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.
abstract: 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, H2O2, 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 H2O2 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.
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", Consejo Nacional de Investigaciones Científicas y Técnicas, Vuelta de Obligado 2490, 1428, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Schlesinger
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", Consejo Nacional de Investigaciones Científicas y Técnicas, Vuelta de Obligado 2490, 1428, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Salomé C
authors.initials: SC
authors.lastname: Vilchez Larrea
authors.affiliation: Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
authors.forename: Teemu
authors.initials: T
authors.lastname: Haikarainen
authors.affiliation: Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
authors.forename: Mohit
authors.initials: M
authors.lastname: Narwal
authors.affiliation: Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
authors.forename: Harikanth
authors.initials: H
authors.lastname: Venkannagari
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", Consejo Nacional de Investigaciones Científicas y Técnicas, Vuelta de Obligado 2490, 1428, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Mirtha M
authors.initials: MM
authors.lastname: Flawiá
authors.affiliation: Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
authors.forename: Lari
authors.initials: L
authors.lastname: Lehtiö
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", Consejo Nacional de Investigaciones Científicas y Técnicas, Vuelta de Obligado 2490, 1428, Ciudad Autónoma de Buenos Aires, Argentina. silvia.villamil@gmail.com.|Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1428, Ciudad Autónoma de Buenos Aires, Argentina. silvia.villamil@gmail.com.
authors.forename: Silvia H
authors.initials: SH
authors.lastname: Fernández Villamil
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D016923:Cell Death; D006026:Glycoside Hydrolases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity*; D008124:Locomotion / Q000187:drug effects; D009153:Mutagens / Q000633:toxicity; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology
chemical_list: D009153:Mutagens; D000246:Adenosine Diphosphate Ribose; D011064:Poly Adenosine Diphosphate Ribose; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase
keywords: Cell death; Genotoxic damage; PAR; PARG; PARP; Trypanosoma brucei