RESUMO
Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.
Assuntos
Proteômica , Proteínas de Protozoários , Trypanosoma , Tripanossomíase , Trypanosoma/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/análise , Proteômica/métodos , Animais , Tripanossomíase/diagnóstico , Tripanossomíase/parasitologia , Detergentes/química , Proteínas de Membrana/química , CavalosRESUMO
The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.
Assuntos
Cálcio/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Protozoários/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Trypanosoma/metabolismo , Animais , Compostos de Boro/farmacologia , Quelantes de Cálcio/química , Biologia Computacional/métodos , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Fura-2/química , Expressão Gênica , Homeostase/genética , Hidroquinonas/farmacologia , Proteínas Sensoras de Cálcio Intracelular/genética , Manganês/metabolismo , Proteínas de Protozoários/genética , Tapsigargina/farmacologia , Canais de Potencial de Receptor Transitório/genética , Trypanosoma/efeitos dos fármacos , Trypanosoma/genética , Tripanossomíase/parasitologiaRESUMO
Trypanosoma evansi is the agent of "surra," a trypanosomosis endemic in many areas worldwide. Trypanosoma proteins released/secreted during infection are attractive biomarkers for disease detection and monitoring. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we performed a comprehensive analysis of the serum proteome of mice infected with T.evansi and detected changes in the abundance of parasite and host serum proteins during infection. Following bioinformatics analysis, 30 T. evansi proteins were identified in the mice serum including known targets such as pyruvate kinase 1, ß-tubulin, actin A, heat shock protein 70, and cyclophilin A. We also identified two exclusive VSG epitopes which are novel putative biomarker targets. In addition, upregulation of 31 mouse proteins, including chitinase-like protein 3 and monocyte differentiation antigen CD14, were observed. Identification of parasite-specific biomarkers in the host serum is critical for the development of reliable serological/ assays for differential diagnosis.
Assuntos
Proteínas de Protozoários/sangue , Trypanosoma/metabolismo , Tripanossomíase/sangue , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Biologia Computacional , Epitopos de Linfócito B , Camundongos , Proteômica , Proteínas de Protozoários/metabolismo , Tripanossomíase/parasitologiaRESUMO
Trypanosoma and Leishmania parasites cause devastating tropical diseases resulting in serious global health consequences. These organisms have complex life cycles with mammalian hosts and insect vectors. The parasites must, therefore, survive in different environments, demanding rapid physiological and metabolic changes. These responses depend upon regulation of gene expression, which primarily occurs posttranscriptionally. Altering the composition or conformation of RNA through nucleotide modifications is one posttranscriptional mechanism of regulating RNA fate and function, and modifications including N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytidine (m5C), N4-acetylcytidine (ac4C), and pseudouridine (Ψ), dynamically regulate RNA stability and translation in diverse organisms. Little is known about RNA modifications and their machinery in Trypanosomatids, but we hypothesize that they regulate parasite gene expression and are vital for survival. Here, we identified Trypanosomatid homologs for writers of m1A, m5C, ac4C, and Ψ and analyze their evolutionary relationships. We systematically review the evidence for their functions and assess their potential use as therapeutic targets. This work provides new insights into the roles of these proteins in Trypanosomatid parasite biology and treatment of the diseases they cause and illustrates that Trypanosomatids provide an excellent model system to study RNA modifications, their molecular, cellular, and biological consequences, and their regulation and interplay.
Assuntos
Transcriptoma , Trypanosoma/genética , Tripanossomíase/parasitologia , Animais , Epigenômica , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma/enzimologia , Trypanosoma/metabolismoRESUMO
Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.
Assuntos
Eucariotos/genética , TATA Box , Proteína de Ligação a TATA-Box , Transcrição Gênica , Eucariotos/metabolismo , Genes de Protozoários , Variação Genética , Giardia/genética , Giardia/metabolismo , Leishmania/genética , Leishmania/metabolismo , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismoRESUMO
BACKGROUND: Since their introduction in the virtual screening field, Receiver Operating Characteristic (ROC) curve-derived metrics have been widely used for benchmarking of computational methods and algorithms intended for virtual screening applications. Whereas in classification problems, the ratio between sensitivity and specificity for a given score value is very informative, a practical concern in virtual screening campaigns is to predict the actual probability that a predicted hit will prove truly active when submitted to experimental testing (in other words, the Positive Predictive Value - PPV). Estimation of such probability is however, obstructed due to its dependency on the yield of actives of the screened library, which cannot be known a priori. OBJECTIVE: To explore the use of PPV surfaces derived from simulated ranking experiments (retrospective virtual screening) as a complementary tool to ROC curves, for both benchmarking and optimization of score cutoff values. METHODS: The utility of the proposed approach is assessed in retrospective virtual screening experiments with four datasets used to infer QSAR classifiers: inhibitors of Trypanosoma cruzi trypanothione synthetase; inhibitors of Trypanosoma brucei N-myristoyltransferase; inhibitors of GABA transaminase and anticonvulsant activity in the 6 Hz seizure model. RESULTS: Besides illustrating the utility of PPV surfaces to compare the performance of machine learning models for virtual screening applications and to select an adequate score threshold, our results also suggest that ensemble learning provides models with better predictivity and more robust behavior. CONCLUSION: PPV surfaces are valuable tools to assess virtual screening tools and choose score thresholds to be applied in prospective in silico screens. Ensemble learning approaches seem to consistently lead to improved predictivity and robustness.
Assuntos
Aprendizado de Máquina , Relação Quantitativa Estrutura-Atividade , 4-Aminobutirato Transaminase/antagonistas & inibidores , 4-Aminobutirato Transaminase/metabolismo , Animais , Anticonvulsivantes/química , Anticonvulsivantes/uso terapêutico , Área Sob a Curva , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Curva ROC , Convulsões/tratamento farmacológico , Convulsões/patologia , Trypanosoma/metabolismoRESUMO
Association of the initiation factor eIF4E with the mRNA cap structure is a key step for translation. Trypanosomatids present six eIF4E homologues, showing a low conservation and also differing significantly from the IF4Es of multicellular eukaryotes. On the mRNA side, while in most eukaryotes the mRNA contains cap-0 (7-methyl-GTP), the trypanosomatid mRNA features a cap-4, which is formed by a cap-0, followed by the AACU sequence containing 2'-O-ribose methylations and base methylations on nucleotides 1 and 4. The studies on eIF4E-cap-4 interaction have been hindered by the difficulty to synthesize this rather elaborated cap-4 sequence. To overcome this problem, we applied a liquid-phase oligonucleotide synthesis strategy and describe for the first time the crystal structure of a trypanosomatid eIF4E (T. cruzi EIF4E5) in complex with cap-4. The TcEIF4E5-cap-4 structure allowed a detailed description of the binding mechanism, revealing the interaction mode for the AACU sequence, with the bases packed in a parallel stacking conformation and involved, together with the methyl groups, in hydrophobic contacts with the protein. This binding mechanism evidences a distinct cap interaction mode in comparison with previously described eIF4E structures and may account for the difference of TcEIF4E5-cap-4 dissociation constant in comparison with other eIF4E homologues.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/química , Trypanosoma cruzi/química , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Metilação de DNA , Humanos , Ligantes , Modelos Moleculares , Nucleotídeos/química , Oligonucleotídeos , Ligação Proteica , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/metabolismo , Schistosoma mansoni/metabolismo , Temperatura , Trypanosoma/metabolismoRESUMO
The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.
Assuntos
Nucléolo Celular/metabolismo , Trypanosoma/metabolismo , Animais , Nucléolo Celular/ultraestrutura , Humanos , Nucleotídeos/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Trypanosoma/genética , Trypanosoma/ultraestruturaRESUMO
Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)2). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)2, most of its functions are replaced by T(SH)2 in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)2 instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)2 is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces GSH-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 × 105 m-1 s-1). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)2 (4.8 × 106 m-1 s-1); however, GSH-mediated reduction was extremely slow (39 m-1 s-1). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)2 in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking GSH reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.
Assuntos
Evolução Biológica , Glutarredoxinas/metabolismo , Compostos de Sulfidrila/metabolismo , Trypanosoma/metabolismo , Animais , Domínio Catalítico , Glutarredoxinas/química , Humanos , Cinética , OxirreduçãoRESUMO
Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07-51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22-1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, ß,γ-imidoadenosine 5'-triphosphate and 5'-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.