Entamoeba histolytica is the etiologic agent of amoebiasis which still causes over 55,000 annual deaths worldwide, and 2.2 million disability‐adjusted life years. To differentiate E. histolytica from ...the non‐pathogenic commensal E. dispar requires costly and laborious diagnostic tools. Apart from housekeeping ncRNA, the E. histolytica ncRNA inventory includes microRNAs and stress‐induced self‐circularized 5ʹ‐externally transcribed spacer rRNAs, therefore we searched for as many circRNA types as we could detect in this parasite with potential value for diagnostic purposes, as drug targets, and as markers between virulent vs non‐virulent strains. CircRNAs are a type of transcripts widely conserved in eukaryotic cells and in E. histolytica we expected to identify potential circRNAs candidates for diagnosis. Using circular RT‐PCR with divergent primers, we identified post‐splicing derived full‐length intronic circles (flicRNAs). In vivo splicing assays and several biochemical approaches revealed that the highly conserved GU‐rich intronic 5'ss is required for flicRNA biogenesis, possibly slowing down intron lariat debranching enzyme activity, thus facilitating intron circularization. Similar approaches, and CLIP analyses using an HA‐tagged Pol II CTD construct, showed that some flicRNAs interact with the transcription machinery and silence their parental genes in cis. We also found cytoplasmic flicRNAs whose functions remain to be uncovered. To expand our searches, we mined the available Entamoeba transcriptomes with the CIRIFull software, and identified 199 exonic and intergenic circular RNAs from the reptile parasite E. invadens, as well as 310 circRNAs from both virulent (HMI:IMSS) and avirulent (Rahman) E. histolytica strains (inclusion criteria: > 2 Back Splice Junctions, with Reverse Overlaps). A small proportion of the E. histolytica circRNAs were differentially expressed between strains, and some of these were validated by circular RT‐PCR. Altogether, our data suggest that E. histolytica expresses a complex repertoire of circRNAs with diverse functions, involved in multiple biochemical pathways.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in ...testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5′UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future.
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.
•E. histolytica harbors all the components for DNA repair using the short and long-patch Base Excision Repair sub-pathways.•Base Excision Repair in E. histolytica has a mixed evolutionary ...origin.•Most DNA glycosylase present in E. histolytica are the product of lateral gene transfer from bacteria.
During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5′-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Amoebiasis is the third leading cause of death among protozoon parasitic diseases in the lower-middle income countries. Understanding the molecular events that control gene expression such as ...transcription factors, their DNA binding mode and target sequences can help to develop new antiamoebic drugs against Entamoeba histolytica. In this paper we performed a genome and structural analysis of a specific transcription factor. The genome of E. histolytica codifies for 9 EhMybSHAQKYF proteins, which are a family within a large group of 34 Myb-DNA-binding domain (Myb-DBD) containing proteins. Here we compared Entamoeba Myb-SHAQKYF proteins with Myb-like proteins from the Reveille (RVE) family, important regulators of plant circadian networks. This comparison could lead to stablish their role in E. histolytica life cycle. We show that the ehmybshaqkyf genes are differentially expressed in trophozoites under basal cell culture conditions. An in-silico analysis predicts that members of this group harbor a highly conserved and structured Myb-DBD and a large portion of intrinsically disordered residues. As the Myb-DBD of these proteins harbors a distinctive QVIRSTHAQKYFF sequence in its putative third α-helix, we consider relevant to determine the three-dimensional (3D) structure of one of them. An NMR structure of the Myb-DBD of EhMybS3 shows that this protein is composed of three α-helices stabilized by a hydrophobic core, similar to Myb proteins of different kingdoms. It is remarkable that despite not sharing similarities in their amino acid sequences, the structure of the Myb-DBD of the EhMybS3 is well conserved in this early branching eukaryote.
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•Genes of ehmybshaqkyf family are differentially expressed in trophozoites of Entamoeba histolytica.•Proteins of EhMYBSHAQKYF family contain large segments of intrinsic disorder.•EhMybS3DBD protein structure is a Helix-Turn-Helix stabilized by a hydrophobic core.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Entamoeba histolytica is the causative agent of human amoebiasis. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of ...intercellular structures such as tight junctions (TJ). We recently showed that the E. histolytica complex EhCPADH112, formed by the EhADH112 adhesin and the EhCP112 cysteine protease, participates during epithelial invasion. To elucidate the particular role of EhADH112 in this process, we overexpressed this adhesion molecule in MDCK epithelial cells. Expression of EhADH112 was proven by RT‐PCR and western blot assays. EhADH112 localized at the plasma membrane of epithelial cells, similar to its location in trophozoites. EhADH112 improved adhesion between epithelial cells induced by an increase in TER and expression of TJ proteins. Importantly, infection of MDCK cells expressing EhADH112 with trophozoites caused a faster drop in TER and more epithelial damage compared to control cells; suggesting that EHADH112 in epithelial cells serves as a ligand for the parasite, facilitating trophozoite entrance into the epithelium. Combined with our previous results that EhADH112 localizes to TJ after secretion by trophozoites, EhADH112 may be a promising new target to prevent amebiasis.
Grant Funding Source: Supported by Institute of Science and Technology of the Federal District (64/2012)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and ...purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using (3H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity.
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•TvDHS catalyzes the posttranslational modification of rTveIF-5A precursor protein.•Only the mature rTveIF-5A was bound to a RNA hairpin ERE structure from cox-2 gene.•The hypusine residue in TveIF-5A was identify using a proteomic method.•Hypusine-containing TveIF-5A shows RNA-binding activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental ...stimulus by biometals such as Zn
2+
. Many of these proteins have the classical C
2
H
2
zinc finger motifs (C
2
H
2
-ZNFm) of approximately 30 amino acids, where a Zn
2+
ion is coordinated by two cysteine and two histidine residues.
Trichomonas vaginalis
is a protozoan parasite than responds to environmental changes including Zn
2+
. Until now has not been described any ZNF that could be involved in the regulation of genic expression of
T. vaginalis
. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from
T. vaginalis
and isolated the gene,
tvznf1
encoding it. TvZNF1 have eight C
2
H
2
-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn
2+
upregulation expression of
tvznf1
gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in
T. vaginalis
.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, ...except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2–GST–sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses.
Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities.
Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite.
We have found numerous new splicing-related factors which could have therapeutic benefit. We also detected all the DExH/A RNA helicases involved in splicing and splicing proofreading control. Still, Entamoeba is very inefficient in splicing fidelity, thus we may have found a possible model system to study these processes.
This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: César López-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.
Nuclear extracts of amoebas transformed with a HA-tagged splicing factor or an MS2-tagged intron were immunoprecipitated using bead-covered HA antibodies or MS2 protein. The recovered precipitates were analyzed by MS/MS. Display omitted
•Epitope-tagged cell lines were used to probeEntamoeba mRNP complexes.•Ectopic protein expression and localization was monitored.•Amoeba transformants nuclear extracts were used for CLIP and proteomic analyses.•Protein datasets were classified into splicing complexes and mRNP components.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental ...stimulus by biometals such as Zn
. Many of these proteins have the classical C
H
zinc finger motifs (C
H
-ZNFm) of approximately 30 amino acids, where a Zn
ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn
. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C
H
-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn
upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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