Iron responsive-like elements in the parasite Entamoeba histolytica.

In Entamoeba histolytica, iron modulates virulence and gene expression via unknown regulatory mechanisms. The existence of a posttranscriptional iron regulatory system parallel with the iron-responsive element (IRE)/iron regulatory protein (IRP) system in the protozoan Trichomonas vaginalis has recently been reported. Due to their evolutionary closeness and the importance of iron for growth and virulence in these protozoa, we hypothesized the existence of an IRE/IRP-like mechanism in E. histolytica. To determine the presence of IRE-like elements in some mRNAs from this parasite, we performed in silico analyses of the 5'- and 3'-UTRs of mRNAs encoding virulence factors and cytoskeleton, ribosomal and metabolism proteins. The Zuker mfold software predicted IRE-like secondary structures in 52 of the 135 mRNAs analysed. However, only nine structures shared sequence similarity with the apical loop sequence (CAGUGN) of the previously reported human IRE-ferritin, whereas the GUU/UUG protozoan-specific motif was detected in 23 stem-loop structures. A new motif, AUU/AUUU, was also observed in 23 structures, suggesting the possible existence of an amoeba-specific motif. Additionally, cross-linking and RNA electrophoretic mobility shift assays showed specific RNA-protein interactions, using as a model two amoebic IRE-like elements from iron-regulated mRNAs and HeLa, T. vaginalis and E. histolytica cytoplasmic proteins. Our data suggest the presence of a posttranscriptional iron regulatory IRE/IRP-like mechanism in E. histolytica.

Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

Structural and thermodynamic folding characterization of triosephosphate isomerases from Trichomonas vaginalis reveals the role of destabilizing mutations following gene duplication.

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α-helix 1 against α-helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a "ball and socket" interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein.

The Circulating miR-107 as a Potential Biomarker Up-Regulated in Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) is a prevalent malignancy in men globally. Current diagnostic methods like PSA testing have limitations, leading to overdiagnosis and unnecessary treatment. Castration-resistant prostate cancer (CRPC) emerges in some patients receiving androgen deprivation therapy (ADT). This study explores the potential of circulating microRNA-107 (miR-107) in liquid biopsies as a prognosis tool to differentiate CRPC from non-castration-resistant PCa (NCRPC). We designed a case-control study to evaluate circulating miR-107 in serum as a potential prognosis biomarker. We analyzed miR-107 expression in liquid biopsies and found significantly higher levels (p < 0.005) in CRPC patients, compared to NCRPC. Notably, miR-107 expression was statistically higher in the advanced stage (clinical stage IV), compared to stages I-III. Furthermore, CRPC patients exhibited significantly higher miR-107 levels (p < 0.05), compared to NCRPC. These findings suggest that miR-107 holds promise as a non-invasive diagnostic biomarker for identifying potential CRPC patients.

The Non-Canonical Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3'-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

Cellular and biochemical characterization of two closely related triosephosphate isomerases from Trichomonas vaginalis.

The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

Gene expression in trypanosomatid parasites.

The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites.

BACKGROUND: The protozoan pathogens Leishmania major, Trypanosoma brucei and Trypanosoma cruzi (the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an in silico analysis of the Tritryps genome sequences. RESULTS: Our analysis indicated the presence of 83, 66 and 120 genes in L. major, T. brucei and T. cruzi, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the L. major genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between T. cruzi and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps. CONCLUSION: A low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.

Characterization of multiple enolase genes from Trichomonas vaginalis. Potential novel targets for drug and vaccine design.

Trichomonas vaginalis is the protist parasite that causes the most common, non-viral sexually transmitted infection called trichomonosis. Enolase is a moonlighting protein that apart from its canonical function as a glycolytic enzyme, serves as a plasminogen receptor on the cell surface of T. vaginalis and, in consequence, it has been stablished as a virulence factor in this parasite. In the Trichomonas vaginalis sequence database there are nine genes annotated as enolase. In this work, we analyzed these genes as well as their products. We found that seven out of nine genes might indeed perform enolase activity, whereas two genes might have been equivocally identified, or they might be pseudogenes. Furthermore, a combination of qRT-PCR and proteomic approaches was used to assess, for the first time, the expression of these genes in the highly virulent mexican isolate of T. vaginalis CNCD-147 at different iron concentrations. We could find peptides corresponding to enolases encoded by genes TVAG_464170, TVAG_043500 and TVAG_329460. Moreover, we identified two distinctive characteristics within enolases from Trichomonas vaginalis. One of them corresponds to three key substitutions within one of the loops of the active site, compared to host enolase. The other, is a unique N-terminal motif, composed of 15 to 18 residues, on all the potentially active enolases, whose function still has to be stablished. Both differential features merit further studies as potential drug and vaccine targets as well as diagnosis markers. These findings offer new possibilities to fight trichomonosis.

Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin.

Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis.

Potential regulatory elements in the Trypanosoma cruzi rRNA gene promoter.

The Trypanosoma cruzi rRNA gene promoter was characterized by deletion and point mutation analyses. A core of 89 bp was identified as the minimal region with full promoter activity. This core region is flanked upstream by a control element that stimulates its activity, and downstream by a novel down regulating region of about 200 bp. A point mutation analysis of the transcription start region evidenced 7 contiguous nucleotides where individual substitutions produced in all cases a defective promoter. It is generally accepted that the anciently speciated trypanosomatids lack strict promoters for protein coding genes transcribed by RNA polymerase II. The occurrence of a well structured rRNA gene promoter in these species suggests an early appearance of the RNA polymerase I promoters in the evolution of eukaryotic cells.

Corrigendum to "α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism".

[This corrects the article DOI: 10.1155/2014/424767.].

Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene.

BACKGROUND: Leishmania and other trypanosomatid parasites possess atypical mechanisms of gene expression, including the maturation of mRNAs by trans-splicing and the involvement of RNA Polymerase III in transcription of all snRNA molecules. Since snRNAs are essential for trans-splicing, we are interested in the study of the sequences that direct their expression. Here we report the characterization of L. major U2 snRNA promoter region. RESULTS: All species of Leishmania possess a single U2 snRNA gene that contains a divergently-oriented tRNA-Ala gene in the upstream region. Between these two genes we found a tRNA-like sequence that possesses conserved boxes A and B. Primer extension and RT-qPCR analyses with RNA from transiently-transfected cells showed that transcription of L. major U2 snRNA is almost abolished when boxes A and B from the tRNA-like are deleted or mutated. The levels of the U2 snRNA were also highly affected when base substitutions were introduced into box B from the tRNA-Ala gene and the first nucleotides of the U2 snRNA gene itself. We also demonstrate that the tRNA-like is transcribed, generating a main transcript of around 109 bases. As pseudouridines in snRNAs are required for splicing in other organisms, we searched for this modified nucleotide in the L. major U2 snRNA. Our results show the presence of six pseudouridines in the U2 snRNA, including one in the Sm site that has not been reported in other organisms. CONCLUSIONS: Four different regions control the transcription of the U2 snRNA gene in L. major: boxes A and B from the neighbor tRNA-like, box B from the upstream tRNA-Ala gene and the first nucleotides of the U2 snRNA. Thus, the promoter region of L. major U2 snRNA is different from any other promoter reported for snRNAs. Pseudouridines could play important roles in L. major U2 snRNA, since they were found in functionally important regions, including the branch point recognition region and the Sm binding site.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

Evidence supporting a major promoter in the Trypanosoma cruzi rRNA gene.

Two clearly separated transcription start points (tsp) have been reported within the Trypanosoma cruzi rDNA (DNA encoding rRNA) gene spacer region. These sites are separated by 270 bp, a distance compatible with the occurrence of two core promoters. To characterize the individual participation of these two elements, a deletion analysis was carried out. Different versions of the promoter regions were assayed in a transient transfection analysis of epimastigotes, using the chloramphenicol acetyl transferase gene (cat) as a reporter. The data indicate that the so-called distal tsp-associated region (relative to the small subunit rRNA 5' terminus coding region) comprises most (80%) if not all of the observed activity. In addition, an associated locus specific repeated element showed a modest upregulating activity, since its presence stimulated the cat reporter gene by about 20%. The data here presented should be valuable in the design of expression vectors for T. cruzi, where the rRNA gene promoter has been an important functional element.

α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

Trichocystatin-2 (TC-2): an endogenous inhibitor of cysteine proteinases in Trichomonas vaginalis is associated with TvCP39.

The causal agent of trichomoniasis is a parasitic protist, Trichomonas vaginalis, which is rich in proteolytic activity, primarily carried out by cysteine proteases (CPs). Some CPs are known virulence factors. T. vaginalis also possesses three genes encoding endogenous cystatin-like CP inhibitors. The aim of this study was to identify and characterize one of these CP inhibitors. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), a cystatin-like peptidase inhibitor dubbed Trichocystatin-2 (TC-2) was identified in the T. vaginalis active degradome in association with TvCP39, a 39kDa CP involved in cytotoxicity. To characterize the TC-2 inhibitor, we cloned and expressed the tvicp-2 gene, purified the recombinant protein (TC-2r), and produced a specific polyclonal antibody (α-TC-2r). This antibody recognized a 10kDa protein band by western blotting. An indirect immunofluorescence assay (IFA) and cell fractionation assays using the α-TC-2r antibody showed that TC-2 was localized in the cytoplasm and lysosomes and that it colocalized with TvCP39. TC-2r showed inhibitory activity against papain, cathepsin-L, and TvCP39 in trichomonad extracts and live parasites but not legumain-like CPs. Live trichomonads treated with TC-2r showed reduced trichomonal cytotoxicity to HeLa cell monolayers in a TC-2r-concentration-dependent manner. In this study, we identified and characterized an endogenous cystatin-like inhibitor in T. vaginalis, TC-2, which is associated with TvCP39 and appears to regulate the cellular damage caused by T. vaginalis.

The effects of environmental factors on the virulence of Trichomonas vaginalis.

This review focused on potential regulatory mechanisms of Trichomonas vaginalis virulence properties, cytoadherence, cytotoxicity, phagocytosis, hemolysis, induction of apoptosis, and immune evasion in response to environmental factors of the human urogenital tract, iron, zinc, and polyamines. Understanding the multifactorial nature of trichomonal pathogenesis and its regulation may help to unravel the survival strategies of trichomonads and to implement prevention policies, opportune diagnosis, and alternative treatments for control of trichomoniasis.