Dataset Information

Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

ABSTRACT: Trypanosoma brucei is a unicellular parasite responsible for African trypanosomiasis or sleeping sickness. It contains a single PARP enzyme opposed to many higher eukaryotes, which have numerous PARPs. PARPs are responsible for a post-translational modification, ADP-ribosylation, regulating a multitude of cellular events. T. brucei PARP, like human PARPs-1-3, is activated by DNA binding and it potentially functions in DNA repair processes. Here we characterized activation requirements, structure and subcellular localization of T. brucei PARP. T. brucei PARP was found to be selectively activated by 5' phosphorylated and 3' phosphorylated DNA breaks. Importantly, the N-terminal region is responsible for high-affinity DNA-binding and required for DNA-dependent enzymatic activation. This module is also required for nuclear localization of the protein in response to oxidative stress. Solution structures of activating and non-activating PARP-DNA complexes were determined with small-angle X-ray scattering revealing distinct differences in their DNA-binding modes.

SUBMITTER: Haikarainen T

PROVIDER: S-EPMC5473844 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

Haikarainen Teemu T Schlesinger Mariana M Obaji Ezeogo E Fernández Villamil Silvia H SH Lehtiö Lari L

Scientific reports 20170616 1

Trypanosoma brucei is a unicellular parasite responsible for African trypanosomiasis or sleeping sickness. It contains a single PARP enzyme opposed to many higher eukaryotes, which have numerous PARPs. PARPs are responsible for a post-translational modification, ADP-ribosylation, regulating a multitude of cellular events. T. brucei PARP, like human PARPs-1-3, is activated by DNA binding and it potentially functions in DNA repair processes. Here we characterized activation requirements, structure ...[more]

PMID: 28623292

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Project description: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.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.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.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.

Dataset Information

Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

Publications

Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

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