RESUMO
Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, which sense bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the leucine-rich repeats domain, cytosolic S1P directly bound to the nucleotide binding domains of NOD1/2, triggering NF-κB activation and inflammatory responses. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of the cytosolic metabolite S1P. We propose S1P, an endogenous metabolite, as a novel NOD1/2 activator and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.
Assuntos
Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/metabolismo , Células THP-1RESUMO
The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for signal transduction induced by the pattern recognition receptors NOD1 and NOD2 (referred to collectively as NOD1/2). Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells. Here, we identified the molecular composition of these complexes. Infection with Shigella flexneri to activate NOD1-RIPK2 revealed that RIPK2 formed dynamic interactions with several cellular proteins, including A20 (also known as TNFAIP3), erlin-1, erlin-2 and 14-3-3. Whereas interaction of RIPK2 with 14-3-3 proteins was strongly reduced upon infection with Shigella, erlin-1 and erlin-2 (erlin-1/2) specifically bound to RIPK2 complexes. The interaction of these proteins with RIPK2 was validated using protein binding assays and immunofluorescence staining. Beside bacterial activation of NOD1/2, depletion of the E3 ubiquitin ligase XIAP and treatment with RIPK2 inhibitors also led to the formation of RIPK2 cytosolic complexes. Although erlin-1/2 were recruited to RIPK2 complexes following XIAP inhibition, these proteins did not associate with RIPK2 structures induced by RIPK2 inhibitors. While the specific recruitment of erlin-1/2 to RIPK2 suggests a role in innate immune signaling, the biological response regulated by the erlin-1/2-RIPK2 association remains to be determined.
Assuntos
Proteína Adaptadora de Sinalização NOD2 , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteínas 14-3-3 , Citosol/metabolismo , Humanos , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ligação Proteica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de SinaisRESUMO
PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.
Assuntos
Deficiência Intelectual , Humanos , Sequenciamento do Exoma , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Alelos , GenótipoRESUMO
Mammalian Nod-like receptor (NLR) proteins contribute to the regulation and induction of innate and adaptive immunity in mammals, although the function of about half of the currently identified NLR proteins remains poorly characterized. Here we analyzed the function of the primate-specific NLRP11 gene product. We show that NLRP11 is highly expressed in immune cells, including myeloid cells, B cells, and some B cell lymphoma lines. Overexpression of NLRP11 in human cells did not trigger key innate immune signaling pathways, including NF-κB and type I interferon responses. NLRP11 harbors a pyrin domain, which is responsible for inflammasome formation in related NLR proteins. However, NLRP11 did not interact with the inflammasome adaptor protein ASC, and it did not trigger caspase-1 activation. By contrast, expression of NLRP11 specifically repressed NF-κB and type I interferon responses, two key innate immune pathways involved in inflammation. This effect was independent of the pyrin domain and ATPase activity of NLRP11. siRNA-mediated knockdown of NLRP11 in human myeloid THP1 cells validated these findings and revealed enhanced lipopolysaccharide and Sendai virus-induced cytokine and interferon responses, respectively, in cells with reduced NLRP11 expression. In summary, our work identifies a novel role of NLRP11 in the regulation of inflammatory responses in human cells.
Assuntos
Linfócitos B/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Mieloides/metabolismo , Proteínas NLR/metabolismo , Substituição de Aminoácidos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/agonistas , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/toxicidade , Masculino , Mutação , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas NLR/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Nuclear factor (NF)-κB transcription factors play major roles in numerous biological processes including development and immunity. Here, we engineered a novel bi-directional NF-κB-responsive reporter, pSGNluc, in which a high-affinity NF-κB promoter fragment simultaneously drives expression of luciferase and GFP. Treatment with TNFα (also known as TNF) induced a strong, dose-dependent luciferase signal in cell culture. The degree of induction over background was comparable to that of other NF-κB-driven luciferase reporters, but the absolute level of expression was at least 20-fold higher. This extends the sensitivity range of otherwise difficult assays mediated exclusively by endogenously expressed receptors, as we show for Nod1 signaling in HEK293 cells. To measure NF-κB activity in the living organism, we established a transgenic zebrafish line carrying the pSGNluc construct. Live in toto imaging of transgenic embryos revealed the activation patterns of NF-κB signaling during embryonic development and as responses to inflammatory stimuli. Taken together, by integrating qualitative and quantitative NF-κB reporter activity, pSGNluc is a valuable tool for studying NF-κB signaling at high spatiotemporal resolution in cultured cells and living animals that goes beyond the possibilities provided by currently available reporters.
Assuntos
Técnicas de Cultura de Células/métodos , Sistemas Computacionais , Genes Reporter , NF-kappa B/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Citocinas/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Inflamação/patologia , Luciferases/metabolismo , Peptidoglicano/metabolismo , Plasmídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fator de Necrose Tumoral alfa/farmacologia , Raios Ultravioleta , Peixe-Zebra/embriologiaRESUMO
Rare diseases (RD) have a prevalence of not more than 1/2000 persons in the European population, and are characterised by the difficulty experienced in obtaining a correct and timely diagnosis. According to Orphanet, 72.5% of RD have a genetic origin although 35% of them do not yet have an identified causative gene. A significant proportion of patients suspected to have a genetic RD receive an inconclusive exome/genome sequencing. Working towards the International Rare Diseases Research Consortium (IRDiRC)'s goal for 2027 to ensure that all people living with a RD receive a diagnosis within one year of coming to medical attention, the Solve-RD project aims to identify the molecular causes underlying undiagnosed RD. As part of this strategy, we developed a phenotypic similarity-based variant prioritization methodology comparing submitted cases with other submitted cases and with known RD in Orphanet. Three complementary approaches based on phenotypic similarity calculations using the Human Phenotype Ontology (HPO), the Orphanet Rare Diseases Ontology (ORDO) and the HPO-ORDO Ontological Module (HOOM) were developed; genomic data reanalysis was performed by the RD-Connect Genome-Phenome Analysis Platform (GPAP). The methodology was tested in 4 exemplary cases discussed with experts from European Reference Networks. Variants of interest (pathogenic or likely pathogenic) were detected in 8.8% of the 725 cases clustered by similarity calculations. Diagnostic hypotheses were validated in 42.1% of them and needed further exploration in another 10.9%. Based on the promising results, we are devising an automated standardized phenotypic-based re-analysis pipeline to be applied to the entire unsolved cases cohort.
Assuntos
Genômica , Doenças Raras , Humanos , Doenças Raras/diagnóstico , Doenças Raras/epidemiologia , Doenças Raras/genética , Fenótipo , Mapeamento CromossômicoRESUMO
Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.
RESUMO
The cellular functions of the serine/threonine protein kinase D (PKD) have been extensively studied within the last decade and distinct roles such as fission of vesicles at the Golgi compartment, coordination of cell migration and invasion, and regulation of gene transcription have been correlated with this kinase family. Here, we highlight the current state of in vivo studies on PKD function with a focus on animal models and discuss the molecular basis of the observed phenotypic characteristics associated with this kinase family.
Assuntos
Proteína Quinase C/fisiologia , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Domínio Catalítico , Crescimento e Desenvolvimento , Humanos , Isoenzimas/fisiologia , Especificidade de Órgãos , Fosforilação , Proteína Quinase C/química , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.
Assuntos
Actinas , NF-kappa B , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Actinas/metabolismo , Transdução de Sinais , Movimento Celular , Células HeLa , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismoRESUMO
Nucleotide-binding and oligomerization domain containing 5 (NLRC5) is the key transcriptional regulator of major histocompatibility (MHC) class I genes. Recent observations suggest a role for NLRC5 in metabolic traits and in transcriptional regulation beyond MHC class I genes. To understand the function of NLRC5 in metabolic disease, we subjected Nlrc5 -/- mice to high-fat diet (HFD) feeding. Female Nlrc5 -/- mice presented with higher weight gain and more adipose tissue (AT) compared to wild-type (WT) animals. Mechanistically, we demonstrate that NLRC5 enhanced the expression of peroxisome proliferator-activated receptor (PPAR) γ target genes in human cells. We identify Sin3A and negative elongation factor (NELF) B as two novel NLRC5 interaction partners and show that Sin3A partly modulates the synergistic transcriptional effect of NLRC5 on PPARγ. Collectively, we show that NLRC5 contributes to weight gain in mice, which involves transcriptional enhancement of PPARγ targets by NLRC5 that is co-regulated by Sin3A.
RESUMO
Skeletal muscle responds to exercise by activation of signalling pathways that co-ordinate gene expression to sustain muscle performance. MEF2 (myocyte enhancer factor 2)-dependent transcriptional activation of MHC (myosin heavy chain) genes promotes the transformation from fast-twitch into slow-twitch fibres, with MEF2 activity being tightly regulated by interaction with class IIa HDACs (histone deacetylases). PKD (protein kinase D) is known to directly phosphorylate skeletal muscle class IIa HDACs, mediating their nuclear export and thus derepression of MEF2. In the present study, we report the generation of transgenic mice with inducible conditional expression of a dominant-negative PKD1kd (kinase-dead PKD1) protein in skeletal muscle to assess the role of PKD in muscle function. In control mice, long-term voluntary running experiments resulted in a switch from type IIb+IId/x to type IIa plantaris muscle fibres as measured by indirect immunofluorescence of MHCs isoforms. In mice expressing PKD1kd, this fibre type switch was significantly impaired. These mice exhibited altered muscle fibre composition and decreased running performance compared with control mice. Our findings thus indicate that PKD activity is essential for exercise-induced MEF2-dependent skeletal muscle remodelling in vivo.
Assuntos
Músculo Esquelético/fisiologia , Canais de Cátion TRPP/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ensaios Enzimáticos , Indução Enzimática , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Atividade Motora , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Corrida , Canais de Cátion TRPP/genéticaRESUMO
NOD-like receptors (NLRs) are a family of pattern recognition receptors, able to respond to conserved microbial structures and endogenous danger signals. The NLR NOD1 responds to bacterial peptidoglycan, leading to recruitment of RIPK2, following activation of NFκB and MAPK pathways. In this chapter, we describe a fluorescent light microscopic approach to analyze the subcellular distribution of NOD1 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri. This method is based on exogenously expressed EGFP-tagged NOD1 and describes a protocol to obtain inducible cell lines with functional NOD1 signaling. The described protocol is useful to study NOD1 function, also in living cells, using live cell imaging and can be adopted for the study of other NLR proteins.
Assuntos
Transdução de Sinais , NF-kappa B/metabolismo , Proteínas NLR , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Shigella flexneri/genéticaRESUMO
The receptor-interacting serine/threonine-protein kinase-2 (RIPK2, RIP2) is a key player in downstream signaling of nuclear oligomerization domain (NOD)-like receptor (NLR)-mediated innate immune response against bacterial infections. RIPK2 is recruited following activation of the pattern recognition receptors (PRRs) NOD1 and NOD2 by sensing bacterial peptidoglycans leading to activation of NF-κB and MAPK pathways and the production of pro-inflammatory cytokines. Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells, also called RIPosomes. These can be induced by Shigella flexneri or by the inhibition of RIPK2 by small compounds, such as GSK583 and gefitinib.In this chapter, we describe fluorescent light microscopic and Western blot approaches to analyze the cytoplasmic aggregation of RIPK2 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri, or by the treatment with RIPK2 inhibitors. This method is based on HeLa cells stably expressing eGFP-tagged RIPK2 and describes a protocol to induce and visualize RIPosome formation. The described method is useful to study the deposition of RIPK2 in speck-like structures, also in living cells, using live cell imaging and can be adopted for the study of other inhibitory proteins or to further analyze the process of RIPosome structure assembly.
Assuntos
NF-kappa B , Transdução de Sinais , Citocinas/metabolismo , Células HeLa , Humanos , Imunidade Inata , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismoRESUMO
For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient's data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe.
Assuntos
Doenças Genéticas Inatas/genética , Disseminação de Informação , Colaboração Intersetorial , Doenças Raras/genética , Conferências de Consenso como Assunto , Europa (Continente) , Doenças Genéticas Inatas/diagnóstico , Testes Genéticos/métodos , Humanos , Doenças Raras/diagnóstico , Sequenciamento do Exoma/métodosRESUMO
The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events. Recently, it was shown that RIPK2 can form higher order molecular structures in vitro. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depended on the caspase activation and recruitment domain of NOD1 or NOD2. Complex formation upon activation required RIPK2 autophosphorylation at Y474 and was influenced by phosphorylation at S176. We found that the E3 ligase X-linked inhibitor of apoptosis (XIAP) counteracts complex formation of RIPK2, accordingly mutation of the XIAP ubiquitylation sites in RIPK2 enhanced complex formation. Taken together, our work reveals novel roles of XIAP in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.
Assuntos
Citosol/metabolismo , Infecções por Enterobacteriaceae/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Escherichia coli Enteropatogênica/patogenicidade , Células HeLa , Humanos , Peso Molecular , NF-kappa B/metabolismo , Fosforilação , Serina , Shigella flexneri/patogenicidade , Transdução de Sinais , Tirosina/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. RESULTS: We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. CONCLUSION: The data presented support an important role for PKD3 during development of these tissues.
Assuntos
Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína Quinase C/genética , Animais , Western Blotting , Tecido Conjuntivo/embriologia , Tecido Conjuntivo/enzimologia , Embrião de Mamíferos/metabolismo , Feminino , Coração Fetal/embriologia , Coração Fetal/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/embriologia , Músculo Esquelético/enzimologia , Tecido Nervoso/embriologia , Tecido Nervoso/enzimologia , Organogênese , GravidezRESUMO
NOD1 belongs to the family of NOD-like receptors, which is a group of well-characterised, cytosolic pattern-recognition receptors. The best-studied function of NOD-like receptors is their role in generating immediate pro-inflammatory and antimicrobial responses by detecting specific bacterial peptidoglycans or by responding to cellular stress and danger-associated molecules. The present study describes a regulatory, peptidoglycan-independent function of NOD1 in anti-inflammatory immune responses. We report that, in human dendritic cells, NOD1 balances IL-10-induced STAT1 and STAT3 activation by a SOCS2-dependent mechanism, thereby suppressing the tolerogenic dendritic cell phenotype. Based on these findings, we propose that NOD1 contributes to inflammation not only by promoting pro-inflammatory processes, but also by suppressing anti-inflammatory pathways.
Assuntos
Células Dendríticas/citologia , Interleucina-10/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Peptidoglicano/imunologia , Animais , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inativação Gênica , Células HEK293 , Humanos , Fenótipo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase-dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity.
Assuntos
Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Movimento Celular/fisiologia , Dendritos/metabolismo , Dendritos/fisiologia , Endocitose/fisiologia , Hipocampo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Receptores de AMPA/metabolismo , Receptores de AMPA/fisiologia , Transdução de Sinais/fisiologia , Membranas Sinápticas/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Sponges (Porifera) are nerve- and muscleless metazoa, but display coordinated motor reactions. Therefore, they represent a valuable phylum to investigate coordination systems, which evolved in a hypothetical Urmetazoon prior to the central nervous system (CNS) of later metazoa. We have chosen the contractile and locomotive species Tethya wilhelma (Demospongiae, Hadromerida) as a model system for our research, using quantitative analysis based on digital time lapse imaging. In order to evaluate candidate coordination pathways, we extracorporeally tested a number of chemical messengers, agonists and antagonists known from chemical signalling pathways in animals with CNS. RESULTS: Sponge body contraction of T. wilhelma was induced by caffeine, glycine, serotonine, nitric oxide (NO) and extracellular cyclic adenosine monophosphate (cAMP). The induction by glycine and cAMP followed patterns varying from other substances. Induction by cAMP was delayed, while glycine lead to a bi-phasic contraction response. The frequency of the endogenous contraction rhythm of T. wilhelma was significantly decreased by adrenaline and NO, with the same tendency for cAMP and acetylcholine. In contrast, caffeine and glycine increased the contraction frequency. The endogenous rhythm appeared irregular during application of caffeine, adrenaline, NO and cAMP. Caffeine, glycine and NO attenuated the contraction amplitude. All effects on the endogenous rhythm were neutralised by the washout of the substances from the experimental reactor system. CONCLUSION: Our study demonstrates that a number of chemical messengers, agonists and antagonists induce contraction and/or modulate the endogenous contraction rhythm and amplitude of our nerveless model metazoon T. wilhelma. We conclude that a relatively complex system of chemical messengers regulates the contraction behaviour through auto- and paracrine signalling, which is presented in a hypothetical model. We assume that adrenergic, adenosynergic and glycinergic pathways, as well as pathways based on NO and extracellular cAMP are candidates for the regulation and timing of the endogenous contraction rhythm within pacemaker cells, while GABA, glutamate and serotonine are candidates for the direct coordination of the contractile cells.