RESUMEN
SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.
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Asma/genética , Interacciones Huésped-Patógeno/genética , Pulmón/virología , Infecciones por Picornaviridae/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Antivirales/farmacología , Asma/patología , Bronquios/patología , Bronquios/fisiología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Isoxazoles/farmacología , Pulmón/fisiología , Masculino , Persona de Mediana Edad , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Infecciones por Picornaviridae/tratamiento farmacológico , Infecciones por Picornaviridae/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Pirrolidinonas/farmacología , Rhinovirus/patogenicidad , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.
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Macrófagos , Microscopía , Membrana Celular , Pulmón , MicrotúbulosRESUMEN
BACKGROUND: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections. METHODS: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified. FINDINGS: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain. INTERPRETATION: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung. FUNDING: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE).
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Bifidobacterium longum/inmunología , Protección Cruzada/inmunología , Interacciones Huésped-Patógeno/inmunología , Virus de la Influenza A/inmunología , Cavidad Nasal/inmunología , Cavidad Nasal/microbiología , Infecciones por Orthomyxoviridae/inmunología , Neumonía Viral/inmunología , Administración Intranasal , Animales , Coinfección/inmunología , Coinfección/microbiología , Coinfección/virología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Mediadores de Inflamación/metabolismo , Ratones , Mortalidad , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/patología , Neumonía Viral/metabolismo , Neumonía Viral/mortalidad , Neumonía Viral/patología , PronósticoRESUMEN
Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at 1 month after infection with influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) that produce increased interleukin-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile that is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population.
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Inmunidad Innata/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones Neumocócicas/inmunología , Animales , RatonesRESUMEN
The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.
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Neoplasias de la Mama/enzimología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositoles/metabolismo , Neoplasias de la Próstata/enzimología , Sistemas de Mensajero Secundario , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Sistemas de Mensajero Secundario/efectos de los fármacos , Factores de TiempoRESUMEN
Influenza A virus (IAV)-induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαß) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV-infected Mx1-positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ-treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non-inflammatory and hence superior treatment option for human IAV infection.
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Interleucinas/uso terapéutico , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Muerte Celular , Citocinas/análisis , Modelos Animales de Enfermedad , Células Epiteliales/fisiología , Humanos , Virus de la Influenza A/aislamiento & purificación , Interferón-alfa/uso terapéutico , Leucocitos/inmunología , Pulmón/patología , Ratones , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Resultado del Tratamiento , Carga ViralRESUMEN
A novel series of quinoline isoxazole BET family bromodomain inhibitors are discussed. Crystallography is used to illustrate binding modes and rationalize their SAR. One member, I-BET151 (GSK1210151A), shows good oral bioavailability in both the rat and minipig as well as demonstrating efficient suppression of bacterial induced inflammation and sepsis in a murine in vivo endotoxaemia model.
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Compuestos Heterocíclicos de 4 o más Anillos/química , Isoxazoles/síntesis química , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Quinolinas/síntesis química , Animales , Sitios de Unión , Cristalografía por Rayos X , Cobayas , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Inflamación/tratamiento farmacológico , Isoxazoles/química , Isoxazoles/farmacología , Ratones , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Quinolinas/química , Quinolinas/farmacología , RatasRESUMEN
Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-associated factor complex (PAFc), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.
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Cromatina/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica/efectos de los fármacos , Proteómica , Transcripción Genética/efectos de los fármacosRESUMEN
Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
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Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inflamación , Macrófagos/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Benzodiazepinas , Células Cultivadas , Epigenómica , Estudio de Asociación del Genoma Completo , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/prevención & control , Estimación de Kaplan-Meier , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/fisiopatología , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Sepsis/tratamiento farmacológico , Sepsis/prevención & control , Choque Séptico/tratamiento farmacológico , Choque Séptico/prevención & controlRESUMEN
T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.
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Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linaje de la Célula , Quinasa 2 de Adhesión Focal/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Quinasa 2 de Adhesión Focal/deficiencia , Quinasa 2 de Adhesión Focal/inmunología , Regulación Neoplásica de la Expresión Génica , Memoria Inmunológica , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Toll-like receptors, which respond to invariant microbial molecules, and receptors for the proinflammatory cytokines tumour necrosis factor and interleukin-1 are crucial for initiation and regulation of innate immune responses. These receptors activate each of the major mitogen-activated protein (MAP) kinase subtypes, extracellular signal-regulated protein kinases, c-Jun amino-terminal kinases and p38 MAP kinases, which are crucial for cell survival and controlling the expression of immune mediators. Here we discuss recent studies characterizing the specific MAP kinase kinase kinases (MAP 3-kinases) that link MAP kinases to receptors involved in innate immunity and the mechanisms by which the activity of MAP 3-kinases is regulated by such receptors.
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Inmunidad Innata/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Animales , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , FosforilaciónRESUMEN
Two members of the NF-kappaB (nuclear factor kappaB)/Rel transcription factor family, NF-kappaB1 and NF-kappaB2, are produced as precursor proteins, NF-kappaB1 p105 and NF-kappaB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-kappaB1 p50 and NF-kappaB2 p52. p105 and p100 are known to function additionally as IkappaBs (inhibitors of NF-kappaB), which retain associated NF-kappaB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-kappaB1 and NF-kappaB2 in immune cells. NF-kappaB2 p100 processing has recently been shown to be stimulated by a subset of NF-kappaB inducers, including lymphotoxin-beta, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-kappaB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-kappaB1 has a distinct function from NF-kappaB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-alpha and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-kappaB activation, p105 functions as a regulator of MAP kinase signalling.
Asunto(s)
Sistema Inmunológico/fisiología , FN-kappa B/fisiología , Animales , Humanos , Subunidad p50 de NF-kappa B , Factor de Transcripción ReIARESUMEN
NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
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Proteínas de Unión al GTP/metabolismo , FN-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Serina , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Fibroblastos , Proteínas de Unión al GTP/genética , Humanos , Quinasa I-kappa B , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Fosforilación , Precursores de Proteínas/efectos de los fármacos , Precursores de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina/metabolismo , Proteínas con Repetición de beta-TransducinaRESUMEN
Stimulation of cells with tumor necrosis factor alpha (TNFalpha) triggers NF-kappaB1 p105 proteolysis, releasing associated Rel subunits to translocate into the nucleus and modulate target gene expression. Phosphorylation of serine 927 within the p105 PEST region by the IkappaB kinase (IKK) complex is required to promote p105 proteolysis in response to TNFalpha stimulation. In this study, the role of the p105 death domain (DD) in signal-induced p105 proteolysis is investigated. Endogenous p105 is shown to interact with the IKK complex in HeLa cells, and transient transfection experiments in 293 cells indicate that each of the catalytic components of the IKK complex, IKK1 and IKK2, can bind to p105. Interaction of p105 with both IKK1 and IKK2 is substantially reduced by deletion of the p105 DD or introduction of a specific point mutation (L841A) into the p105 DD homologous to the lpr mutation in Fas. Phosphorylation of immunoprecipitated p105 on serine 927 by purified recombinant IKK1 or IKK2 protein in vitro is dramatically reduced in both DD mutants relative to wild type. Furthermore, both of the DD mutations significantly impair the ability of low concentrations of IKK2 to induce p105 serine 927 phosphorylation and proteolysis in transiently transfected 3T3 cells. However, high levels of transiently expressed IKK2 bypass the requirement for the p105 DD to induce p105 serine 927 phosphorylation. Finally, p105 serine 927 phosphorylation by the endogenous IKK complex after TNFalpha stimulation and subsequent p105 proteolysis is blocked in both p105 DD mutants when stably expressed in HeLa cells. Thus, the p105 DD acts as a docking site for IKK, increasing its local concentration in the vicinity of the p105 PEST region and facilitating efficient serine 927 phosphorylation.