RESUMO
Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.
Assuntos
Entamoeba histolytica , Parasitos , Animais , Humanos , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Entamoeba histolytica/genética , Parasitos/metabolismo , Filogenia , Genômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Human alpha-L-iduronidase (IDUA) is a 653 amino acid protein involved in the sequential degradation of glycos-amino-glycans (GAG), heparan sulfate (HS), and dermatan sulfate (DS). Some variants in the IDUA gene produce a deficient enzyme that causes un-degraded DS and HS to accumulate in multiple tissues, leading to an organ dysfunction known as muco-poly-saccharidosis type I (MPS I). Molecular and catalytic activity assays of new or rare variants of IDUA do not predict the phenotype that a patient will develop. Therefore, it is of interest to describe the molecular docking analysis, to locate binding regions of DS to IDUA to better understand the effect of a variant on MPS I development. The results presented herein demonstrate the presence of a polar/acidic catalytic site and a basic region in the putative binding site of DS to IDUA. Further, synthetic substrate docking with the enzyme could help in the predictions of the MPS I phenotype.
RESUMO
The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.
Assuntos
Entamoeba histolytica , Parasitos , Animais , Entamoeba histolytica/genética , Resposta ao Choque Térmico , Simulação de Acoplamento Molecular , Fatores de TranscriçãoRESUMO
Hypertension (HTN) causes end-organ damage and is a major cause of morbidity and mortality globally. Recent studies suggested blood cells participate in the maintenance of HTN. Platelets-anucleated cell fragments derived from megakaryocytes-exert diverse functions, including their well-characterized role in the formation of hemostatic clots. However, platelets from patients with HTN exhibit altered membrane lipid and protein compositions that impact platelet function and lead to formation of aggregates and vascular obstructions. Here, for the first time, we have identified, by proteomic analyses, the most relevant 11 proteins that show the greatest difference in their expression in platelets derived from patients with HTN, in comparison with those from normotensive individuals. These proteins are involved in cytoskeletal organization and the coagulation cascade that contributes to platelet activation, release of granule contents, and aggregation, which culminate in thrombus formation. These results have important implications in our understanding of the molecular mechanisms associated with the development of HTN, and in consequence, the development of new strategies to counteract the cardiovascular disorders associated with constitutive activation of platelets in HTN.
Assuntos
Hipertensão , Trombose , Plaquetas , Humanos , Hipertensão/metabolismo , Megacariócitos/metabolismo , Ativação Plaquetária , Proteômica , Trombose/metabolismoRESUMO
Pomegranate juice (Punica granatum) has been used since ancient times in traditional medicine (Unani Medicine, Ayurveda); its main compounds are anthocyanins and ellagic acid, which have anti-inflammatory, antioxidant, hepatoprotective, and cardiovascular health effects. The objective was to evaluate the effect of pomegranate juice on inflammation, blood pressure, and vascular and physiological markers associated with obesity induced by a high-fat diet in a murine model. The results show that pomegranate juice reduces the concentration of low-density lipoprotein cholesterol (cLDL) 39% and increases the concentration of high-density lipoprotein cholesterol (cHDL) by 27%, leading to a 12%-18% decrease in the risk of cardiovascular diseases (CVD). In addition to reducing blood pressure by 24%, it also had an antiatherogenic effect by decreasing sE-selectin levels by 42%. On the other hand, the juice significantly increased adiponectin levels in adipose tissue, decreased levels of inflammation markers (tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor-1 (PAI-1), interleukin-17A (IL-17A), interleukin-6 (IL-6), interleukin-1ß (IL-1ß)), and inhibited the monocyte chemoattractant protein-1 (MCP-1). Pomegranate juice requires clinical studies to prove its immunoregulatory and therapeutic effects on cardiovascular and atherogenic risks.
Assuntos
Tecido Adiposo/metabolismo , Doenças Cardiovasculares/prevenção & controle , Sucos de Frutas e Vegetais , Fatores de Risco de Doenças Cardíacas , Inflamação , Obesidade/fisiopatologia , Punica granatum , Adiponectina/metabolismo , Tecido Adiposo/imunologia , Animais , Biomarcadores/análise , Pressão Sanguínea , Doenças Cardiovasculares/etiologia , Quimiocina CCL2/antagonistas & inibidores , Citocinas/metabolismo , Dieta Hiperlipídica , Ingestão de Energia , Sucos de Frutas e Vegetais/análise , Lipídeos/sangue , Masculino , Obesidade/complicações , Ratos , Ratos WistarRESUMO
Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 µmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.
Assuntos
Anormalidades Múltiplas/genética , Dermatan Sulfato/química , Heparitina Sulfato/química , Iduronidase/química , Mucopolissacaridose I/genética , Mutação Puntual , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/terapia , Domínio Catalítico , Cristalografia por Raios X , Dermatan Sulfato/metabolismo , Terapia de Reposição de Enzimas/métodos , Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Lactente , Masculino , Simulação de Dinâmica Molecular , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
The infection of Capsicum annuum cv. mirasol by Candidatus Phytoplasma trifolii (16SrVI) causes devastating crop losses in northern Mexico. This study addresses the metabolomics profiling of mirasol chili peppers (Capsicum annuum cv. mirasol) infected by Candidatus Phytoplasma trifolii. For this study, 25 diseased fruits and 25 healthy fruits were used. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) revealed dramatic changes in the content of 42 metabolites which were identified in diseased and healthy mirasol chili peppers. The endogenous levels of fructose, glucose and formic acid were substantially decreased in the diseased chili peppers. In the same group of samples, high concentrations of alanine, asparagine, fumaric acid, sucrose and threonine were observed. The content of Choline didnt present a significant difference. This evidence supports the fact that Candidatus Phytoplasma trifolii infection reduces de CO2 fixation into carbohydrates, decreases invertase activity, and inhibits glycolysis in the diseased plant tissues. The levels of ascorbic acid, capsaicin and dihydrocapsaicinin in diseased fruits were dramatically decreased, suggesting that Candidatus Phytoplasma trifolii can reduce the pungency and the nutraceutical value of mirasol chili peppers.
Assuntos
Capsicum/metabolismo , Espectroscopia de Ressonância Magnética , Metaboloma , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Capsicum/microbiologia , Regulação da Expressão Gênica de Plantas , Análise de Componente PrincipalRESUMO
Computational identification of potential epitopes with an immunogenic capacity challenges immunological research. Several methods show considerable success, and together with experimental studies, the efficiency of the algorithms to identify potential peptides with biological activity has improved. Herein, an epitope was designed by combining bioinformatics, docking, and molecular dynamics simulations. The hemagglutinin protein of the H1N1 influenza pandemic strain served as a template, owing to the interest of obtaining a scheme of immunization. Afterward, we performed enzyme-linked immunosorbent assay (ELISA) using the epitope to analyze if any antibodies in human sera before and after the influenza outbreak in 2009 recognize this peptide. Also, a plaque reduction neutralization test induced by virus-neutralizing antibodies and the IgG determination showed the biological activity of this computationally designed peptide. The results of the ELISAs demonstrated that the serum of both prepandemic and pandemic recognized the epitope. Moreover, the plaque reduction neutralization test evidenced the capacity of the designed peptide to neutralize influenza virus in Madin-Darby canine cells.
Assuntos
Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Influenza Humana/metabolismo , Peptídeos/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular , Biologia Computacional , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/imunologia , Simulação de Dinâmica Molecular , Peptídeos/química , CoelhosRESUMO
All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132-138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.
Assuntos
Cisteína/genética , Mutação/genética , Saccharomyces cerevisiae/enzimologia , Serina/genética , Triose-Fosfato Isomerase/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Saccharomyces cerevisiae/genética , Serina/química , Serina/metabolismo , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
Enolase is a multifunctional protein that participates in glycolysis and gluconeogenesis and can act as a plasminogen receptor on the cell surface of several organisms, among other functions. Despite its participation in a variety of biological and pathophysiological processes, its stability and folding/unfolding reaction have not been fully explored. In this paper we present, the urea and GdnHCl-induced denaturation of enolase studied by means of fluorescence and circular dichroism spectroscopies. We found that enolase unfolds through a highly reversible pathway, populating a stable intermediate species in a range of experimental conditions. The refolding reaction also exhibits an intermediate state that might have a slightly more compact conformation compared to the unfolding intermediate. The thermodynamic parameters associated with the unfolding reaction are presented and discussed.