Estudio de la nucleasa EndoG en el protozoo "Leishmania infantum"caracterización e implicaciones en la supervivencia y muerte del parásito

Abstract

It is increasingly accepted that single-celled organisms, such as Leishmania parasites, are able to undergo a cell death process that resembles apoptosis in metazoans and is induced by a variety of stimuli. However, the molecular mechanisms that participate and regulate this death process are still very poorly described, and very few of the participating molecules have been identified. Because DNA degradation is probably the most frequently characterized event during programmed cell death in Leishmania parasites, we have focused on identifying a candidate nuclease responsible for this effect during the cell death process. The results presented in this thesis demonstrate that Leishmania infantum promastigotes express a nuclease similar to the Endonuclease G of higher eukaryotes which, according to its predicted structure, belongs to the ββα-metal superfamily of nucleases. Its optimal pH and its cation dependence resembles that of EndoGs present in other organisms and, similarly to them, it is inhibited by moderate concentrations of K+, Na+ and Ca+2. Moreover, L. infantum nuclease processes each DNA strand independently, as described for other Endonucleases G, and shows, like yeast Nuc1p, endo-exonuclease activity. The activity of the recombinant protein can be inhibited by addition of the general nuclease inhibitor aurintricarboxylic acid (ATA) or the reducing agent β-mercaptoethanol. This latter result is probably due to the presence of a disulfide bound between Cys100 and Cys116 that appears to be crucial for the nuclease activity. This activity can also be inhibited by the synthetic thymidine analogue LEI-49, which also shows higher anti-Leishmania activity against wild-type parasites than against parasites overexpressing EndoG. L. infantum EndoG contains a signal peptide that causes its translocation to the mitochondrion where it is maintained under normal growth conditions. However, under the pressure of a death stimulus such as edelfosine or miltefosine treatment, L. infantum EndoG is released from the single mitochondrion and translocates to the nucleus, where it is thought to participate in the process of DNA degradation that is associated with an apoptotic process. Our results also demonstrate that overexpression of the nuclease in edelfosine- and miltefosine-treated promastigotes causes a significant increase in the percentage of TUNEL-positive parasites, whilst this overexpression has no effect on the normal growth of the parasites. On the other hand, parasites expressing low levels of the protein grow at a lower rate than do wild-type parasites under healthy growth conditions, and show a higher resistance to death than wild-type parasites under a cell-death stimulus, as revealed by the decrease in the percentage of TUNEL-positive parasites treated with edelfosine. Mutation analysis reveals that the amino acids present in the catalytic center of the nuclease are not equally relevant for the nuclease activity. Thus, His214 and Arg212 are crucial because they are involved in the catalytic mechanism and in substrate recognition, respectively, whereas Ser211 is not. Finally, we have identified a putative inhibitory domain in L. infantum EndoG that could be playing a regulatory role in the catalytic activity of this important enzyme.