RESUMO
Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explaining these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.
Assuntos
Dexametasona/farmacologia , Lipopolissacarídeos/imunologia , NF-kappa B/metabolismo , Receptor 4 Toll-Like/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , Animais , Sequência de Bases , Sítios de Ligação/efeitos dos fármacos , Bovinos , Sequência Conservada , Cavalos , Humanos , Receptores de Lipopolissacarídeos/genética , Antígeno 96 de Linfócito/genética , Camundongos , Pan troglodytes , Regiões Promotoras Genéticas , Ratos , SuínosRESUMO
BACKGROUND: Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The Schistosoma mansoni genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the S. mansoni predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets. RESULTS: We have identified 252 ePKs, which corresponds to 1.9% of the S. mansoni predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that S. mansoni has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in S. mansoni or belong to an expanded family in this parasite. Only 16 S. mansoni ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite. CONCLUSIONS: Our approach has improved the functional annotation of 40% of S. mansoni ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of S. mansoni in response to diverse environments during the parasite development, vector interaction, and host infection.
Assuntos
Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Proteômica , Schistosoma mansoni/enzimologia , Schistosoma mansoni/parasitologia , Animais , Domínio Catalítico , Cadeias de Markov , Filogenia , Proteínas Quinases/química , Proteoma/química , Proteoma/classificação , Proteoma/metabolismo , Schistosoma mansoni/citologia , Transdução de SinaisRESUMO
BACKGROUND: Malaria has a devastating impact on worldwide public health in many tropical areas. Studies on vector immunity are important for the overall understanding of the parasite-vector interaction and for the design of novel strategies to control malaria. A member of the fibrinogen-related protein family, fbn9, has been well studied in Anopheles gambiae and has been shown to be an important component of the mosquito immune system. However, little is known about this gene in neotropical anopheline species. METHODS: This article describes the identification and characterization of the fbn9 gene partial sequences from four species of neotropical anopheline primary and secondary vectors: Anopheles darlingi, Anopheles nuneztovari, Anopheles aquasalis, and Anopheles albitarsis (namely Anopheles marajoara). Degenerate primers were designed based on comparative analysis of publicly available Aedes aegypti and An. gambiae gene sequences and used to clone putative homologs in the neotropical species. Sequence comparisons and Bayesian phylogenetic analyses were then performed to better understand the molecular diversity of this gene in evolutionary distant anopheline species, belonging to different subgenera. RESULTS: Comparisons of the fbn9 gene sequences of the neotropical anophelines and their homologs in the An. gambiae complex (Gambiae complex) showed high conservation at the nucleotide and amino acid levels, although some sites show significant differentiation (non-synonymous substitutions). Furthermore, phylogenetic analysis of fbn9 nucleotide sequences showed that neotropical anophelines and African mosquitoes form two well-supported clades, mirroring their separation into two different subgenera. CONCLUSIONS: The present work adds new insights into the conserved role of fbn9 in insect immunity in a broader range of anopheline species and reinforces the possibility of manipulating mosquito immunity to design novel pathogen control strategies.
Assuntos
Anopheles/genética , Fibrinogênio/genética , Sequência de Aminoácidos , Animais , Anopheles/classificação , Anopheles/imunologia , Anopheles/parasitologia , Sequência de Bases , Brasil , Clonagem Molecular , Evolução Molecular , Genes de Insetos , Imunoglobulinas/genética , Insetos Vetores , Malária/parasitologia , Filogenia , Análise de SequênciaRESUMO
Comparative genomics has shown that protein families vary significantly within and across organisms in both number and functional composition. In the present work, we tested how the diversity at the family level reflects biological differences among organisms and contributes to their unique characteristics. For this purpose, we collected sequence-similar proteins of three selected families from model bacteria: Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa. Protein relationships were identified using a phylogenomic approach to connect the functional diversity of enzymes to the metabolic capabilities of these organisms. All protein families studied have distinct functional compositions across the selected bacteria as supported by our Bayesian analysis. Some conserved functional features among family members included a shared reaction mechanism, cofactor usage, and/or ligand specificity. Many observations of the presence/absence of protein functions matched current knowledge of the physiology and biochemistry of the bacteria. In some cases, new functional predictions were made to family members previously uncharacterized. We believe that genome comparisons at the protein family level would also be useful in predicting metabolic diversity for organisms that are relatively unknown or currently uncultured in the laboratory.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Pseudomonas aeruginosa/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Escherichia coli/genética , Genômica/métodos , Filogenia , Pseudomonas aeruginosa/genética , Fosfato de Piridoxal/metabolismo , Piruvato Descarboxilase/classificação , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Especificidade da Espécie , Tiamina Pirofosfato , Transaminases/classificação , Transaminases/genética , Transaminases/metabolismoRESUMO
Helminths include free-living and parasitic Platyhelminthes and Nematoda which infect millions of people worldwide. Some Platyhelminthes species of blood flukes (Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni) and liver flukes (Clonorchis sinensis and Opisthorchis viverrini) are known to be involved in human cancers. Other helminths are likely to be carcinogenic. Our main goals are to summarize the current knowledge of human cancers caused by Platyhelminthes, point out some helminth and human biomarkers identified so far, and highlight the potential contributions of phylogenetics and molecular evolution to cancer research. Human cancers caused by helminth infection include cholangiocarcinoma, colorectal hepatocellular carcinoma, squamous cell carcinoma, and urinary bladder cancer. Chronic inflammation is proposed as a common pathway for cancer initiation and development. Furthermore, different bacteria present in gastric, colorectal, and urogenital microbiomes might be responsible for enlarging inflammatory and fibrotic responses in cancers. Studies have suggested that different biomarkers are involved in helminth infection and human cancer development; although, the detailed mechanisms remain under debate. Different helminth proteins have been studied by different approaches. However, their evolutionary relationships remain unsolved. Here, we illustrate the strengths of homology identification and function prediction of uncharacterized proteins from genome sequencing projects based on an evolutionary framework. Together, these approaches may help identifying new biomarkers for disease diagnostics and intervention measures. This work has potential applications in the field of phylomedicine (evolutionary medicine) and may contribute to parasite and cancer research.
RESUMO
The availability of the genomic data of diverse parasites provides an opportunity to identify new drug candidates against neglected tropical diseases affecting people worldwide. Histone modifying enzymes (HMEs) are potential candidates since they play key roles in the regulation of chromatin modifications, thus globally regulating gene expression. Furthermore, aberrant epigenetic states are often associated with human diseases, leading to great interest in HMEs as therapeutic targets. Our work focused on two families of protein lysine deacetylases (HDACs and sirtuins). First, we identified potential homologues in the predicted proteomes of selected taxa by using hidden Markov model profiles. Then, we reconstructed the evolutionary relationships of protein sequences by Bayesian inference and maximum likelihood method. In addition, we constructed homology models for five parasite HDACs to provide information for experimental validation and structure-based optimization of inhibitors. Our results showed that parasite genomes code for diverse HDACs and sirtuins. The evolutionary pattern of protein deacetylases with additional experimental data points to these enzymes as common drug targets among parasites. This work has improved the functional annotation of approximately 63% HDACs and 51% sirtuins in the selected taxa providing insights for experimental design. Homology models pointed out structural conservation and differences among parasite and human homologues and highlight potential candidates for further inhibitor development. Some of these parasite proteins are undergoing RNA interference or knockout analyses to validate the function of their corresponding genes. In the future, we will investigate the main functions performed by these proteins, related phenotypes, and their potential as therapeutic targets.
Assuntos
Anti-Helmínticos/química , Antiprotozoários/química , Genoma , Proteínas de Helminto/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Proteínas de Protozoários/química , Animais , Anti-Helmínticos/farmacologia , Antiprotozoários/farmacologia , Bases de Dados Genéticas , Epigênese Genética , Evolução Molecular , Expressão Gênica , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Helmintíase/tratamento farmacológico , Helmintíase/parasitologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Leishmania/efeitos dos fármacos , Leishmania/enzimologia , Leishmania/genética , Simulação de Acoplamento Molecular , Doenças Negligenciadas , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Conformação Proteica , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/parasitologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Schistosoma/efeitos dos fármacos , Schistosoma/enzimologia , Schistosoma/genética , Homologia Estrutural de Proteína , Trypanosoma/efeitos dos fármacos , Trypanosoma/enzimologia , Trypanosoma/genéticaRESUMO
BACKGROUND: Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; http://egenbio.lsu.edu) to begin to address this. DESCRIPTION: EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. CONCLUSION: EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity.
Assuntos
Biodiversidade , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Evolução Molecular , Animais , Biologia Computacional , Genômica , Humanos , Internet , Análise de Sequência de DNA , Design de SoftwareRESUMO
The cystatin family comprises cysteine protease inhibitors distributed in 3 subfamilies (I25A-C). Family members lacking cystatin activity are currently unclassified. Little is known about the evolution of Schistosoma cystatins, their physiological roles, and expression patterns in the parasite life cycle. The present study aimed to identify cystatin homologs in the predicted proteome of three Schistosoma species and other Platyhelminthes. We analyzed the amino acid sequence diversity focused in the identification of protein signatures and to establish evolutionary relationships among Schistosoma and experimentally validated human cystatins. Gene expression patterns were obtained from different developmental stages in Schistosoma mansoni using microarray data. In Schistosoma, only I25A and I25B proteins were identified, reflecting little functional diversification. I25C and unclassified subfamily members were not identified in platyhelminth species here analyzed. The resulting phylogeny placed cystatins in different clades, reflecting their molecular diversity. Our findings suggest that Schistosoma cystatins are very divergent from their human homologs, especially regarding the I25B subfamily. Schistosoma cystatins also differ significantly from other platyhelminth homologs. Finally, transcriptome data publicly available indicated that I25A and I25B genes are constitutively expressed thus could be essential for schistosome life cycle progression. In summary, this study provides insights into the evolution, classification, and functional diversification of cystatins in Schistosoma and other Platyhelminthes, improving our understanding of parasite biology and opening new frontiers in the identification of novel therapeutic targets against helminthiases.
RESUMO
Schistosomes are digenean blood flukes of aves and mammals comprising 23 species. Some species are causative agents of human schistosomiasis, the second major neglected disease affecting over 230 million people worldwide. Modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large-scale analyses of parasites and hosts, opening new frontiers in biological research with potential biomedical and biotechnological applications. Nuclear genomes of the three most socioeconomically important species (S. haematobium, S. japonicum, and S. mansoni) have been sequenced and are under intense investigation. Mitochondrial genomes of six Schistosoma species have also been completely sequenced and analysed from an evolutionary perspective. Furthermore, DNA barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes. Despite the efforts in the characterization of Schistosoma genomes and transcriptomes, many questions regarding the biology and evolution of this important taxon remain unanswered. This paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics. It also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies.
RESUMO
Schistosoma mansoni, one of the causative agents of schistosomiasis, has a complex life cycle infecting over 200 million people worldwide. Such a successful and prolific parasite life cycle has been shown to be dependent on the adaptive interaction between the parasite and hosts. Tyrosine kinases (TKs) play a key role in signaling pathways as demonstrated by a large body of experimental work in eukaryotes. Furthermore, comparative genomics have allowed the identification of TK homologs and provided insights into the functional role of TKs in several biological systems. Finally, TK structural biology has provided a rational basis for obtaining selective inhibitors directed to the treatment of human diseases. This paper covers the important aspects of the phospho-tyrosine signaling network in S. mansoni, Caenorhabditis elegans, and humans, the main process of functional diversification of TKs, that is, protein-domain shuffling, and also discusses TKs as targets for the development of new anti-schistosome drugs.