Metabolism of Poly-ADP-ribose in Trypanosomatids

Abstract

The poly-ADP-ribosylation reaction is a post-translational modification involved invarious cellular processes including DNA repair, DNA replication, gene expression,maintenance of genomic stability, cell death and cellular differentiation. Poly(ADPribose)polymerase (PARP) is an abundant nuclear protein that is potently activated by DNA strand nicks and breaks. This enzyme transfers ADP-ribose units from NAD+ to several proteins implicated in chromatin architecture and DNA metabolism, but the main acceptor protein is PARP itself. The homopolymer of ADP-ribose is mainly catabolized by poly(ADP-ribose)glycohydrolase (PARG), a mechanism through which PARP and other modified proteins may be restored to their native state. Poly-ADP-ribose metabolism has been studied in several organisms, but limited information is available about this process in trypanosomatids. In our laboratory, we have demonstrated the presence of PARP in the trypanosomatids Crithidia fasciculata, Trypanosoma cruzi and Trypanosoma brucei, all of which showed properties characterizing other representatives of the PARP group. By using the genome project of T. cruzi and T. brucei we identified TcPARP and TbPARP as well as TcPARG and TbPARG sequences and found that they presented several conserved structural characteristics and only showed slight differences when compared with PARPs and PARGs from other organisms. In addition, we characterized the purified C. fasciculata PARP and recombinant TcPARP biochemically and found that both enzymes were strongly activated by nicked DNA. We also found that histones increase PARP activity and demonstrated the covalent attachment of [32P]-ADPribose moieties to histones, which are catalyzed by PARP. In further experiments, we demonstrated that C. fasciculata PARP and recombinant TcPARP require no magnesium or any other metal ion cofactor for their activity and are inhibited by typical PARP inhibitors: nicotinamide, 3-aminobenzamide, theophylline and thymidine. TcPARP and TcPARG were localized to the cell nucleus. Our findings demonstrated that covalent poly-ADP-ribosylation of PARP itself or DNA topoisomerase I resulted in the inhibitionof their activities, which were restored after removing the ADP-ribose polymers. DNAdamaging agents, which trigger different repair mechanisms, induce poly-ADP-ribosesynthesis in the nucleus, as was established by our group in epimastigotes of T. cruzi.The role of poly-ADP-ribose metabolism in trypanosomatids remains to be furtherinvestigated, and unraveling it could be important for the development of new therapies against these parasites.