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1.
Genes Dev ; 33(21-22): 1591-1612, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31601616

RESUMO

Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. Here, we developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. We observe extensive genome shuffling between these species with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.

2.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
3.
Methods Mol Biol ; 1969: 181-203, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877678

RESUMO

Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/normas , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/imunologia , Controle de Qualidade , Humanos , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Polissacarídeos Bacterianos/classificação
4.
Genome Biol ; 19(1): 150, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266094

RESUMO

BACKGROUND: Partially methylated domains are extended regions in the genome exhibiting a reduced average DNA methylation level. They cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. We present a comprehensive comparative analysis of partially methylated domains in human and mouse cells, to identify structural and functional features associated with them. RESULTS: Partially methylated domains are present in up to 75% of the genome in human and mouse cells irrespective of their tissue or cell origin. Each cell type has a distinct set of partially methylated domains, and genes expressed in such domains show a strong cell type effect. The methylation level varies between cell types with a more pronounced effect in differentiating and replicating cells. The lowest level of methylation is observed in highly proliferating and immortal cancer cell lines. A decrease of DNA methylation within partially methylated domains tends to be linked to an increase in heterochromatic histone marks and a decrease of gene expression. Characteristic combinations of heterochromatic signatures in partially methylated domains are linked to domains of early and middle S-phase and late S-G2 phases of DNA replication. CONCLUSIONS: Partially methylated domains are prominent signatures of long-range epigenomic organization. Integrative analysis identifies them as important general, lineage- and cell type-specific topological features. Changes in partially methylated domains are hallmarks of cell differentiation, with decreased methylation levels and increased heterochromatic marks being linked to enhanced cell proliferation. In combination with broad histone marks, partially methylated domains demarcate distinct domains of late DNA replication.

5.
Sci Rep ; 8(1): 10872, 2018 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-30022098

RESUMO

The biological interpretation of gene lists with interesting shared properties, such as up- or down-regulation in a particular experiment, is typically accomplished using gene ontology enrichment analysis tools. Given a list of genes, a gene ontology (GO) enrichment analysis may return hundreds of statistically significant GO results in a "flat" list, which can be challenging to summarize. It can also be difficult to keep pace with rapidly expanding biological knowledge, which often results in daily changes to any of the over 47,000 gene ontologies that describe biological knowledge. GOATOOLS, a Python-based library, makes it more efficient to stay current with the latest ontologies and annotations, perform gene ontology enrichment analyses to determine over- and under-represented terms, and organize results for greater clarity and easier interpretation using a novel GOATOOLS GO grouping method. We performed functional analyses on both stochastic simulation data and real data from a published RNA-seq study to compare the enrichment results from GOATOOLS to two other popular tools: DAVID and GOstats. GOATOOLS is freely available through GitHub: https://github.com/tanghaibao/goatools .

6.
Nucleic Acids Res ; 46(W1): W11-W16, 2018 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-29901812

RESUMO

Galaxy HiCExplorer is a web server that facilitates the study of the 3D conformation of chromatin by allowing Hi-C data processing, analysis and visualization. With the Galaxy HiCExplorer web server, users with little bioinformatic background can perform every step of the analysis in one workflow: mapping of the raw sequence data, creation of Hi-C contact matrices, quality assessment, correction of contact matrices and identification of topological associated domains (TADs) and A/B compartments. Users can create publication ready plots of the contact matrix, A/B compartments, and TADs on a selected genomic locus, along with additional information like gene tracks or ChIP-seq signals. Galaxy HiCExplorer is freely usable at: https://hicexplorer.usegalaxy.eu and is available as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer.

7.
Cell Syst ; 6(6): 752-758.e1, 2018 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-29953864

RESUMO

The primary problem with the explosion of biomedical datasets is not the data, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences. This project is accessible at https://training.galaxyproject.org.

8.
Nat Commun ; 9(1): 189, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335486

RESUMO

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de .


Assuntos
Cromatina/ultraestrutura , Mapeamento Cromossômico/métodos , Cromossomos de Insetos/ultraestrutura , Drosophila melanogaster/genética , Genoma de Inseto , Animais , Evolução Biológica , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Cromossomos de Insetos/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/ultraestrutura , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Humanos , Camundongos , Conformação Molecular , Motivos de Nucleotídeos , Software , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Nat Struct Mol Biol ; 23(6): 580-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27183194

RESUMO

Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Quinases Ciclina-Dependentes/genética , Compensação de Dosagem (Genética) , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Deleção de Genes , Masculino , Mutação , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Serina/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional
10.
Nucleic Acids Res ; 44(W1): W160-5, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27079975

RESUMO

We present an update to our Galaxy-based web server for processing and visualizing deeply sequenced data. Its core tool set, deepTools, allows users to perform complete bioinformatic workflows ranging from quality controls and normalizations of aligned reads to integrative analyses, including clustering and visualization approaches. Since we first described our deepTools Galaxy server in 2014, we have implemented new solutions for many requests from the community and our users. Here, we introduce significant enhancements and new tools to further improve data visualization and interpretation. deepTools continue to be open to all users and freely available as a web service at deeptools.ie-freiburg.mpg.de The new deepTools2 suite can be easily deployed within any Galaxy framework via the toolshed repository, and we also provide source code for command line usage under Linux and Mac OS X. A public and documented API for access to deepTools functionality is also available.


Assuntos
Biologia Computacional/estatística & dados numéricos , Drosophila melanogaster/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/estatística & dados numéricos , Software , Animais , Sequência de Bases , Biologia Computacional/métodos , Gráficos por Computador , Humanos , Armazenamento e Recuperação da Informação , Internet , Alinhamento de Sequência
11.
Mol Cell ; 60(1): 146-62, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26431028

RESUMO

Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. We propose that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Montagem e Desmontagem da Cromatina , Análise Citogenética , Compensação de Dosagem (Genética) , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Cromossomo X/genética
12.
Biologicals ; 42(6): 312-5, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25218518

RESUMO

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.


Assuntos
Anticorpos Monoclonais/química , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/química , Neisseria meningitidis/metabolismo , Polissacarídeos/química , Animais , Calibragem , Ensaio de Imunoadsorção Enzimática , Limite de Detecção , Infecções Meningocócicas/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Sorogrupo , Especificidade da Espécie
13.
Mol Cell ; 55(2): 277-90, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-24981170

RESUMO

Heterochromatin is required to restrict aberrant expression of retrotransposons, but it remains poorly defined due to the underlying repeat-rich sequences. We dissected Suv39h-dependent histone H3 lysine 9 trimethylation (H3K9me3) by genome-wide ChIP sequencing in mouse embryonic stem cells (ESCs). Refined bioinformatic analyses of repeat subfamilies indicated selective accumulation of Suv39h-dependent H3K9me3 at interspersed repetitive elements that cover ∼5% of the ESC epigenome. The majority of the ∼8,150 intact long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs), but only a minor fraction of the >1.8 million degenerate and truncated LINEs/ERVs, are enriched for Suv39h-dependent H3K9me3. Transcriptional repression of intact LINEs and ERVs is differentially regulated by Suv39h and other chromatin modifiers in ESCs but governed by DNA methylation in committed cells. These data provide a function for Suv39h-dependent H3K9me3 chromatin to specifically repress intact LINE elements in the ESC epigenome.


Assuntos
Células-Tronco Embrionárias/enzimologia , Retrovirus Endógenos/genética , Inativação Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Metiltransferases/fisiologia , Proteínas Repressoras/fisiologia , Animais , Células Cultivadas , Metilação de DNA , Camundongos , Processamento de Proteína Pós-Traducional
14.
Elife ; 3: e02024, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24842875

RESUMO

Histone acetyl transferases (HATs) play distinct roles in many cellular processes and are frequently misregulated in cancers. Here, we study the regulatory potential of MYST1-(MOF)-containing MSL and NSL complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. We find that both complexes influence transcription by targeting promoters and TSS-distal enhancers. In contrast to flies, the MSL complex is not exclusively enriched on the X chromosome, yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix, the major repressor of Xist lncRNA. MSL depletion leads to decreased Tsix expression, reduced REX1 recruitment, and consequently, enhanced accumulation of Xist and variable numbers of inactivated X chromosomes during early differentiation. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs.DOI: http://dx.doi.org/10.7554/eLife.02024.001.


Assuntos
Células-Tronco Embrionárias/citologia , Histona Acetiltransferases/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Ligação Proteica , RNA Longo não Codificante/genética , Inativação do Cromossomo X
15.
Nucleic Acids Res ; 42(Web Server issue): W187-91, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24799436

RESUMO

We present a Galaxy based web server for processing and visualizing deeply sequenced data. The web server's core functionality consists of a suite of newly developed tools, called deepTools, that enable users with little bioinformatic background to explore the results of their sequencing experiments in a standardized setting. Users can upload pre-processed files with continuous data in standard formats and generate heatmaps and summary plots in a straight-forward, yet highly customizable manner. In addition, we offer several tools for the analysis of files containing aligned reads and enable efficient and reproducible generation of normalized coverage files. As a modular and open-source platform, deepTools can easily be expanded and customized to future demands and developments. The deepTools webserver is freely available at http://deeptools.ie-freiburg.mpg.de and is accompanied by extensive documentation and tutorials aimed at conveying the principles of deep-sequencing data analysis. The web server can be used without registration. deepTools can be installed locally either stand-alone or as part of Galaxy.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Gráficos por Computador , Sequenciamento de Nucleotídeos em Larga Escala/normas , Internet
16.
Cell Rep ; 3(6): 2142-54, 2013 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-23746450

RESUMO

Human cells contain five canonical, replication-dependent somatic histone H1 subtypes (H1.1, H1.2, H1.3, H1.4, and H1.5). Although they are key chromatin components, the genomic distribution of the H1 subtypes is still unknown, and their role in chromatin processes has thus far remained elusive. Here, we map the genomic localization of all somatic replication-dependent H1 subtypes in human lung fibroblasts using an integrative DNA adenine methyltransferase identification (DamID) analysis. We find in general that H1.2 to H1.5 are depleted from CpG-dense regions and active regulatory regions. H1.1 shows a DamID binding profile distinct from the other subtypes, suggesting a unique function. H1 subtypes can mark specific domains and repressive regions, pointing toward a role for H1 in three-dimensional genome organization. Our work integrates H1 subtypes into the epigenome maps of human cells and provides a valuable resource to refine our understanding of the significance of H1 and its heterogeneity in the control of genome function.


Assuntos
Histonas/classificação , Histonas/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Genômica/métodos , Histonas/química , Histonas/metabolismo , Humanos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico
17.
Methods Mol Biol ; 910: 33-53, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22821591

RESUMO

Complex biological systems comprise a large number of interacting molecules. The identification and detailed characterization of the functions of the involved genes and proteins are crucial for modeling and understanding such systems. To interrogate the various cellular processes, high-throughput techniques such as the Affymetrix Exon Array or RNA interference (RNAi) screens are powerful experimental approaches for functional genomics. However, they typically yield long gene lists that require computational methods to further analyze and functionally annotate the experimental results and to gain more insight into important molecular interactions. Here, we focus on bioinformatics software tools for the functional interpretation of exon expression data to discover alternative splicing events and their impact on gene and protein architecture, molecular networks, and pathways. We additionally demonstrate how to explore large lists of candidate genes as they also result from RNAi screens. In particular, our exemplary application studies show how to analyze the function of human genes that play a major role in human stem cells or viral infections.


Assuntos
Éxons/genética , Interferência de RNA , Processamento Alternativo , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Software
18.
Bioinformatics ; 28(2): 269-76, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22180409

RESUMO

MOTIVATION: Numerous annotations are available that functionally characterize genes and proteins with regard to molecular process, cellular localization, tissue expression, protein domain composition, protein interaction, disease association and other properties. Searching this steadily growing amount of information can lead to the discovery of new biological relationships between genes and proteins. To facilitate the searches, methods are required that measure the annotation similarity of genes and proteins. However, most current similarity methods are focused only on annotations from the Gene Ontology (GO) and do not take other annotation sources into account. RESULTS: We introduce the new method BioSim that incorporates multiple sources of annotations to quantify the functional similarity of genes and proteins. We compared the performance of our method with four other well-known methods adapted to use multiple annotation sources. We evaluated the methods by searching for known functional relationships using annotations based only on GO or on our large data warehouse BioMyn. This warehouse integrates many diverse annotation sources of human genes and proteins. We observed that the search performance improved substantially for almost all methods when multiple annotation sources were included. In particular, our method outperformed the other methods in terms of recall and average precision.


Assuntos
Algoritmos , Biologia Computacional/métodos , Genes , Proteínas/fisiologia , Bases de Dados Genéticas , Humanos , Internet , Anotação de Sequência Molecular , Proteínas/genética , Vocabulário Controlado
19.
Cell Host Microbe ; 9(1): 32-45, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21238945

RESUMO

Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.


Assuntos
Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular , Técnicas de Silenciamento de Genes , Hepatócitos/química , Hepatócitos/enzimologia , Hepatócitos/virologia , Humanos , Fígado/química , Fígado/enzimologia , Fígado/virologia , Antígenos de Histocompatibilidade Menor , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
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