the mechanism by which BRCA1 is recruited to DNA damage sites remains elusive. BRCA1 forms a Ring-domain heterodimer with BARD1, a major partner of BRCA1 that contains tandem BRCT motifs. Here, we identify the BRCTs of BARD1 as a poly(ADP-ribose) (PAR)-binding module. The binding of the BARD1 BRCTs to PAR targets the BRCA1/BARD1 heterodimer to DNA damage sites. Thus, our study uncovers a PAR-dependent mechanism of rapid recruitment of BRCA1/BARD1 to DNA damage sites.

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Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi

Cited by 24 publications

References 81 publications

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

Function of BRCA1 in the DNA Damage Response Is Mediated by ADP-Ribosylation

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