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1.
Cell ; 149(5): 1008-22, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22579044

RESUMO

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.


Assuntos
Replicação do DNA , Embrião de Mamíferos/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Animais , Instabilidade Cromossômica , DNA Polimerase Dirigida por DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
Development ; 150(15)2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37306293

RESUMO

Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors. This crucial event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate. Using optic vesicle organoids to model the onset of the EF, we generate time-course transcriptomic data allowing us to identify dynamic gene expression programmes that characterize this cellular-state transition. Integrating this with chromatin accessibility data suggests a direct role of canonical EF transcription factors in regulating these gene expression changes, and highlights candidate cis-regulatory elements through which these transcription factors act. Finally, we begin to test a subset of these candidate enhancer elements, within the organoid system, by perturbing the underlying DNA sequence and measuring transcriptomic changes during EF activation.


Assuntos
Olho , Fatores de Transcrição , Animais , Olho/metabolismo , Fatores de Transcrição/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Organoides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/genética
3.
EMBO J ; 38(9)2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30988016

RESUMO

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.


Assuntos
Processamento Alternativo , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células-Tronco Neurais/patologia
4.
Development ; 146(19)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31511252

RESUMO

Topologically associating domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD - creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used to investigate the changes in chromatin conformation that result from these manipulations. Our data suggest that these substantial alterations in TAD structure have no readily detectable effect on Shh expression patterns or levels of Shh expression during development - except where enhancers are deleted - and result in no detectable phenotypes. Only in the case of a larger deletion at one TAD boundary could ectopic influence of the Shh limb enhancer be detected on a gene (Mnx1) in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Animais , Pareamento de Bases/genética , Fator de Ligação a CCCTC , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Extremidades/embriologia , Genoma , Proteínas Hedgehog/metabolismo , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Deleção de Sequência/genética
5.
EMBO J ; 35(8): 831-44, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26903602

RESUMO

Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Imunidade Inata/genética , Proteínas de Membrana/imunologia , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/genética , Nucleotidiltransferases/imunologia , Ribonuclease H/genética , Ribonucleases/genética , Animais , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Autoimunidade/genética , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ribonuclease H/metabolismo
7.
PLoS Genet ; 11(12): e1005673, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26646717

RESUMO

Human ß-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six ß-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-ß in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-ß. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-ß, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , RNA Helicases DEAD-box/genética , Psoríase/genética , Receptor 3 Toll-Like/genética , beta-Defensinas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Medula Óssea , Quimiocina CXCL10/genética , RNA Helicases DEAD-box/metabolismo , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon , Lipossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Poli I-C/administração & dosagem , Psoríase/patologia , Receptor 3 Toll-Like/antagonistas & inibidores , beta-Defensinas/metabolismo
8.
Development ; 141(8): 1715-25, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24715461

RESUMO

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.


Assuntos
Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Cromossomos de Mamíferos/química , Análise Mutacional de DNA , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/embriologia , Membro Posterior/metabolismo , Hibridização in Situ Fluorescente , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Conformação de Ácido Nucleico , Fenótipo , Mutação Puntual/genética , Deleção de Sequência/genética
9.
PLoS Genet ; 9(10): e1003826, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204287

RESUMO

ß-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine ß-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that ß-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.


Assuntos
Deleção Cromossômica , Infertilidade Masculina/genética , Espermatozoides/metabolismo , beta-Defensinas/genética , Animais , Cromossomos/genética , Cromossomos/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Maturação do Esperma/genética , Espermatozoides/patologia
10.
Antimicrob Agents Chemother ; 54(5): 1922-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20176896

RESUMO

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.


Assuntos
Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Fragmentos de Peptídeos/química , beta-Defensinas/química , Animais , Antibacterianos/farmacologia , Dimerização , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , beta-Defensinas/farmacologia
11.
Eur J Immunol ; 39(5): 1353-60, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19404978

RESUMO

Beta-defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. In other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine beta-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4(+) T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines restored (Defr1 Y5C) or with only a single cysteine (Defr1-1c(V)) chemoattract CD4(+) T cells with reduced activity, but do not chemoattract DC. Beta-defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of beta-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiotaxia/imunologia , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Receptores CCR6/imunologia , beta-Defensinas/imunologia , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Defensinas/genética
12.
Nat Genet ; 51(1): 96-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478443

RESUMO

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Nanismo/genética , Mutação com Ganho de Função/genética , Microcefalia/genética , Proteínas do Grupo Polycomb/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , DNA Metiltransferase 3A , Metilases de Modificação do DNA/genética , Feminino , Células HeLa , Histonas/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos/genética , Ligação Proteica/genética , Sequências Reguladoras de Ácido Nucleico/genética
13.
BMC Evol Biol ; 5: 32, 2005 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-15904491

RESUMO

BACKGROUND: We have examined the evolution of the genes at the major human beta-defensin locus and the orthologous loci in a range of other primates and mouse. For the first time these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as the ancient past. We have used a combination of maximum likelihood based tests and a maximum parsimony based sliding window approach to give a detailed view of the varying modes of selection operating at this locus. RESULTS: We provide evidence for strong positive selection soon after the duplication of these genes within an ancestral mammalian genome. Consequently variable selective pressures have acted on beta-defensin genes in different evolutionary lineages, with episodes both of negative, and more rarely positive selection, during the divergence of primates. Positive selection appears to have been more common in the rodent lineage, accompanying the birth of novel, rodent-specific beta-defensin genes. These observations allow a fuller understanding of the evolution of mammalian innate immunity. In both the rodent and primate lineages, sites in the second exon have been subject to positive selection and by implication are important in functional diversity. A small number of sites in the mature human peptides were found to have undergone repeated episodes of selection in different primate lineages. Particular sites were consistently implicated by multiple methods at positions throughout the mature peptides. These sites are clustered at positions predicted to be important for the specificity of the antimicrobial or chemoattractant properties of beta-defensins. Surprisingly, sites within the prepropeptide region were also implicated as being subject to significant positive selection, suggesting previously unappreciated functional significance for this region. CONCLUSIONS: Identification of these putatively functional sites has important implications for our understanding of beta-defensin function and for novel antibiotic design.


Assuntos
Evolução Molecular , Seleção Genética , beta-Defensinas/genética , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Evolução Biológica , Análise por Conglomerados , Éxons , Variação Genética , Genoma , Íntrons , Funções Verossimilhança , Camundongos , Modelos Genéticos , Modelos Moleculares , Peptídeos/química , Filogenia , Reação em Cadeia da Polimerase , Primatas , Fatores de Tempo
14.
J Cyst Fibros ; 3 Suppl 2: 59-62, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15463928

RESUMO

We describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.


Assuntos
Técnicas de Preparação Histocitológica/métodos , Membranas Artificiais , Mucosa Respiratória/patologia , Animais , Células Cultivadas , Células Epiteliais , Camundongos , Modelos Animais , Técnicas de Patch-Clamp/métodos , Manejo de Espécimes/métodos , Traqueia/citologia , Traqueia/patologia
15.
Mol Immunol ; 47(6): 1378-82, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20022113

RESUMO

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.


Assuntos
Fatores Quimiotáticos/química , Fatores Quimiotáticos/metabolismo , Isoleucina/metabolismo , Leucina/metabolismo , Receptores CCR6/metabolismo , beta-Defensinas/química , beta-Defensinas/metabolismo , Substituição de Aminoácidos/efeitos dos fármacos , Antibacterianos/farmacologia , Linhagem Celular , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Deleção de Sequência , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , beta-Defensinas/farmacologia
16.
Protein Pept Lett ; 16(6): 668-76, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19519528

RESUMO

Human beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed (1)H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.


Assuntos
Anti-Infecciosos/metabolismo , Escherichia coli/genética , beta-Defensinas/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Receptores CCR6/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , beta-Defensinas/química , beta-Defensinas/genética , beta-Defensinas/farmacologia
17.
Infect Immun ; 70(6): 3053-60, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12010997

RESUMO

Beta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung. Defb1, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate Defb1-deficient (Defb1(tm1Hgu)/Defb1(tm1Hgu) [Defb1(-/-)]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against Staphylococcus aureus than against Escherichia coli or Pseudomonas aeruginosa. Defb1(-/-) mice were found, however, to be effective in the clearance of the cystic fibrosis relevant pathogen S. aureus from the airways after nebulization. Although no overt deleterious phenotype was evident in the Defb1(-/-) mice, the number of mutant mice found to harbor bacteria of the Staphylococcus species in the bladder was significantly higher (P = 0.008) than that of controls, suggesting a role for these peptides in resistance to urinary tract infection.


Assuntos
Antibacterianos , Pulmão/microbiologia , Proteínas/imunologia , Staphylococcus aureus/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Lavagem Broncoalveolar , Modelos Animais de Doenças , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Rim/patologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Miocárdio/patologia , Peptídeos , Proteínas/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Regulação para Cima , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , beta-Defensinas/metabolismo
18.
Mamm Genome ; 13(8): 445-51, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12226710

RESUMO

Beta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity. We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes. In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins. This defensin-related gene (Defr1) is most highly expressed in testis and heart. The genomic organization is highly similar to Defb3, 4, 5, and 6, and the exon 1 sequence is very highly conserved. A synthetic Defr1 peptide displayed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Burkholderia cepacia. The antimicrobial activity of Defr1 against S. aureus, E.coli, and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. This is the first report of a functional beta defensin that lacks one of the conserved cysteine residues in its predicted mature peptide. This study has major implications for the structure and functions of these important host defense molecules.


Assuntos
beta-Defensinas/genética , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , beta-Defensinas/farmacologia
19.
Mol Biol Evol ; 20(3): 460-70, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12644567

RESUMO

Beta-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six beta-defensin exon 2-like sequences and subsequently found full-length expressed transcripts for these novel genes. Expression was high in brain and reproductive tissues. Eleven beta-defensins could be grouped into two clear subgroups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, however, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Examination of the gene sequences of orthologs in other rodents also revealed an excess of nucleotide changes that altered amino acids in the mature peptide region. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding noncoding DNA show little divergence within subgroups. The focus for rapid sequence divergence is localized in the DNA encoding the mature peptide and this is driven by accelerated positive selection. This mechanism of evolution is consistent with the role of this gene family as defense against bacterial pathogens and the sequence changes have implications for novel antibiotic design.


Assuntos
Camundongos Endogâmicos C57BL/genética , Sinais Direcionadores de Proteínas/genética , beta-Defensinas/genética , Animais , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Evolução Molecular , Feminino , Variação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL/classificação , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , beta-Defensinas/classificação
20.
Hum Mol Genet ; 11(3): 243-51, 2002 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11823443

RESUMO

The majority of cystic fibrosis patients produce a mutant form of CFTR (DeltaF508) which has been shown to be mislocalized in both humans and mice. G480C, another clinically 'severe' mutation, has also been demonstrated to be defective in its intracellular processing, but when allowed to traffic in Xenopus oocytes showed similar channel characteristics to that of wild-type CFTR. We have replicated the G480C mutation in the murine Cftr gene using the 'hit and run' double recombination procedure. As expected, the G480C cystic fibrosis mouse model expresses the G480C mutant transcript at a level comparable to that of wild-type CFTR: The homozygous mutant mice were fertile, had normal survival, weight, tooth colour and no evidence of caecal blockage, despite mild goblet cell hypertrophy in the intestine. Analysis of the mutant protein revealed that the majority of G480C CFTR was abnormally processed and no G480C CFTR-specific immunostaining in the apical membranes of intestinal cells was detected. The bioelectric phenotype of these mice revealed organ-specific electrophysiological effects. In contrast to DeltaF508 'hit and run' homozygotes, the classic defect of forskolin-induced chloride ion transport is not replicated in the caecum, but the response to low chloride in the nose is clearly defective in the G480C mutant animals. The mild phenotype of these G480C mutant animals combined with the defective chloride transport in the nose uniquely provides a valuable resource to test novel pharmacological agents aimed at improving trafficking and correcting the electrophysiological defect in the respiratory tract.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Mutação , Animais , Modelos Animais de Doenças , Eletrofisiologia , Feminino , Homozigoto , Masculino , Camundongos , Fenótipo , Processamento de Proteína Pós-Traducional
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