RESUMEN
Population genetics continues to identify genetic variants associated with diseases of the immune system and offers a unique opportunity to discover mechanisms of immune regulation. Multiple genetic variants linked to severe fungal infections and autoimmunity are associated with caspase recruitment domain-containing protein 9 (CARD9). We leverage the CARD9 R101C missense variant to uncover a biochemical mechanism of CARD9 activation essential for antifungal responses. We demonstrate that R101C disrupts a critical signaling switch whereby phosphorylation of S104 releases CARD9 from an autoinhibited state to promote inflammatory responses in myeloid cells. Furthermore, we show that CARD9 R101C exerts dynamic effects on the skin cellular contexture during fungal infection, corrupting inflammatory signaling and cell-cell communication circuits. Card9 R101C mice fail to control dermatophyte infection in the skin, resulting in high fungal burden, yet show minimal signs of inflammation. Together, we demonstrate how translational genetics reveals molecular and cellular mechanisms of innate immune regulation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Micosis , Animales , Ratones , Fosforilación , Proteínas Adaptadoras de Señalización CARD/metabolismo , Transducción de Señal , Inflamación , AntifúngicosRESUMEN
Posttranslational modifications are efficient means to rapidly regulate protein function in response to a stimulus. Although ubiquitination events and the E3 ubiquitin ligases involved are increasingly characterized in many signaling pathways, their regulation by deubiquitinating enzymes remains less understood. The C-type lectin receptor (CLR) signaling adaptor CARD9 was previously reported to be activated via TRIM62-mediated ubiquitination. In this study, we identify the deubiquitinase USP15 as a novel regulator of CARD9, demonstrating that USP15 constitutively associates with CARD9 and removes TRIM62-deposited ubiquitin marks. Furthermore, USP15 knockdown and knockout specifically enhance CARD9-dependent CLR signaling in both mouse and human immune cells. Altogether, our study identifies a novel regulator of innate immune signaling and provides a blueprint for the identification of additional deubiquitinases that are likely to control these processes.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Células HEK293 , Humanos , Ratones , Unión Proteica , Procesamiento Proteico-Postraduccional , Transducción de Señal , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
BACKGROUND AND OBJECTIVES: Although intravenous lipid emulsion has been proved a powerful antidote for local anesthetic toxicity, there are few pharmacokinetic data on using lipid infusion as a pretreatment for other clinical applications. We assessed the influence of lipid pretreatment on the pharmacodynamics and pharmacokinetics of levobupivacaine. METHODS: Altogether, 12 patients undergoing below-knee surgery for a fracture were randomly assigned to 2 groups (6 patients per group): pretreatment with 1.5 mL/kg lipid infusion (lipid group) or saline infusion (control subjects) followed by complete femoral and sciatic nerve block with 0.375% levobupivacaine (2.5 mg/kg). Total and free (non-protein bound) plasma levobupivacaine concentrations and triglycerides in the lipid group were determined. RESULTS: Results were given as means ± SD. Total and free maximum plasma levobupivacaine concentrations were lower in the lipid group than in control subjects (865 ± 98 vs 1145 ± 177 µg/L and 56.8 ± 7.5 vs 78.2 ± 13.7 µg/L, respectively; P < 0.01). Apparent volume of distribution and clearance were higher in the lipid group than in control subjects (211 ± 35 vs 170 ± 21 L and 35.1 ± 8.0 vs 25.8 ± 2.6 L/h, respectively; P < 0.05). Triglyceride level was significantly higher at the end of lipid infusion than baseline values (7.59 ± 1.32 vs 1.34 ± 0.39 mmol/L; P < 0.01). CONCLUSIONS: Lipid pretreatment increased the apparent volume of distribution and clearance and decreased the maximum total and free levobupivacaine concentrations, thus offering a reasonable explanation for the effects of lipids on local anesthesia-related toxicity in humans. Rapid lipid infusion induced hypertriglyceridemia without other apparent risks in this study. CLINICAL TRIAL REGISTRATION: This study was registered at the Chinese Clinical Trial Registry, identifier ChiCTR-TRC-14005203.
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Anestésicos Locales/sangre , Bloqueo Nervioso Autónomo/métodos , Emulsiones Grasas Intravenosas/administración & dosificación , Nervio Femoral/efectos de los fármacos , Fracturas Óseas/sangre , Levobupivacaína/sangre , Nervio Ciático/efectos de los fármacos , Adulto , Anestésicos Locales/administración & dosificación , Femenino , Nervio Femoral/fisiología , Fracturas Óseas/cirugía , Humanos , Levobupivacaína/administración & dosificación , Masculino , Persona de Mediana Edad , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Dolor Postoperatorio/sangre , Dolor Postoperatorio/prevención & control , Cuidados Preoperatorios/métodos , Nervio Ciático/fisiologíaRESUMEN
Polymorphisms in C1orf106 are associated with increased risk of inflammatory bowel disease (IBD). However, the function of C1orf106 and the consequences of disease-associated polymorphisms are unknown. Here we demonstrate that C1orf106 regulates adherens junction stability by regulating the degradation of cytohesin-1, a guanine nucleotide exchange factor that controls activation of ARF6. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. Consistent with this model, C1orf106-/- mice exhibit defects in the intestinal epithelial cell barrier, a phenotype observed in IBD patients that confers increased susceptibility to intestinal pathogens. Furthermore, the IBD risk variant increases C1orf106 ubiquitination and turnover with consequent functional impairments. These findings delineate a mechanism by which a genetic polymorphism fine-tunes intestinal epithelial barrier integrity and elucidate a fundamental mechanism of cellular junctional control.
Asunto(s)
Uniones Adherentes/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Fosfoproteínas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Células CACO-2 , Células HEK293 , Humanos , Inmunoprecipitación , Mucosa Intestinal/patología , Ratones , Ratones Mutantes , Fosfoproteínas/genética , Polimorfismo Genético , Proteolisis , Riesgo , Ubiquitinación/genéticaRESUMEN
Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/antagonistas & inhibidores , Proteínas Adaptadoras de Señalización CARD/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Variación Genética , Enfermedades Inflamatorias del Intestino/genética , Evaluación Preclínica de Medicamentos , Marcadores Genéticos , Ensayos Analíticos de Alto Rendimiento , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Unión Proteica , Sensibilidad y Especificidad , Proteínas de Motivos Tripartitos/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/fisiología , Candidiasis Invasiva/inmunología , Receptores de Angiotensina/fisiología , Receptores de Endotelina/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Candidiasis Invasiva/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Genes Dominantes , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Humanos , Enfermedades Inflamatorias del Intestino/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores de Angiotensina/química , Receptores de Angiotensina/deficiencia , Receptores de Endotelina/química , Receptores de Endotelina/deficiencia , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Organismos Libres de Patógenos Específicos , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
BACKGROUND: Placenta growth factor (PlGF), a dimeric glycoprotein with 53% homology to VEGF, binds to VEGF receptor-1 (Flt-1), but not to VEGF receptor-2 (Flk-1), and may function by modulating VEGF activity. We previously have showed that PlGF displays prognostic value in colorectal cancer (CRC) but the mechanism remains elucidated. RESULTS: Overexpression of PlGF increased the invasive/migration ability and decreased apoptosis in CRC cells showing Flt-1 expression. Increased migration was associated with increasing MMP9 via p38 MAPK activation. Tumors grew faster, larger; with higher vascularity from PlGF over-expression cells in xenograft assay. In two independent human CRC tissue cohorts, PlGF, MMP9, and Flt-1 expressions were higher in the advanced than the localized disease group. PlGF expression correlated with MMP9, and Flt-1 expression. CRC patients with high PlGF and high Flt-1 expression in tissue had poor prognosis. CONCLUSION: PlGF/Flt-1 signaling plays an important role in CRC progression, blocking PlGF/Flt-1 signaling maybe an alternative therapy for CRC.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Gestacionales/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/patología , Femenino , Humanos , Metaloproteinasa 9 de la Matriz/genética , Fosforilación , Factor de Crecimiento Placentario , Transducción de Señal , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND & AIMS: Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection. METHODS: We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis. RESULTS: Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria. CONCLUSIONS: The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citrobacter rodentium , Colitis/metabolismo , Infecciones por Enterobacteriaceae/metabolismo , Mucosa Intestinal/metabolismo , Células Th17/metabolismo , Animales , Biomarcadores/metabolismo , Proteínas Adaptadoras de Señalización CARD , Colitis/etiología , Colitis/inmunología , Colitis/patología , Citocinas/metabolismo , Sulfato de Dextran , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunidad Innata , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Profiling studies of mRNA and microRNA, particularly microarray-based studies, have been extensively used to create compendia of genes that are preferentially expressed in the immune system. In some instances, functional studies have been subsequently pursued. Recent efforts such as the Encyclopedia of DNA Elements have demonstrated the benefit of coupling RNA sequencing analysis with information from expressed sequence tags (ESTs) for transcriptomic analysis. However, the full characterization and identification of transcripts that function as modulators of human immune responses remains incomplete. In this study, we demonstrate that an integrated analysis of human ESTs provides a robust platform to identify the immune transcriptome. Beyond recovering a reference set of immune-enriched genes and providing large-scale cross-validation of previous microarray studies, we discovered hundreds of novel genes preferentially expressed in the immune system, including noncoding RNAs. As a result, we have established the Immunogene database, representing an integrated EST road map of gene expression in human immune cells, which can be used to further investigate the function of coding and noncoding genes in the immune system. Using this approach, we have uncovered a unique metabolic gene signature of human macrophages and identified PRDM15 as a novel overexpressed gene in human lymphomas. Thus, we demonstrate the utility of EST profiling as a basis for further deconstruction of physiologic and pathologic immune processes.
Asunto(s)
Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Sistema Inmunológico/metabolismo , Animales , Análisis por Conglomerados , Biología Computacional/métodos , Proteínas de Unión al ADN/genética , Bases de Datos de Ácidos Nucleicos , Redes Reguladoras de Genes , Genómica , Humanos , Enfermedades del Sistema Inmune/genética , Linfoma de Células B/genética , Ratones , Anotación de Secuencia Molecular , ARN Largo no Codificante/genética , Reproducibilidad de los Resultados , Factores de Transcripción/genética , TranscriptomaRESUMEN
Using measured capacitance-voltage curves with different gate lengths and current-voltage characteristics at low drain-to-source voltage for the AlGaN/AlN/GaN heterostructure field-effect transistors (HFETs) of different drain-to-source distances, we found that the dominant scattering mechanism in AlGaN/AlN/GaN HFETs is determined by the ratio of gate length to drain-to-source distance. For devices with small ratio (here, less than 1/2), polarization Coulomb field scattering dominates electron mobility. However, for devices with large ratio (here, more than 1/2), longitudinal optical (LO) phonon scattering and interface roughness scattering are dominant. The reason is closely related to polarization Coulomb field scattering.
RESUMEN
BACKGROUND: Toll-like receptors (TLRs) are expressed by a variety of cells, including intestinal epithelia. However, the full spectrum of regulators modulating innate responses via TLRs has not been delineated. Tribbles (Trib) have been identified as a highly conserved family of kinase-like proteins. We sought to clarify the role of Trib2 in the TLR signaling pathway. METHODS: Trib2 mRNA and protein levels were analyzed by quantitative polymerase chain reaction (PCR) and western blot, respectively. Immunohistochemical staining was used to determine the expression of Trib2 in human tissue. Involvement of Trib2 in nuclear factor kappa B (NF-κB) pathways was assessed in epithelial cells by NF-κB reporter assay. Proteins that interacted with Trib2 were identified by mass spectrometry and confirmed by immunoprecipitation. The domain essential for Trib2 function was mapped using truncated constructs. RESULTS: Trib2 expression is decreased in active inflamed tissue from patients with inflammatory bowel disease (IBD). Trib2 is expressed in human and mouse colonic epithelium as well as immune cells, and its expression in epithelium is inducible in a ligand-dependent manner by TLR5 ligand stimulation. Trib2 inhibits TLR5-mediated activation of NF-κB downstream of TRAF6. Trib2 selectively modulates mitogen-activated protein kinase (MAPK) pathways p38 and Jun N-terminal kinase (JNK) but not p44/p42 (ERK1/2). NF-κB2 (p100) was identified as a Trib2 binding partner in regulating the TLR5 signaling pathway that leads to inhibition of NF-κB activity. Residues 158-177 in the Trib2 kinase-like domain are required for Trib2 function. CONCLUSIONS: These observations indicate that Trib2 is a novel regulator in the TLR5 signaling pathway and altered expression of Trib2 may play a role in IBD.
Asunto(s)
Regulación de la Expresión Génica , Enfermedades Inflamatorias del Intestino/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptor Toll-Like 5/metabolismo , Animales , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Células Cultivadas , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Luciferasas/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Receptor Toll-Like 5/genéticaRESUMEN
The identification of transcriptional regulatory networks, which control tissue-specific development and function, is of central importance to the understanding of lymphocyte biology. To decipher transcriptional networks in T-cell development and differentiation we developed a browsable expression atlas and applied a novel quantitative method to define gene sets most specific to each of the represented cell subsets and tissues. Using this system, body atlas size datasets can be used to examine gene enrichment profiles from a cell/tissue perspective rather than gene perspective, thereby identifying highly enriched genes within a cell type, which are often key to cellular differentiation and function. A systems analysis of transcriptional regulators within T cells during different phases of development and differentiation resulted in the identification of known key regulators and uncharacterized coexpressed regulators. ZBTB25, a BTB-POZ family transcription factor, was identified as a highly T cell-enriched transcription factor. We provide evidence that ZBTB25 functions as a negative regulator of nuclear factor of activated T cells (NF-AT) activation, such that RNA interference mediated knockdown resulted in enhanced activation of target genes. Together, these findings suggest a novel mechanism for NF-AT mediated gene expression and the compendium of expression data provides a quantitative platform to drive exploration of gene expression across a wide range of cell/tissue types.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Factores de Transcripción NFATC/genética , Proteínas Nucleares/genética , Linfocitos T/citología , Diferenciación Celular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Genes , Humanos , Leucemia de Células T/genética , Linfoma de Células B/genética , Factores de Transcripción NFATC/análisis , Factores de Transcripción NFATC/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismoRESUMEN
Autophagy is a conserved cellular process required for the removal of defective organelles, protein aggregates, and intracellular pathogens. We used a network analysis strategy to identify novel human autophagy components based upon the yeast interactome centered on the core yeast autophagy proteins. This revealed the potential involvement of 14 novel mammalian genes in autophagy, several of which have known or predicted roles in membrane organization or dynamics. We selected one of these membrane interactors, FNBP1L (formin binding protein 1-like), an F-BAR-containing protein (also termed Toca-1), for further study based upon a predicted interaction with ATG3. We confirmed the FNBP1L/ATG3 interaction biochemically and mapped the FNBP1L domains responsible. Using a functional RNA interference approach, we determined that FNBP1L is essential for autophagy of the intracellular pathogen Salmonella enterica serovar Typhimurium and show that the autophagy process serves to restrict the growth of intracellular bacteria. However, FNBP1L appears dispensable for other forms of autophagy induced by serum starvation or rapamycin. We present a model where FNBP1L is essential for autophagy of intracellular pathogens and identify FNBP1L as a differentially used molecule in specific autophagic contexts. By using network biology to derive functional biological information, we demonstrate the utility of integrated genomics to novel molecule discovery in autophagy.
Asunto(s)
Autofagia/inmunología , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Saccharomyces cerevisiae/inmunología , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/genética , Línea Celular , Biología Computacional , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Espacio Intracelular/inmunología , Unión Proteica , ARN Interferente Pequeño/genética , Saccharomyces cerevisiae/genética , Salmonella typhimurium/inmunología , Especificidad por Sustrato , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Quimiocina CCL20/metabolismo , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Lisofosfolípidos/farmacología , Animales , Asma/inmunología , Bronquios/anatomía & histología , Proteínas Adaptadoras de Señalización CARD/genética , Células Cultivadas , Quimiocina CCL20/genética , Citocinas/genética , Células Epiteliales/citología , Humanos , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZalphaA, then the yeast expression vector pPICZalpha-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 microg/mL) of YPD plate and medium. When temperature was 30 degrees C, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L (1.25 g/L). The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1-H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu(2+)-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of > 96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.
Asunto(s)
Pichia/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Calicreínas de Tejido/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía por Intercambio Iónico/métodos , Vectores Genéticos/genética , Humanos , Riñón/metabolismo , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Calicreínas de Tejido/genéticaRESUMEN
Apical Membrane Antigen 1 (AMA1) and Merozoite Surface Protein 1 (MSP1) were produced as a recombinant fusion protein and formulated with the adjuvant Montanide ISA 720 with the aim of replicating the structure present in the parasite protein. A previous trial with this construct demonstrated the vaccine was safe and immunogenic but was associated with injection site reactogenicity. This Phase 1a dose-escalating, double blind, randomized, controlled trial of PfCP2.9/Montanide ISA 720 was conducted to evaluate alternative dose levels and vaccination schedules, with a pre-formulated vaccine that had undergone more in-depth and frequent quality control and stability analysis. The trial was conducted in seventy healthy Chinese malaria-naïve volunteers between January 2006 and January 2007. The objective was to assess the safety, reactogenicity and immunogenicity of 5, 20 and 50microg of PfCP2.9/ISA 720 under 2 different schedules. The most common adverse event was injection site tenderness (53%). The frequency and severity of adverse events was similar in both vaccination schedules. Antibody responses were induced and remained elevated throughout the study in volunteers receiving vaccine (p<0.001). Although high antibody titers as measured by ELISA to the PfCP2.9 immunogen were observed, biological function of these antibodies was not reflected by the in vitro inhibition of parasite growth, and there was limited recognition of fixed parasites in an immunofluorescence assay. At all three dose levels and both schedules, this formulation of PfCP2.9/ISA 720 is well tolerated, safe and immunogenic; however no functional activity against the parasite was observed.
Asunto(s)
Proteínas de Unión al Calcio/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Proteína 1 de Superficie de Merozoito/inmunología , Proteínas Quinasas/inmunología , Proteínas Protozoarias/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos/farmacología , Adolescente , Adulto , Anticuerpos Antiprotozoarios/análisis , Anticuerpos Antiprotozoarios/biosíntesis , Química Farmacéutica , Relación Dosis-Respuesta Inmunológica , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Estudios de Seguimiento , Humanos , Esquemas de Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/genética , Masculino , Persona de Mediana Edad , Proteínas Mutantes Quiméricas/inmunología , Pichia/química , Pichia/inmunología , Tamaño de la Muestra , Adulto JovenRESUMEN
BACKGROUND: The P. falciparum chimeric protein 2.9 (PfCP-2.9) consisting of the sequences of MSP1-19 and AMA-1 (III) is a malaria vaccine candidate that was found to induce inhibitory antibodies in rabbits and monkeys. This was a phase I randomized, single-blind, placebo-controlled, dose-escalation study to evaluate the safety and immunogenicity of the PfCP-2.9 formulated with a novel adjuvant Montanide ISA720. Fifty-two subjects were randomly assigned to 4 dose groups of 10 participants, each receiving the test vaccine of 20, 50, 100, or 200 microg respectively, and 1 placebo group of 12 participants receiving the adjuvant only. METHODS AND FINDINGS: The vaccine formulation was shown to be safe and well-tolerated, and none of the participants withdrew. The total incidence of local adverse events (AEs) was 75%, distributed among 58% of the placebo group and 80% of those vaccinated. Among the vaccinated, 65% had events that were mild and 15% experienced moderate AEs. Almost all systemic adverse reactions observed in this study were graded as mild and required no therapy. The participants receiving the test vaccine developed detectable antibody responses which were boosted by the repeated vaccinations. Sixty percent of the vaccinated participants had high ELISA titers (>1:10,000) of antigen-specific antibodies which could also recognize native parasite proteins in an immunofluorescence assay (IFA). CONCLUSION: This study is the first clinical trial for this candidate and builds on previous investigations supporting PfCP-2.9/ISA720 as a promising blood-stage malaria vaccine. Results demonstrate safety, tolerability (particularly at the lower doses tested) and immunogenicity of the formulation. Further clinical development is ongoing to explore optimizing the dose and schedule of the formulation to decrease reactogenicity without compromising immunogenicity. TRIAL REGISTRATION: Chinese State Food and Drug Administration (SFDA) 2002SL0046; Controlled-Trials.com ISRCTN66850051 [66850051].
Asunto(s)
Antígenos de Protozoos/química , Vacunas contra la Malaria/química , Malaria Falciparum/prevención & control , Manitol/análogos & derivados , Proteínas de la Membrana/química , Proteína 1 de Superficie de Merozoito/química , Ácidos Oléicos/química , Plasmodium falciparum/metabolismo , Subunidades de Proteína/química , Proteínas Protozoarias/química , Adolescente , Adulto , Animales , Femenino , Humanos , Vacunas contra la Malaria/farmacología , Masculino , Manitol/química , Manitol/farmacología , Persona de Mediana Edad , Ácidos Oléicos/farmacología , Proteínas Recombinantes de Fusión/química , SeguridadRESUMEN
PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/fisiología , Comunicación Celular/fisiología , Proteínas de la Membrana/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Línea Celular , Línea Celular Tumoral , Polaridad Celular/fisiología , Homólogo 1 de la Proteína Discs Large , Endocitosis/fisiología , Células Epiteliales/fisiología , Células HeLa , Humanos , Dominios PDZ/fisiología , Unión Proteica/fisiologíaRESUMEN
Wound healing is a dynamic and complex biologic process that could be accelerated by growth factors. To investigate the efficacy of topical recombinant human acidic fibroblast growth factor (rh-aFGF) treatment in deep partial-thickness burn or skin graft donor sites, we designed a randomized, multicenter, double-blind, and placebo-controlled clinical trial. The healing rate, fully healed rate, and healing time were evaluated to assess the efficacy of rh-aFGF application. Laboratory examinations and abnormal signs were used to assess the side and toxic effects. The results showed that the healing rate of burn wounds and skin graft donor sites treated by rh-aFGF was significantly higher than that by placebo, and the mean healed time of burn wounds and skin graft donor sites in the rh-aFGF group was significantly the shorter than that in the placebo group. In conclusion, topical administration of rh-aFGF can accelerate the wound healing process and shorten the healed time. It is a potential therapeutic application for promoting healing of deep partial-thickness burns or skin graft donor sites.
Asunto(s)
Quemaduras/tratamiento farmacológico , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Adulto , Unidades de Quemados , Método Doble Ciego , Femenino , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Humanos , Masculino , Placebos , Trasplante de Piel , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
PDZ domain-containing proteins and their interaction partners are mutated in numerous human diseases and function in complexes regulating epithelial polarity, ion channels, cochlear hair cell development, vesicular sorting, and neuronal synaptic communication. Among several properties of a collection of documented PDZ domain-ligand interactions, we discovered embedded in a large-scale expression data set the existence of a significant level of co-regulation between PDZ domain-encoding genes and these ligands. From this observation, we show how integration of expression data, a comparative genomics catalog of 899 mammalian genes with conserved PDZ-binding motifs, phylogenetic analysis, and literature mining can be utilized to infer PDZ complexes. Using molecular studies we map novel interaction partners for the PDZ proteins DLG1 and CARD11. These results provide insight into the diverse roles of PDZ-ligand complexes in cellular signaling and provide a computational framework for the genome-wide evaluation of PDZ complexes.