RESUMO
Entamoeba histolytica is a protozoan parasite that belongs to the Amoebozoa supergroup whose study related to the nucleocytoplasmic transport of proteins through the nucleus is poorly studied. In this work, we have performed in silico predictions of the potential nuclear localization signals (NLS) corresponding to the proteome of 8201 proteins from Entamoeba histolytica annotated in the AmoebaDB database. We have found the presence of monopartite nuclear localization signals (MNLSs), bipartite nuclear localization signals (BNLSs), and non-canonical monopartite NLSs with lengths exceeding 20 amino acid residues. Additionally, we detected a new type of NLS consisting of multiple juxtaposed bipartite NLSs (JNLSs) that have not been described in any eukaryotic organism. Also, we have generated consensus sequences for the nuclear import of proteins with the NLSs obtained. Docking experiments between EhImportin α and an MNLS, BNLS, and JNLS outlined the interacting residues between the Importin and cargo proteins, emphasizing their putative roles in nuclear import. By transfecting HA-tagged protein constructs, we assessed the nuclear localization of MNLS (U1A and U2AF1), JMNLS (U2AF2), and non-canonical NLS (N-terminus of Pol ll) in vivo. Our data provide the basis for understanding the nuclear transport process in E. histolytica.
RESUMO
Entamoeba histolytica, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in Entamoeba invadens, responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in E. invadens encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and splicing. Transcriptomic data analysis revealed expression signatures of eimyb genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that E. invadens Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.
Assuntos
Amebíase , Entamoeba histolytica , Entamoeba , Humanos , Entamoeba/genética , Entamoeba/metabolismo , Entamoeba histolytica/genética , Perfilação da Expressão Gênica , Regulação da Expressão GênicaRESUMO
Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Cromossômicas não Histona/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucléolo Celular/metabolismoRESUMO
Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (TcRPA31-EGFP), we describe its nuclear distribution during growth and metacyclogenesis. Our findings indicate that TcRPA31-EGFP alters its nuclear presence from concentrated nucleolar localization in exponentially growing epimastigotes to a dispersed granular distribution in the nucleoplasm of stationary epimastigotes and metacyclic trypomastigotes. These changes likely reflect a structural redistribution of the Pol I transcription machinery in quiescent cellular stages where downregulation of rRNA synthesis is known to occur. In addition, and related to the nuclear internalization of this protein, the presence of a classical bipartite-type nuclear localization signal was identified towards its C-terminal end. The functionality of this motif was demonstrated by its partial or total deletion in recombinant versions of the tagged fluorescent protein. Moreover, ivermectin inhibited the nuclear localization of the labelled chimaera, suggesting the involvement of the importin α/ß transport system.