Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model.

Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.

Proteomic and Functional Analysis of the Effects of Quinoxaline Derivatives on Entamoeba histolytica.

Quinoxalines are heterocyclic compounds that contain a benzene ring and a pyrazine ring. The oxidation of both nitrogen of the pyrazine ring results in quinoxaline derivatives (QdNO), which exhibit a variety of biological properties, including antiparasitic activity. However, its activity against Entamoeba histolytica, the protozoan that causes human amebiasis, is poorly understood. Recently, our group reported that various QdNOs produce morphological changes in E. histolytica trophozoites, increase reactive oxygen species, and inhibit thioredoxin reductase activity. Notably, T-001 and T-017 derivatives were among the QdNOs with the best activity. In order to contribute to the characterization of the antiamebic effect of QdNOs, in this work we analyzed the proteomic profile of E. histolytica trophozoites treated with the QdNOs T-001 and T-017, and the results were correlated with functional assays. A total number of 163 deregulated proteins were found in trophozoites treated with T-001, and 131 in those treated with T-017. A set of 21 overexpressed and 24 under-expressed proteins was identified, which were mainly related to cytoskeleton and intracellular traffic, nucleic acid transcription, translation and binding, and redox homeostasis. Furthermore, T-001 and T-017 modified the virulence of trophozoites, since they altered their erythrophagocytosis, migration, adhesion and cytolytic capacity. Our results show that in addition to alter reactive oxygen species, and thioredoxin reductase activity, T-001 and T-017 affect essential functions related to the actin cytoskeleton, which eventually affects E. histolytica virulence and survival.