RESUMO
Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.
Assuntos
Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Ureia/metabolismo , Ureia/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doença de Chagas/microbiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fase G1/efeitos dos fármacos , Heme/metabolismo , Humanos , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Testes de Sensibilidade Parasitária , Fosforilação , Ratos , Ureia/análogos & derivados , eIF-2 Quinase/metabolismoRESUMO
Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Proteínas dos Microfilamentos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/parasitologia , Citoplasma/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.
Assuntos
Cromatina/química , Código das Histonas , Estágios do Ciclo de Vida , Proteômica/métodos , Acetilação , Ciclo Celular , Regulação da Expressão Gênica , Metilação , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Trypanosoma cruziRESUMO
The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating 'slender' bloodstream forms differentiate to non-dividing 'stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/parasitologia , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/química , Camundongos , Mutação , Iniciação Traducional da Cadeia Peptídica , Fosforilação , Proteínas de Protozoários/química , Treonina/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologiaRESUMO
Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of cardiomyopathy in Latin America. It is estimated that 10%-30% of all infected individuals will acquire chronic chagasic cardiomyopathy (CCC). The etiology of CCC is multifactorial and involves parasite genotype, host genetic polymorphisms, immune response, signaling pathways and autoimmune progression. Herein we verified the impact of the recombinant form of P21 (rP21), a secreted T. cruzi protein involved in host cell invasion, on progression of inflammatory process in a polyester sponge-induced inflammation model. Results indicated that rP21 can recruit immune cells induce myeloperoxidase and IL-4 production and decrease blood vessels formation compared to controls in vitro and in vivo. In conclusion, T. cruzi P21 may be a potential target for the development of P21 antagonist compounds to treat chagasic cardiomyopathy.
Assuntos
Cardiomiopatias/etiologia , Doença de Chagas/patologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma cruzi/metabolismo , Animais , Cardiomiopatias/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Quimiotaxia/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/prevenção & controle , Interleucina-4/metabolismo , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Peroxidase/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D (1)H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.
Assuntos
Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma cruzi , Animais , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Redobramento de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solubilidade , Trypanosoma cruzi/genética , Células VeroRESUMO
The invasion of host cells by the intracellular protozoan Trypanosoma cruzi requires interactions with host cell molecules, and the replication of the parasite requires escape from a parasitophorous vacuole into the host cell cytosol. Galectin-3, a member of ß-galactosidase-binding lectin family, has numerous extracellular and intracellular functions. In this study, we investigated the role of galectin-3 during the invasion and intracellular trafficking of T. cruzi extracellular amastigotes (EAs). Endogenous galectin-3 from mouse peritoneal macrophages accumulated around the pathogen during cell invasion by EAs. In addition, galectin-3 accumulated around parasites after their escape from the parasitophorous vacuole. Thus, galectin-3 behaved as a novel marker of phagolysosome lysis during the infection of host cells by T. cruzi.