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1.
Article in English | MEDLINE | ID: mdl-28293543

ABSTRACT

Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E. histolytica. The aim of this study was to identify the possible cellular events where EhTSN is involved. Here, we found that EhTSN in nucleus is located in euchromatin and close to, but not into, heterochromatin. We also showed the association of EhTSN with proteins involved in transcription and that the knockdown of EhTSN provokes a diminishing in the mRNA level of the EhRabB gene, which in its promoter region contains the URE1 motif, confirming that EhTSN participates in transcription regulation. In cytoplasm, this protein was found linked to the membrane of small vesicles and to plasma membrane. Through pull-down assays and mass spectrometry we identity thirty two candidate proteins to interact with EhTSN. These proteins participate in transcription, metabolism, signaling, and stress response, among other cellular processes. Interaction of EhTSN with some candidate proteins involved in metabolism, and signaling was validated by co-immunoprecipitation or co-localization. Finally we showed the co-localization of EhTSN and HSP70 in putative stress granules during heat shock and that the knockdown of EhTSN increases the cell death during heat shock treatment, reinforcing the hypothesis that EhTSN has a role during stress response. All data support the proposal that EhTSN is a multifunctional protein of E. histolytica.


Subject(s)
Entamoeba histolytica/enzymology , Entamoeba histolytica/genetics , Entamoeba histolytica/physiology , Gene Expression Regulation , Micrococcal Nuclease/genetics , Physiological Phenomena , Cloning, Molecular , Cytoplasm/metabolism , DNA, Protozoan/chemistry , Entamoeba histolytica/ultrastructure , Escherichia coli/genetics , Gene Knockdown Techniques , Genes, Protozoan , Heat-Shock Response , Microscopy, Immunoelectron , Protein Binding , Protozoan Proteins/genetics , RNA, Messenger , Transcription Factors/metabolism
2.
Int J Clin Exp Pathol ; 10(10): 10334-10342, 2017.
Article in English | MEDLINE | ID: mdl-31966368

ABSTRACT

Epithelial-mesenchymal transition (EMT) is a biological process involved in different steps of tumor progression and metastasis of breast cancer cells. Epidemiological studies suggest a link between obesity and the progression of breast cancer. Leptin is an adipocyte-secreted hormone which can promote cell migration and invasion as part of EMT in breast cancer cells. We investigated the effect of leptin on expression of EMT markers in MCF10A cells, as well as, the role of FAK and ERK in this process. We found that leptin induces morphological changes from an epithelial phenotype towards a mesenchymal phenotype and promotes cell migration in MCF10A cells. Moreover, leptin induces an increase in vimentin expression, changes in the cellular localization of E-cadherin and increase in FAK and ERK phosphorylation. Furthermore, using FAK and ERK chemical inhibitors we show that leptin regulates EMT markers in a FAK and ERK dependent manner. In conclusion, leptin promotes vimentin expression and cell migration in a FAK and ERK dependent pathway in the non-tumorigenic epithelial cell line MCF10A.

3.
Exp Parasitol ; 135(1): 79-86, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23800535

ABSTRACT

Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.


Subject(s)
Calcium-Transporting ATPases/isolation & purification , Entamoeba histolytica/enzymology , Amino Acid Sequence , Animals , Calcium/metabolism , Calcium-Transporting ATPases/chemistry , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Entamoeba histolytica/chemistry , Entamoeba histolytica/genetics , Fluorescent Antibody Technique , Microscopy, Confocal , Microscopy, Immunoelectron , Phylogeny , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/immunology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/isolation & purification , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
4.
Parasitol Res ; 112(4): 1631-40, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23400794

ABSTRACT

It has been described that the pathogenicity of Entamoeba histolytica is influenced by environmental conditions and that transcription profile changes occur during invasion, suggesting that gene expression may be involved in the virulence of this parasite. However, the molecular mechanisms that are implicated in the control of gene expression in this microorganism are poorly understood. Here, we showed that the expression of the EhRabB protein, a small GTPase involved in phagocytosis, is modified through the interaction with red blood cells. By ELISA, Western blot, and immunofluorescence assays, we observed that the expression of EhRabB diminished after 5 min of the interaction of trophozoites with red blood cells, but protein level was recovered at subsequent times. In the EhRabB amino acid sequence, we found two lysine residues that could be target for ubiquitin modification and trigger the degradation of this GTPase at early times of phagocytosis. The analysis of the expression of the EhrabB mRNA showed that the interaction of trophozoites with red blood cells produces a drastic diminishing in its half-life. In addition, promoter assays using the chloramphenicol acetyltransferase reporter gene and electrophoretic mobility shift assays experiments showed that the URE1 motif located in the promoter region of EhrabB is involved in the expression regulation of this gene during phagocytosis. Moreover, the immunolocalization of the URE1-binding protein during phagocytosis indicated that the transcription of the EhrabB gene is determined, at least in part, by the translocation of this transcription factor to nuclei. These results suggested that the expression of particular genes of this parasite is controlled at several stages.


Subject(s)
Entamoeba histolytica/physiology , Gene Expression Regulation , Phagocytosis , rab GTP-Binding Proteins/biosynthesis , Blotting, Western , Entamoeba histolytica/genetics , Enzyme-Linked Immunosorbent Assay , Erythrocytes/parasitology , Fluorescent Antibody Technique , Gene Expression Profiling , RNA Stability , RNA, Messenger/biosynthesis , Time Factors , Transcription, Genetic
5.
Int J Parasitol ; 41(7): 775-82, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21447339

ABSTRACT

Transcription initiation is the most regulated stage for the control of gene expression. This event requires that a complex of proteins called transcription factors bind to DNA through cis-regulatory elements located in the gene promoters. However, little is known about transcription regulation in Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis. Some genes encoding for proteins involved in the parasite pathogenicity contain specific upstream regulatory elements (URE1-URE5) in their promoters. Here, we identified the protein that specifically binds to the URE1 sequence (EhURE1BP). This protein contains five SNase domains and one Tudor motif, and has 21% identity and 36% similarity to the multifunctional eukaryotic protein known as the protein containing Tudor and staphyloccocal nuclease-like domains (TSN). To obtain antibodies against EhURE1BP, the recombinant protein was expressed and immunised in rabbits. Western blot and immunofluorescence assays showed that EhURE1BP is located in both nuclei and cytoplasm. Electrophoretic mobility shift assays and supershift assays demonstrated that EhURE1PB specifically binds to URE1 and that the C-terminus that includes the Tudor motif contains the DNA-binding domain of this protein. Results suggest that this TSN-like protein is the transcription factor that activates the transcription of some pathogenicity-related genes of E. histolytica.


Subject(s)
DNA-Binding Proteins/metabolism , Entamoeba histolytica/physiology , Gene Expression Regulation , Protozoan Proteins/metabolism , Regulatory Elements, Transcriptional , Amino Acid Motifs , Blotting, Western , Cell Nucleus/chemistry , Cytoplasm/chemistry , DNA, Protozoan/genetics , DNA, Protozoan/metabolism , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Microscopy, Fluorescence , Protein Binding , Protein Structure, Tertiary , Protozoan Proteins/genetics , Sequence Homology, Amino Acid
6.
J Biomed Biotechnol ; 2010: 726045, 2010.
Article in English | MEDLINE | ID: mdl-20204171

ABSTRACT

Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.


Subject(s)
Eukaryota/genetics , Gene Expression Regulation , Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Gene Expression Profiling/methods , Genes, Protozoan , Genome, Protozoan , Plasmodium/genetics , Plasmodium/metabolism , Trichomonas vaginalis/genetics , Trichomonas vaginalis/metabolism , Trypanosoma/genetics , Trypanosoma/metabolism
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