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

BackgroundPoly(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.MethodsThe 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.ResultsAbolition 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.ConclusionsPARP 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 materialThe online version of this article (doi:10.1186/s13071-016-1461-1) contains supplementary material, which is available to authorized users.

Poly(ADP-Ribose) Polymerases in Host-Pathogen Interactions, Inflammation, and Immunity

“…Knockdown of host cell PARG also abrogated T. cruzi infection (485). In Trypanosoma brucei, PARG knockdown led to increased sensitivity to oxidative stress (486). Hence, PARG functionality is also essential for establishing trypanosomal infection.…”

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

“…We have previously shown that Tb PARP is catalytically activated by damaged DNA and it most likely functions in DNA-damage response and DNA-repair like hPARPs-1-3 6 . While hPARP-1 utilizes three zinc-fingers for DNA binding, hPARPs-2 and 3 have a positively charged N-terminus that is utilized for DNA interaction.…”

“…Interestingly, cytotoxicity induced by hydrogen peroxide was reduced both by down-regulation and inhibition of the catalytic activity of the enzyme suggesting a role for Tb PARP in cell death pathways. In addition, inhibition of the catalytic activity of Tb PARP did not inhibit the growth of T. brucei procyclic and bloodstream forms 6 . These results are in contrast to what have been observed for the related parasite T. cruzi 7 .…”

“…We have previously shown that Tb PARP is enzymatically activated by DNA and migrates to the nucleus after a genomic stress 6 . Interestingly, cytotoxicity induced by hydrogen peroxide was reduced both by down-regulation and inhibition of the catalytic activity of the enzyme suggesting a role for Tb PARP in cell death pathways.…”

The DNA damage response is developmentally regulated in the African trypanosome

“…1C). Genetic and biochemical studies clearly demonstrate the action of Nucleotide Excision Repair (NER) [[27], [28], [29]], Base Excision Repair (BER) [[30], [31], [32], [33], [34], [35], [36], [37], [38]] and Mismatch Repair (MMR) in T. brucei [[39], [40], [41], [42], [43], [44]]. In contrast, further T. brucei DNA repair pathways are less well explored, including Direct Reversion Repair (DR) [45], Interstrand Crosslink Repair (ICL repair) [29,46] and Translesion Synthesis (TLS) [47].…”