Date Published: April 15, 2015
Publisher: Public Library of Science
Author(s): Carla Ritagliati, Victoria L. Alonso, Romina Manarin, Pamela Cribb, Esteban C. Serra, Alvaro Acosta-Serrano. http://doi.org/10.1371/journal.pntd.0003725
Abstract: BackgroundTrypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi.MethodologyDm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity.ConclusionTcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.
Partial Text: Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. A limited set of acetyltransferases and deacetylases, and of the acetyl-lysine “reading” domain (bromodomain) are the principal components of the acetylation/deacetylation machinery. Among them, protein deacetylases are enzymes that catalyze the removal of acetyl groups from the ε-amino group of lysine residues and are classified into four classes. Sirtuins, the class III (NAD+-dependent) protein deacetylases, are homologous to the yeast transcriptional repressor, Sir2 . Sir2, as well as all sirtuins, deacetylates lysine residues in a unique chemical reaction that consumes nicotinamide adenine dinucleotide (NAD+) and generates nicotinamide, O-acetyl-ADP-ribose (OAADRr), and the deacetylated substrate .
We present herein the first experimental characterization of Trypanosoma cruzi sirtuins TcSIR2RP1 and TcSIR2RP3. The expression of these enzymes is developmentally regulated throughout T. cruzi life cycle. TcSIR2RP1 is highly expressed in epimastigotes and amastigotes, but at lower levels in trypomastigotes. On the other hand, TcSIR2RP3 expression levels are higher in epimastigotes than in amastigotes, and it seems not to be expressed in trypomastigotes. The fact that the two sirtuins are differentially expressed along the life cycle of the parasite suggests that acetylation levels could play a role in the regulation of the biology of the parasite forms. It is remarkable that the life cycle pattern expression of Tetracycline induced/T7 transcribed sirtuins is similar to those of the wild type enzymes. This observation suggests that the protein levels could be regulated by a post-transcriptional mechanism independent of the 3´ and 5’ non-coding regions, which are absent in the pTc-INDEX-GW constructions or by a post-translational mechanism.