RESUMEN
Introduction: The protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice. Methods: To further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy. Results: After a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes. Discussion: The rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.
RESUMEN
In many eukaryotes, multiple protein kinases are situated in the plasma membrane where they respond to extracellular ligands. Ligand binding elicits a signal that is transmitted across the membrane, leading to activation of the cytosolic kinase domain. Humans have over 100 receptor protein kinases. In contrast, our search of the Trypanosoma brucei kinome showed that there were only ten protein kinases with predicted transmembrane domains, and unlike other eukaryotic transmembrane kinases, seven are predicted to bear multiple transmembrane domains. Most of the ten kinases, including their transmembrane domains, are conserved in both Trypanosoma cruzi and Leishmania species. Several possess accessory domains, such as Kelch, nucleotide cyclase, and forkhead-associated domains. Surprisingly, two contain multiple regions with predicted structural similarity to domains in bacterial signaling proteins. A few of the protein kinases have previously been localized to subcellular structures such as endosomes or lipid bodies. We examined the localization of epitope-tagged versions of seven of the predicted transmembrane kinases in T. brucei bloodstream forms and show that five localized to the endoplasmic reticulum. The last two kinases are enzymatically active, integral membrane proteins associated with the flagellum, flagellar pocket, or adjacent structures as shown by both fluorescence and immunoelectron microscopy. Thus, these kinases are positioned in structures suggesting participation in signal transduction from the external environment.
Asunto(s)
Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Trypanosoma brucei brucei/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Microscopía Inmunoelectrónica , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Proteínas Quinasas/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on posttranscriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (ß-d-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP) tagging and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in the regulation of transcription in other eukaryotes. The downregulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional readthrough at the 3' end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt the progression of the RNA polymerase. This decoupling of transcription termination from the splicing of individual genes enables the parasites' unique reliance on polycistronic transcription and posttranscriptional regulation of gene expression.IMPORTANCELeishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent of those involved in the control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.
Asunto(s)
Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Leishmania/genética , Proteínas Protozoarias/genética , Terminación de la Transcripción Genética , Cromatina/metabolismo , ADN Protozoario/metabolismo , Regulación de la Expresión Génica , ARN MensajeroRESUMEN
Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy.
Asunto(s)
Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Protozoarias/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Toxoplasmosis Animal/tratamiento farmacológico , Toxoplasmosis Cerebral/tratamiento farmacológico , Administración Oral , Animales , Área Bajo la Curva , Femenino , Técnicas In Vitro , Ratones , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/sangre , Pirazoles/farmacología , Pirimidinas/sangre , Pirimidinas/farmacologíaRESUMEN
In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC50s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.
Asunto(s)
Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Naftalenos/farmacología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Toxoplasma/efectos de los fármacos , Toxoplasmosis/tratamiento farmacológico , Animales , Resistencia a Medicamentos/genética , Quinasa 2 de Adhesión Focal/genética , Humanos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.
Asunto(s)
Apicoplastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Biogénesis de Organelos , Tiorredoxinas/metabolismo , Toxoplasma/fisiología , Secuencia de Aminoácidos , Biomarcadores/metabolismo , Secuencia Conservada , Evolución Molecular , Técnicas de Silenciamiento del Gen , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tiorredoxinas/química , Tiorredoxinas/genética , Toxoplasma/citología , Toxoplasma/crecimiento & desarrolloRESUMEN
Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.
Asunto(s)
Antiprotozoarios/farmacología , Apicomplexa/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Infecciones por Protozoos/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Apicomplexa/enzimología , Bencimidazoles/química , Humanos , Imidazoles/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Infecciones por Protozoos/prevención & control , Piridinas/químicaRESUMEN
In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite's second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion. While inducible knockdown of NDH2 had a modest growth effect in bloodstream forms, NDH2 null mutants, as well as inducible knockdowns in a complex I deficient background, showed a greater reduction in growth. Altering the NAD+/NADH balance would affect numerous processes directly and indirectly, including acetate production. Indeed, loss of NDH2 led to reduced levels of acetate, which is required for several essential pathways in bloodstream form T. brucei and which may have contributed to the observed growth defect. In conclusion our study shows that NDH2 is important, but not essential, in proliferating bloodstream forms of T. brucei, arguing that the mitochondrial NAD+/NADH balance is important in this stage, even though the mitochondrion itself is not actively engaged in the generation of ATP.
Asunto(s)
Acetatos/metabolismo , NADH Deshidrogenasa/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismo , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Estadios del Ciclo de Vida , Mitocondrias/metabolismo , Mutación , NADH Deshidrogenasa/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Trypanosoma brucei brucei/genéticaRESUMEN
Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5µM range but interfere with intracellular division at 2.5µM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis.
Asunto(s)
Encefalomielitis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/efectos de los fármacos , Sarcocystis/enzimología , Sarcocistosis/tratamiento farmacológico , Animales , Línea Celular , Chlorocebus aethiops , Encefalomielitis/parasitología , Femenino , Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/parasitología , Caballos , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Sarcocystis/efectos de los fármacos , Temperatura , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimologíaRESUMEN
New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses antitoxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human ether-a-go-go-related gene (hERG) inhibitory activity, associated with long Q-T syndrome, and consequently presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These properties make 32 a promising lead for the development of a new antitoxoplasmosis therapy.
Asunto(s)
Antiprotozoarios/farmacología , Sistema Nervioso Central/efectos de los fármacos , Diseño de Fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Toxoplasma/efectos de los fármacos , Toxoplasmosis/tratamiento farmacológico , Administración Oral , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/química , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Haplorrinos , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Toxoplasma/enzimología , Toxoplasmosis/metabolismoRESUMEN
While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low-abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites including trypanosomes and Plasmodium. It has been used to identify new protein-coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation.
Asunto(s)
Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/genética , Ribosomas/genética , Animales , Perfilación de la Expresión Génica , Orden Génico , Genoma de Protozoos/genética , Humanos , Estadios del Ciclo de Vida/genética , ProteómicaRESUMEN
BACKGROUND: Trypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model. RESULTS: We refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated. CONCLUSIONS: Levels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism.
Asunto(s)
Estabilidad del ARN , ARN Mensajero/genética , ARN Protozoario/genética , Proteínas Ribosómicas/metabolismo , Trypanosoma brucei brucei/genética , Semivida , Modelos Genéticos , Proteínas Protozoarias/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Transcripción Genética , TranscriptomaRESUMEN
Human African trypanosomiasis a fatal disease for which no vaccines exist and treatment regimens are difficult. Here, we evaluate a Trypanosoma brucei protein kinase, AEK1, as a potential drug target. Conditional knockouts confirmed AEK1 essentiality in bloodstream forms. For chemical validation, we overcame the lack of AEK1 inhibitors by creating parasites expressing a single, functional analog-sensitive AEK1 allele. Analog treatment of mice infected with this strain delayed parasitemia and death, with one-third of animals showing no parasitemia. These studies validate AEK1 as a drug target and highlight the need for further understanding of its function.
Asunto(s)
Parasitemia/parasitología , Proteínas Quinasas/metabolismo , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/parasitología , Adenosina Trifosfato/metabolismo , Alelos , Animales , Técnicas de Inactivación de Genes , Humanos , Ratones , Parasitemia/sangre , Parasitemia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/genética , Tripanocidas/administración & dosificación , Tripanocidas/efectos adversos , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/sangre , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure-activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.
RESUMEN
Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we make use of existing Trypanosoma brucei ribosome profiling data to provide evidence of ribosome occupancy (and likely translation) of mRNAs from 225 currently unannotated coding sequences (CDSs). A small number of these putative genes correspond to extra copies of previously annotated genes, but 85% are novel. The median size of these novels CDSs is small (81 aa), indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs confirmed here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as protein-coding genes. Nonetheless, approximately one-third of the new CDSs were found only in T. brucei subspecies. Using ribosome footprints, RNA-Seq and spliced leader mapping data, we updated previous work to definitively revise the start sites for 414 CDSs as compared to the current gene models. The data pointed to several regions of the genome that had sequence errors that altered coding region boundaries. Finally, we consolidated this data with our previous work to propose elimination of 683 putative genes as protein-coding and arrive at a view of the translatome of slender bloodstream and procyclic culture form T. brucei.
Asunto(s)
Codón Iniciador/genética , Genes Protozoarios , ARN Lider Empalmado/genética , Ribosomas/metabolismo , Trypanosoma brucei brucei/genética , Evolución Molecular , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ARNRESUMEN
Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.
Asunto(s)
Apicoplastos/metabolismo , Aparato de Golgi/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Vesículas Transportadoras/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Transporte de ProteínasRESUMEN
BACKGROUND: Trypanosoma brucei subspecies infect humans and animals in sub-Saharan Africa. This early diverging eukaryote shows many novel features in basic biological processes, including the use of polycistronic transcription to generate all protein-coding mRNAs. Therefore we hypothesized that translational control provides a means to tune gene expression during parasite development in mammalian and fly hosts. RESULTS: We used ribosome profiling to examine genome-wide protein synthesis in animal-derived slender bloodstream forms and cultured procyclic (insect midgut) forms. About one-third of all CDSs showed statistically significant regulation of protein production between the two stages. Of these, more than two-thirds showed a change in translation efficiency, but few appeared to be controlled by this alone. Ribosomal proteins were translated poorly, especially in animal-derived parasites. A disproportionate number of metabolic enzymes were up-regulated at the mRNA level in procyclic forms, as were variant surface glycoproteins in bloodstream forms. Comparison with cultured bloodstream forms from another strain revealed stage-specific changes in gene expression that transcend strain and growth conditions. Genes with upstream ORFs had lower mean translation efficiency, but no evidence was found for involvement of uORFs in stage-regulation. CONCLUSIONS: Ribosome profiling revealed that differences in the production of specific proteins in T. brucei bloodstream and procyclic forms are more extensive than predicted by analysis of mRNA abundance. While in vivo and in vitro derived bloodstream forms from different strains are more similar to one another than to procyclic forms, they showed many differences at both the mRNA and protein production level.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Biosíntesis de Proteínas , Proteínas Protozoarias/genética , Proteínas Ribosómicas/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Animales , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/genética , ARN Protozoario/genética , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.
RESUMEN
Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and ß) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.