INVESTIGADORES
VILCHEZ LARREA Salome Catalina
congresos y reuniones científicas
Título:
Characterization of poly(ADP-ribose)polymerase and ADP-ribose polymer metabolism in Trypanosoma brucei
Autor/es:
MARIANA SCHLESINGER; SALOMÉ VILCHEZ LARREA; GUILLERMO D. ALONSO; MIRTHA M. FLAWIÁ; SILVIA H. FERNÁNDEZ VILLAMIL
Lugar:
Mar del Plata
Reunión:
Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011
Institución organizadora:
Sociedad Argentina de Protozoología
Resumen:
Poly(ADP-ribose) polymerase (PARP) catalyses the synthesis of poly(ADP-ribose) (PAR) from NAD+ to modify its target proteins, modulating their activities and functions. The intracellular production of PAR polymers is induced by DNA strand breaks (DSB). Trypanosoma  brucei evades the immune system by antigenic variation of surface glycoproteins (VSG), a process which requires transient DSB. In order to study in the future whether TbPARP is involved in such a process, in the present work we introduce the characterization of the protein.  In silico analysis showed the presence of one 1722 bp TbPARP gene in the T. brucei genome, confirmed by Southern-blot. Moreover, sequence alignments of the catalytic domain displayed the characteristic a-helix, 6 b-sheets and H-Y-E triad. TbPARP was cloned and expressed in a bacterial system to produce specific antibodies and for further biochemical characterization. Recombinant TbPARP was activated in vitro by nicked DNA, being its most active form a dimer. In contrast to other PARPs, it does not require Mg2+ or other metal ions; instead, it is inhibited by many divalent cations. In vivo TbPARP activity was also tested in parasites by measuring PAR generation after a genotoxic insult. Polymers were detected by using anti-PAR antibodies after 10-minute H2O2 treatment, decreasing 90 minutes later. In addition, the basal state of PAR polymers was determined along the parasite life cycle. Expression of the 65 kDa enzyme in the procyclic and bloodstream stages was demonstrated by Western-blot, as well as its subcellular localization by IFI. Finally, TbPARP down regulation by RNAi in procyclics exhibited that this enzyme is not essential in this stage of the parasite, at least under regular growing conditions.