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A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

ABSTRACT: The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5'-triphosphate, transfer of guanosine through a 5'-5' triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2'-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m(7)GpppAmpUp or m(7)GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.

SUBMITTER: Simoes-Barbosa A

PROVIDER: S-EPMC2904801 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

Simoes-Barbosa Augusto A Hirt Robert P RP Johnson Patricia J PJ

PLoS pathogens 20100715 7

The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5'-triphosphate, transfer of guanosine through a 5'-5' triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2'-O-ribose methylated in met ...[more]

PMID: 20664792

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Project description:Cytoadherence and migration are crucial for pathogens to establish colonization in the host. In contrast to a nonadherent isolate of Trichomonas vaginalis, an adherent one expresses more actin-related machinery proteins with more active flagellate-amoeboid morphogenesis, amoeba migration, and cytoadherence, activities that were abrogated by an actin assembly blocker. By immunoprecipitation coupled with label-free quantitative proteomics, an F-actin capping protein (T. vaginalis F-actin capping protein subunit α [TvFACPα]) was identified from the actin-centric interactome. His-TvFACPα was detected at the barbed end of a growing F-actin filament, which inhibited elongation and possessed atypical activity in binding G-actin in in vitro assays. TvFACPα partially colocalized with F-actin at the parasite pseudopod protrusion and formed a protein complex with α-actin through its C-terminal domain. Meanwhile, TvFACPα overexpression suppressed F-actin polymerization, amoeboid morphogenesis, and cytoadherence in this parasite. Ser2 phosphorylation of TvFACPα enriched in the amoeboid stage of adhered trophozoites was reduced by a casein kinase II (CKII) inhibitor. Site-directed mutagenesis and CKII inhibitor treatment revealed that Ser2 phosphorylation acts as a switching signal to alter TvFACPα actin-binding activity and the consequent actin cytoskeleton behaviors. Through CKII signaling, TvFACPα also controls the conversion of adherent trophozoites from amoeboid migration to the flagellate form with axonemal motility. Together, CKII-dependent Ser2 phosphorylation regulates TvFACPα binding to actin to fine-tune cytoskeleton dynamics and drive crucial behaviors underlying host colonization by T. vaginalis. IMPORTANCE Trichomoniasis is one of the most prevalent nonviral sexually transmitted diseases. T. vaginalis cytoadherence to urogenital epithelium cells is the first step in the colonization of the host. However, studies on the mechanisms of cytoadherence have focused mainly on the role of adhesion molecules, and their effects are limited when analyzed by loss- or gain-of-function assays. This study proposes an extra pathway in which the actin cytoskeleton mediated by a capping protein α-subunit may play roles in parasite morphogenesis, cytoadherence, and motility, which are crucial for colonization. Once the origin of the cytoskeleton dynamics could be manipulated, the consequent activities would be controlled as well. This mechanism may provide new potential therapeutic targets to impair this parasite infection and relieve the increasing impact of drug resistance on clinical and public health.

Project description:Mitochondrial processing peptidases are heterodimeric enzymes (alpha/betaMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an alpha/beta heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single betaMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas alphaMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric alpha/betaMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.

Project description:Backgroundγ-Carboxymuconolactone decarboxylase (CMD) participates in the β-ketoadipate pathway, which catalyzes aromatic compounds to produce acetyl- or succinyl-CoA, in prokaryotes and yeast. Our previous study demonstrated that expression of a CMD homologue that contains two signatures (dualCMD) is negatively regulated by iron in Trichomonas vaginalis. However, we were not able to identify the components of the β-ketoadipate pathway in the parasite's genome. These observations prompted us to investigate the biological functions of this novel CMD homologue in T. vaginalis.MethodsThe specific anti-TvCMD1 antibody was generated, and the expression of TvCMD1 in T. vaginalis cultured under iron-rich and iron-deficient were evaluated. Phylogenetic, metabolomic and substrate induction (protocatechuate and benzoate) analysis were conducted to clarify the function of dualCMD in trichomonad cells. Subcellular localization of TvCMD1 was observed by confocal microscopy. The cell cycle-related role of TvCMD1 was assessed by treating cells with G2/M inhibitor nocodazole.ResultsWe confirmed that T. vaginalis is not able to catabolize the aromatic compounds benzoate and protocatechuate, which are known substrates of the β-ketoadipate pathway. Using immunofluorescence microscopy, we found that TvCMD1 is spatially associated with the basal body, a part of the cytoskeletal organizing center in T. vaginalis. TvCMD1 accumulated upon treatment with the G2/M inhibitor nocodazole. Additionally, TvCMD1 was expressed and transported to/from the basal body during cytokinesis, suggesting that TvCMD1 plays a role in cell division.ConclusionWe demonstrated that TvCMD1 is unlikely to participate in the β-ketoadipate pathway and demonstrated that it is a novel basal body-localizing (associated) protein. This model sheds light on the importance of genes that are acquired laterally in the coevolution of ancient protists, which surprisingly functions in cell cycle regulation of T. vaginalis.

Dataset Information

A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

Publications

A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

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