In this paper, we explored the presence of GATA in Entamoeba histolytica and their function as regulators of phagocytosis‐related genes. Bioinformatics analyses evidenced a single 579 bp sequence ...encoding for a protein (EhGATA), smaller than GATA factors of other organisms. EhGATA appeared phylogenetically close to Dictyostelium discoideum and Schistosoma mansoni GATA proteins. Its sequence predicts the presence of a zinc‐finger DNA binding domain and an AT‐Hook motif; it also has two nuclear localization signals. By transmission electron and confocal microscopy, anti‐EhGATA antibodies revealed the protein in the cytoplasm and nucleus, and 65% of nuclear signal was in the heterochromatin. EhGATA recombinant protein and trophozoites nuclear extracts bound to GATA‐DNA consensus sequence. By in silico scrutiny, 1,610 gene promoters containing GATA‐binding sequences appeared, including Ehadh and Ehvps32 promoters, whose genes participate in phagocytosis. Chromatin immunoprecipitation assays showed that EhGATA interact with Ehadh and Ehvps32 promoters. In EhGATA‐overexpressing trophozoites (NeoGATA), the Ehadh and Ehvps32 mRNAs amount was modified, strongly supporting that EhGATA could regulate their transcription. NeoGATA trophozoites exhibited rounded shapes, high proliferation rates, and diminished erythrophagocytosis. Our results provide new insights into the role of EhGATA as a noncanonical transcription factor, regulating genes associated with phagocytosis.
EhGATA is a noncanonical GATA protein family member. It contains a GATA domain and an AT‐hook motif. This protein localizes at trophozoites cytoplasm and chromatin regions. EhGATA binds to DNA, acting as a transcription factor, probably regulating the expression of genes involved proliferation, adhesion, and phagocytosis. EhGATA regulates the transcription of the Ehadh and Ehvps32 genes and possibly others involved in phagocytosis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Circular RNAs (circRNAs) are non‑coding single‑stranded covalently closed RNA molecules that are considered important as regulators of gene expression at the transcriptional and post‑transcriptional ...levels. These molecules have been implicated in the initiation and progression of multiple human diseases, ranging from cancer to inflammatory and metabolic diseases, including diabetes mellitus and its vascular complications. The present article aimed to review the current knowledge on the biogenesis and functions of circRNAs, as well as their role in cell processes associated with diabetic nephropathy. In addition, novel potential interactions between circRNAs expressed in renal cells exposed to high‑glucose concentrations and the transcription factors c‑Jun and c‑Fos are reported.
Zinc is an essential micronutrient that plays an important role as a co-factor to several proteins, including zinc-responsive transcription factors.
Trichomonas vaginalis
is able to survive in the ...presence of high zinc concentrations in the male urogenital tract. Several genes in
T. vaginalis
have been shown to respond to changes in zinc concentrations, however, the zinc-dependent mechanism remains undetermined. Recently, we identified in
T. vaginalis
the zinc finger protein, TvZNF1, which is an ortholog of the mammal metal transcription factor (MTF1). We searched for several of the zinc-responsive genes in
T. vaginalis
to determine whether if they contain metal response elements (MRE),
cis
-acting DNA elements that specifically bind MTF1. Six highly conserved over-represented sequence motifs (TvMREs), which share similarity with other eukaryotic MREs, were identified in the zinc-responsive genes in
T. vaginalis
. We also demonstrated that some of the TvMREs assemble as divalent complexes either as two closely spaced TvMREs or as two overlapping TvMREs forming a palindromic-like sequence: TGCC(N3)GGCA. Electrophoretic mobility shift assays were used to detect the zinc-dependent binding of TvZNF1 and nuclear proteins from
T. vaginalis
to this specific palindromic motif. Our results support a novel mechanism used by
T. vaginalis
for the transcriptional regulation of associated zinc-responsive genes through a MTF1/MRE-like system.
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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
(
) exhibits a remarkable capacity to respond to thermal shock stress through a sophisticated genetic regulation mechanism. This process is carried out via Heat Shock Response Elements (HSEs), which ...are recognized by Heat Shock Transcription Factors (EhHSTFs), enabling fine and precise control of gene expression. Our study focused on screening for HSEs in the promoters of the
genome, specifically analyzing six HSEs, including Ehpgp5, EhrabB1, EhrabB4, EhrabB5, Ehmlbp, and Ehhsp100. We discovered 2578 HSEs, with 1412 in promoters of hypothetical genes and 1166 in coding genes. We observed that a single promoter could contain anywhere from one to five HSEs. Gene ontology analysis revealed the presence of HSEs in essential genes for the amoeba, including cysteine proteinases, ribosomal genes, Myb family DNA-binding proteins, and Rab GTPases, among others. Complementarily, our molecular docking analyses indicate that these HSEs are potentially recognized by EhHSTF5, EhHSTF6, and EhHSTF7 factors in their trimeric conformation. These findings suggest that
has the capability to regulate a wide range of critical genes via HSE-EhHSTFs, not only for thermal stress response but also for vital functions of the parasite. This is the first comprehensive study of HSEs in the genome of
, significantly contributing to the understanding of its genetic regulation and highlighting the complexity and precision of this mechanism in the parasite's survival.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
According to their oncogenic properties, Human Papillomaviruses (HPVs) are classified into two types: Low-Risk (LR-HPVs) and High-Risk Human Papillomaviruses (HR-HPVs). The immune system naturally ...controls the majority of HPV infections; however, when the HR-HPV infection is persistent, the risk of developing cervical cancer increases. Previous studies indicate that multiple-infection or coinfection with HR-HPV occurs frequently and can potentiate the development of cervical lesions. This study aimed to establish the HPV coinfection rate in squamous intraepithelial lesions from Mexican patients. For HPV detection, we performed PCR on 55 cervical lesions diagnosed by colposcopy. We detected the presence of HPV infection in 87.27% (48/55) of the lesions; interestingly, HPV coinfection was observed in 70.83% (34/48) of these samples. We also evaluated HPV infection in adjacent areas without morphological changes from 25 samples. The results showed that 80% (20/25) of these were HPV-positive and, curiously, all presented HPV-16 infection. In conclusion, our results revealed a high prevalence of HPV coinfection in cervical lesions in Mexican patients, and these results contribute to future research focused on the role that HPV coinfection plays in the development of cervical cancer.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression.
, the causative agent of ...dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis
, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5'ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5'ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5'-3'ss ligation points. Different from the reported functions of ciRNAs, the 5'ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in
. Furthermore, introns of
virulence-related genes are also processed as flicRNAs.
Proliferating cell nuclear antigen (PCNA) coordinates multienzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of ...DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the interdomain connector loop (IDCL) of PCNA through its PCNA‐interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N‐terminal deletions to establish that DNA binding and nick‐sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box–IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. As the rate constants for single‐turnover ligation for the full‐length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C‐terminal mutants of PCNA are not able to stimulate nick‐sealing activity of Entamoeba histolytica DNA ligase I. Our data support the notion that the C‐terminal region of PCNA may be involved in promoting an allosteric transition in E. histolytica DNA ligase I from a spread‐shaped to a ring‐shaped structure. This study suggests that the ring‐shaped PCNA is a binding platform able to stabilize coevolved protein–protein interactions, in this case an interaction with DNA ligase I.
PCNA is able to stimulate the nick‐sealing reaction of DNA ligase I. DNA ligase with deletion of the PCNA‐interacting peptide (PIP box) is still stimulated by PCNA and a DNA ligase with compromised DNA binding properties is rescued upon PCNA addition. This indicates that this stimulation is not solely dependent on the PIP box.
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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.
, 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
, 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
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
. Transcriptomic data analysis revealed expression signatures of
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
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.
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