RESUMO
Neisseria meningitidis (the meningococcus) remains an important cause of human disease, including meningitis and sepsis. Adaptation to the host environment includes many interactions with specific cell surface receptors, resulting in intracellular signalling and cytoskeletal rearrangements that contribute to pathogenesis. Here, we assessed the interactions between meningococci and Fibroblast Growth Factor Receptor 1-IIIc (FGFR1-IIIc): a receptor specific to endothelial cells of the microvasculature, including that of the blood-brain barrier. We show that the meningococcus recruits FGFR1-IIIc onto the surface of human blood microvascular endothelial cells (HBMECs). Furthermore, we demonstrate that expression of FGFR1-IIIc is required for optimal invasion of HBMECs by meningococci. We show that the ability of N. meningitidis to interact with the ligand-binding domain of FGFR1-IIIc is shared with the other pathogenic Neisseria species, N. gonorrhoeae, but not with commensal bacteria including non-pathogenic Neisseria species.
Assuntos
Neisseria meningitidis , Barreira Hematoencefálica , Células Endoteliais , Humanos , Neisseria gonorrhoeae , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.
Assuntos
Acrocefalossindactilia/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Regulação para Cima , Acrocefalossindactilia/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Éxons , Humanos , Camundongos , Camundongos Endogâmicos , Modelos Animais , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis. We show that the open reading frames of active and evolutionary young LINE-1s are 5caC-enriched in round spermatids and 5caC is eliminated from LINE-1s and spermiogenesis-specific genes during spermatid maturation, being simultaneously retained at promoters and introns of developmental genes. Our results reveal an association of 5caC with activity of LINE-1 retrotransposons suggesting a potential direct role for this DNA modification in fine regulation of their transcription.
Assuntos
Citosina/análogos & derivados , Elementos Nucleotídeos Longos e Dispersos , Fases de Leitura Aberta , Espermátides/metabolismo , Animais , Citosina/metabolismo , Masculino , Camundongos , Espermátides/citologia , Espermatogênese , Transcrição GênicaRESUMO
BACKGROUND: DNA methylation (5-methylcytosine (5mC)) patterns are often altered in cancers. Ten-eleven translocation (Tet) proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). In addition to their presumptive specific biological roles, these oxidised forms of 5mC may serve as intermediates in demethylation process. According to several reports, 5hmC levels are strongly decreased in cancers; however, the distribution of 5fC and 5caC in malignant tissue has not been studied. FINDINGS: Here, we examine the levels of 5hmC and 5caC in 28 samples of normal breast tissue, 59 samples of invasive human breast cancer and 74 samples of gliomas using immunochemistry. In agreement with previous reports, we show that 71 % of normal breast samples exhibit strong 5hmC signal, compared with only 18 % of breast cancer samples with equivalent levels of 5hmC staining. Unexpectedly, although 5caC is not detectable in normal breast tissue, 27 % of breast cancer samples exhibit significant staining for this modification (p < 0.001). Surprisingly, the presence of immunochemically detectable 5caC is not associated with the intensity of 5hmC signal in breast cancer tissue. In gliomas, we show that 5caC is detectable in 45 % of tumours. CONCLUSIONS: We demonstrate that, unlike 5hmC, the levels of 5caC are elevated in a proportion of breast cancers and gliomas. Our results reveal another level of complexity to the cancer epigenome, suggesting that active demethylation and/or 5caC-dependent transcriptional regulation are pre-activated in some tumours and may contribute to their pathogenesis. Larger studies to evaluate the clinicopathological significance of 5caC in cancers are warranted.
RESUMO
5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.
Assuntos
Linhagem da Célula , Citosina/análogos & derivados , Metilação de DNA , Células-Tronco Neurais/metabolismo , Animais , Células Cultivadas , Citosina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Neurais/citologia , Neurogênese , Timina DNA Glicosilase/metabolismoRESUMO
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Galectina 3/metabolismo , Regulação da Expressão Gênica , Neisseria meningitidis/metabolismo , Receptores de Laminina/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Reagentes de Ligações Cruzadas/química , Humanos , Ligação de Hidrogênio , Integrinas/metabolismo , Lactose/química , Ligantes , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Multimerização ProteicaRESUMO
Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.
Assuntos
Citocinas/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Interleucina-8/metabolismo , Meningites Bacterianas/microbiologia , Neisseria meningitidis/patogenicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , Ligantes , Meningites Bacterianas/metabolismo , Camundongos , Camundongos Transgênicos , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts.
Assuntos
Núcleo Celular/metabolismo , Citosina/análogos & derivados , Eucromatina/metabolismo , Ovário/metabolismo , 5-Metilcitosina/análogos & derivados , Ambystoma mexicanum , Animais , Células Cultivadas , Citosina/metabolismo , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Camundongos , Microscopia Confocal , Ovário/patologiaRESUMO
Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Porinas/química , Porinas/metabolismo , Receptores de Laminina/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation. PRINCIPAL FINDINGS: Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour. CONCLUSIONS: This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an 'in vivo' role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.
Assuntos
Proteínas de Membrana/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Células Cultivadas , Hipocampo/citologia , Humanos , Sistema de Sinalização das MAP Quinases , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Neuritos/química , Neuritos/ultraestrutura , Neuroblastoma/patologia , Neurônios/ultraestrutura , Fosforilação/fisiologia , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.
Assuntos
Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Humanos , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Receptores de Superfície Celular/química , Especificidade por Substrato , Tirosina/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we sample candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding; structural and regulatory components of the cytoskeleton; known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.
Assuntos
Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Células HeLa , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/isolamento & purificação , Análise de Sequência de Proteína , Proteínas rab1 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/deficiência , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Células Cultivadas , Extremidades/embriologia , Extremidades/fisiologia , Gânglios Espinais/embriologia , Gânglios Espinais/fisiologia , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica , Especificidade de Órgãos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, we show that hSpry2 is a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways.