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
[This corrects the article DOI: 10.3389/fcimb.2018.00214.].
RESUMO
Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.
Assuntos
Entamoeba histolytica/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Western Blotting , Núcleo Celular/química , Biologia Computacional , Citoplasma/química , Ensaio de Desvio de Mobilidade Eletroforética , Entamoeba histolytica/química , Entamoeba histolytica/genética , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Humanos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Telômeros/genéticaRESUMO
The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolytica encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H2O2 treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H2O2 treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.
Assuntos
Dano ao DNA , DNA Ligases/metabolismo , Reparo do DNA , Entamoeba histolytica/enzimologiaRESUMO
The molecular mechanisms involved during the infection of Rhipicephalus microplus midgut cells by Babesia bigemina are of great relevance and currently unknown. In a previous study, we found a voltage-dependent anion channel (VDAC)-like protein (BmVDAC) that may participate during parasite invasion of midgut cells. In this work, we investigated BmVDAC expression at both mRNA and protein levels and examined BmVDAC localization in midgut cells of ticks infected with B. bigemina at different times post-repletion. Based on the RT-PCR results, Bmvdac expression levels were significantly higher in infected ticks compared to uninfected ones, reaching their highest values at 24h post-repletion (p<0.0001). Similar results were obtained at the protein level (p<0.0001). Interestingly, BmVDAC immunolocalization showed that there was an important differential expression and redistribution of BmVDAC protein between the midgut cells of infected and uninfected ticks, which was more evident 24h post-repletion of infected ticks. This is the first report of BmVDAC upregulation and immunolocalization in R. microplus midgut cells during B. bigemina infection. Further studies regarding the function of BmVDAC during the infection may provide new insights into the molecular mechanisms between B. bigemina and its tick vector and could result in its use as an anti-tick and transmission-blocking vaccine candidate.
Assuntos
Babesia/fisiologia , Regulação da Expressão Gênica/fisiologia , Rhipicephalus/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Rhipicephalus/microbiologia , Regulação para Cima , Canais de Ânion Dependentes de Voltagem/genéticaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that function as negative regulators of gene expression by either inhibiting translation or inducing deadenylation-dependent degradation of target transcripts. Notably, deregulation of miRNAs expression is associated with the initiation and progression of human cancers where they act as oncogenes or tumor suppressors contributing to tumorigenesis. Abnormal miRNA expression may provide potential diagnostic and prognostic tumor biomarkers and new therapeutic targets in cancer. Recently, several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs. Here, we present a review of the current knowledge of miRNAs in cancer with a special focus on metastamiRs. In addition we discuss their potential use as novel specific markers for cancer progression.