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Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei

BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast t...

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Autores principales: Schlesinger, Mariana, Vilchez Larrea, Salomé C., Haikarainen, Teemu, Narwal, Mohit, Venkannagari, Harikanth, Flawiá, Mirtha M., Lehtiö, Lari, Fernández Villamil, Silvia H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806436/
https://www.ncbi.nlm.nih.gov/pubmed/27007296
http://dx.doi.org/10.1186/s13071-016-1461-1
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author Schlesinger, Mariana
Vilchez Larrea, Salomé C.
Haikarainen, Teemu
Narwal, Mohit
Venkannagari, Harikanth
Flawiá, Mirtha M.
Lehtiö, Lari
Fernández Villamil, Silvia H.
author_facet Schlesinger, Mariana
Vilchez Larrea, Salomé C.
Haikarainen, Teemu
Narwal, Mohit
Venkannagari, Harikanth
Flawiá, Mirtha M.
Lehtiö, Lari
Fernández Villamil, Silvia H.
author_sort Schlesinger, Mariana
collection PubMed
description BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. METHODS: The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H(2)O(2,) was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. RESULTS: Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H(2)O(2) treatment when compared to their wild type counterparts(.) PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. CONCLUSIONS: PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1461-1) contains supplementary material, which is available to authorized users.
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spelling pubmed-48064362016-03-24 Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei Schlesinger, Mariana Vilchez Larrea, Salomé C. Haikarainen, Teemu Narwal, Mohit Venkannagari, Harikanth Flawiá, Mirtha M. Lehtiö, Lari Fernández Villamil, Silvia H. Parasit Vectors Research BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. METHODS: The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H(2)O(2,) was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. RESULTS: Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H(2)O(2) treatment when compared to their wild type counterparts(.) PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. CONCLUSIONS: PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1461-1) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-23 /pmc/articles/PMC4806436/ /pubmed/27007296 http://dx.doi.org/10.1186/s13071-016-1461-1 Text en © Schlesinger et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Schlesinger, Mariana
Vilchez Larrea, Salomé C.
Haikarainen, Teemu
Narwal, Mohit
Venkannagari, Harikanth
Flawiá, Mirtha M.
Lehtiö, Lari
Fernández Villamil, Silvia H.
Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title_full Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title_fullStr Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title_full_unstemmed Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title_short Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei
title_sort disrupted adp-ribose metabolism with nuclear poly (adp-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic trypanosoma brucei
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806436/
https://www.ncbi.nlm.nih.gov/pubmed/27007296
http://dx.doi.org/10.1186/s13071-016-1461-1
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