RESUMO
Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.
Assuntos
Polarização de Fluorescência , Ensaios de Triagem em Larga Escala , Proteínas de Protozoários , Tripanossomicidas , Trypanosoma cruzi , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Ensaios de Triagem em Larga Escala/métodos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as α-tubulin and ß-tubulin. Also, I immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.
Assuntos
Trypanosoma cruzi , Tubulina (Proteína) , Animais , Estágios do Ciclo de Vida , Microscopia , Isoformas de Proteínas , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Doença de Chagas/parasitologia , Histonas/metabolismo , Mamíferos/metabolismo , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genéticaRESUMO
The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this review article, we present an update of the most important known acetylated proteins in trypanosomatids, analyzing the acetylomes available. Also, we summarize the information available regarding acetyltransferases and deacetylases in trypanosomes and their potential use as chemotherapeutic targets.
Assuntos
Lisina , Processamento de Proteína Pós-Traducional , Trypanosoma , Acetilação , Acetiltransferases/metabolismo , Proteínas/metabolismo , Trypanosoma/efeitos dos fármacosRESUMO
There is an urgent need to develop safer and more effective drugs for Chagas disease, as the current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli ß-galactosidase gene, lacZ, we have adapted and validated an easy, quick and reliable in vitro assay suitable for high-throughput screening for candidate compounds with anti-T. cruzi activity. In vitro studies were conducted to determine trypomastigotes sensitivity to BZ and NFX from Dm28c/pLacZ strain by comparing the conventional labour-intensive microscopy counting method with the colourimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (lytic concentration 50%) were 41.36 and 17.99 µM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 µM, for the colourimetric method, respectively. The optimal conditions for the amastigote development inhibitory assay were established considering the parasite-host relationship (i.e. multiplicity of infection) and interaction time, the time for colourimetric readout and the incubation time with the ß-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition obtained with the colourimetric assay were 2.31 µM for BZ and 0.97 µM for NFX, similar to the reported values for the Dm28c wild strain (2.80 and 1.5 µM, respectively). In summary, a colourimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up, obtaining biologically meaningful sensibility values with the reference compounds on both trypomastigotes and amastigotes forms. This development could be applied to high-throughput screening programmes aiming to identify compounds with anti-T. cruzi in vitro activity.
RESUMO
Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.
Assuntos
Trypanosoma cruzi , Tubulina (Proteína) , Acetilação , Animais , Divisão Celular , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.
Assuntos
Estruturas do Núcleo Celular/ultraestrutura , Proteínas HMGB/metabolismo , Trypanosoma cruzi , Pontos de Checagem do Ciclo Celular , Nucléolo Celular , Citocinese , Proteínas HMGB/farmacologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Trypanosoma cruzi/ultraestrutura , VirulênciaRESUMO
Bromodomains recognize and bind acetyl-lysine residues present in histone and non-histone proteins in a specific manner. In the last decade they have raised as attractive targets for drug discovery because the miss-regulation of human bromodomains was discovered to be involved in the development of a large spectrum of diseases. However, targeting eukaryotic pathogens bromodomains continues to be almost unexplored. We and others have reported the essentiality of diverse bromodomain- containing proteins in protozoa, offering a new opportunity for the development of antiparasitic drugs, especially for Trypansoma cruzi, the causative agent of Chagas' disease. Mammalian bromodomains were classified in eight groups based on sequence similarity but parasitic bromodomains are very divergent proteins and are hard to assign them to any of these groups, suggesting that selective inhibitors can be obtained. In this review, we describe the importance of lysine acetylation and bromodomains in T. cruzi as well as the current knowledge on mammalian bromodomains. Also, we summarize the myriad of small-molecules under study to treat different pathologies and which of them have been tested in trypanosomatids and other protozoa. All the information available led us to propose that T. cruzi bromodomains should be considered as important potential targets and the search for smallmolecules to inhibit them should be empowered.
Assuntos
Doença de Chagas/tratamento farmacológico , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Acetilação , Animais , Linhagem Celular Tumoral , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Lisina/química , Domínios Proteicos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/química , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.
RESUMO
Target-directed dynamic combinatorial chemistry (DCC) has emerged as a strategy for the identification of inhibitors of relevant therapeutic targets. In this contribution, we use this strategy for the identification of a high-affinity binder of a parasite target, the Trypanosoma cruzi bromodomain-containing protein TcBDF3. This protein is essential for viability of T. cruzi, the protozoan parasite that causes Chagas disease. A small dynamic library of acylhydrazones was prepared from aldehydes and acylhydrazides at neutral pH in the presence of aniline. The most amplified library member shows (a) high affinity for the template, (b) interesting antiparasitic activity against different parasite forms, and (c) low toxicity against Vero cells. In addition, parasites are rescued from the compound toxicity by TcBDF3 overexpression, suggesting that the toxicity of this compound is due to the TcBDF3 inhibition, i.e., the binding event that initially drives the molecular amplification is reproduced in the parasite, leading to selective toxicity.
RESUMO
A set of chemically engineered extracts enriched in compounds including N-N and N-O fragments in their structures was prepared. Bromodomain binding screening and bioguided fractionation led to the identification of one oxime hit that interacts with TcBDF3 with affinity in the submicromolar range and that shows interesting antiparasitic properties against the different life cycle stages of T. cruzi.
Assuntos
Antiparasitários/química , Doença de Chagas/tratamento farmacológico , Óleos Voláteis/química , Extratos Vegetais/química , Óleos de Plantas/química , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiparasitários/isolamento & purificação , Antiparasitários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Escherichia coli/genética , Oximas/química , Oximas/farmacologia , Extratos Vegetais/isolamento & purificação , Óleos de Plantas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Células VeroRESUMO
OBJECTIVES: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease. METHODS: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content. The final preparation was subjected to an in vivo palatability assay. Mice were infected and treated orally in a murine model. RESULTS: The results showed that benznidazole solubility increased up to 18.38 mg/ml in the optimised co-solvent system. The final formulation remained stable at all three temperatures tested, with suitable drug content and no significant variability. Palatability of the preparation was improved by taste masking of BZL. In vivo studies showed that both parasitaemia and mortality diminished, particularly at a dose of 40 mg/kg/day. CONCLUSION: Quality by design was a suitable approach to formulate a co-solvent system of benznidazole. The in vivo studies confirmed the suitability of the optimised such solutions to diminish both parasitaemia and mortality. Thus, this novel alternative should be taken into account for further clinical evaluation in all age ranges.
Assuntos
Doença de Chagas/parasitologia , Formas de Dosagem , Nitroimidazóis/administração & dosagem , Solventes , Tripanossomicidas/administração & dosagem , Trypanosoma cruzi , Álcoois , Animais , Doença de Chagas/tratamento farmacológico , Doença de Chagas/mortalidade , Química Farmacêutica , Criança , Chlorocebus aethiops , Contraindicações , Humanos , Camundongos Endogâmicos BALB C , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Polietilenoglicóis , Propilenoglicol , Solubilidade , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Células Vero , ÁguaRESUMO
BACKGROUND: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. PRINCIPAL FINDINGS: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1ß and IFN-γ gene expression. Also, TGF-ß and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. CONCLUSIONS: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.
Assuntos
Doença de Chagas/imunologia , Proteínas HMGB/imunologia , Mediadores da Inflamação/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Animais , Núcleo Celular/metabolismo , Doença de Chagas/genética , Doença de Chagas/parasitologia , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/imunologia , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin. In the present work we show that parasites overexpressing TcBDF3 exhibit altered differentiation patterns and are less susceptible to treatment with bromodomain inhibitors. We also demonstrate that recombinant TcBDF3 is able to bind to these inhibitors in vitro in a concentration-dependant manner. In parallel, the overexpression of a mutated version of TcBDF3 negatively affects growth of epimastigotes. Recent results, including the ones presented here, suggest that bromodomain inhibitors can be conceived as a new type of anti-parasitic drug against trypanosomiasis.
Assuntos
Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/genética , Tripanossomíase/genética , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Antiprotozoários/química , Antiprotozoários/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Humanos , Estágios do Ciclo de Vida/genética , Mutação , Ligação Proteica , Domínios Proteicos/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Tripanossomíase/tratamento farmacológico , Tripanossomíase/parasitologia , Tubulina (Proteína)/genéticaRESUMO
Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication.
Assuntos
Líquido Intracelular/metabolismo , Proteínas de Membrana/biossíntese , Microcorpos/metabolismo , Neuraminidase/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/metabolismo , Animais , Chlorocebus aethiops , Líquido Intracelular/parasitologia , Microcorpos/parasitologia , Células VeroRESUMO
BACKGROUND: Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. METHODOLOGY: Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. CONCLUSION: TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.
Assuntos
Descoberta de Drogas/métodos , Leishmania/crescimento & desenvolvimento , Sirtuínas/antagonistas & inibidores , Sirtuínas/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Acetilação , Animais , Doença de Chagas/tratamento farmacológico , Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida/fisiologia , Proteínas Mitocondriais/metabolismo , Sirtuínas/genéticaRESUMO
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
Assuntos
Expressão Gênica/genética , Vetores Genéticos/genética , Plasmídeos , Mapeamento por Restrição/métodos , Trypanosoma cruzi/genética , Western Blotting , Etiquetas de Sequências Expressas/metabolismo , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida/genética , Mutagênese Insercional , Tetraciclina/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
Assuntos
Expressão Gênica/genética , Vetores Genéticos/genética , Plasmídeos , Mapeamento por Restrição/métodos , Trypanosoma cruzi/genética , Western Blotting , Etiquetas de Sequências Expressas/metabolismo , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida/genética , Mutagênese Insercional , Tetraciclina/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Bromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. The Trypanosoma cruzi genome encodes at least four bromodomain factors (TcBDFs). We describe here bromodomain factor 3 (TcBDF3), a bromodomain-containing protein localized in the cytoplasm. TcBDF3 cytolocalization was determined, using purified antibodies, by Western blot and immunofluorescence analyses in all life cycle stages of T. cruzi. In epimastigotes and amastigotes, it was detected in the cytoplasm, the flagellum, and the flagellar pocket, and in trypomastigotes only in the flagellum. Subcellular localization of TcBDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry, and expression of TcBDF3 fused to cyan fluorescent protein (CFP). Tubulin can acquire different posttranslational modifications, which modulate microtubule functions. Acetylated α-tubulin has been found in the axonemes of flagella and cilia, as well as in the subpellicular microtubules of trypanosomatids. TcBDF3 and acetylated α-tubulin partially colocalized in isolated cytoskeletons and flagella from T. cruzi epimastigotes and trypomastigotes. Interaction between the two proteins was confirmed by coimmunoprecipitation and far-Western blot assays with synthetic acetylated α-tubulin peptides and recombinant TcBDF3.
Assuntos
Flagelos/metabolismo , Estágios do Ciclo de Vida , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/metabolismo , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Citoplasma/metabolismo , Flagelos/ultraestrutura , Microtúbulos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
In the past ten years the number of acetylated proteins reported in literature grew exponentially. Several authors have proposed that acetylation might be a key component in most eukaryotic signaling pathways, as important as phosphorylation. The enzymes involved in this process are starting to emerge; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this paper we overview the most important known acetylated proteins and the advances made in the identification of new acetylated proteins using high-resolution mass spectrometry. Also, we summarize what is known so far about the acetyltransferases and deacetylases in eukaryotes, focusing on trypanosomes and their potential use as chemotherapeutic targets.