RESUMEN
STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein.
Asunto(s)
Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT2/fisiología , Transducción de Señal , Animales , Sitios de Unión , Núcleo Celular/metabolismo , ADN/metabolismo , Dimerización , Expresión Génica/fisiología , Humanos , Interferón gamma/metabolismo , Interferón gamma/fisiología , Fosforilación , Unión Proteica , Conformación Proteica , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismoRESUMEN
CD4(+)CD25(hi) FOXP3(+) regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.
Asunto(s)
Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Receptor Toll-Like 2/metabolismo , Adulto , Estudios de Casos y Controles , Diferenciación Celular/efectos de los fármacos , Citocinas/biosíntesis , Femenino , Humanos , Inmunomodulación , Inmunofenotipificación , Lipoproteínas/farmacología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Células Th17/citología , Receptor Toll-Like 2/agonistas , Adulto JovenRESUMEN
The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Cadherinas/metabolismo , Calnexina/metabolismo , Proteínas Portadoras/genética , Células Cultivadas , Corteza Cerebral/citología , Embrión de Mamíferos , Hipocampo/citología , Proteínas de Andamiaje Homer , Humanos , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Transfección , Ubiquitinación/fisiologíaRESUMEN
Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.
Asunto(s)
Encéfalo/enzimología , Cuerpos de Inclusión/enzimología , Cuerpos de Lewy/enzimología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/genética , Neuronas/enzimología , Complejo de la Endopetidasa Proteasomal/deficiencia , Animales , Encéfalo/patología , Femenino , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Cuerpos de Lewy/genética , Cuerpos de Lewy/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Degeneración Nerviosa/patología , Neuronas/patología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/fisiologíaRESUMEN
Neuropathological investigations have identified major hallmarks of chronic neurodegenerative disease. These include protein aggregates called Lewy bodies in dementia with Lewy bodies and Parkinson's disease. Mutations in the alpha-synuclein gene have been found in familial disease and this has led to intense focused research in vitro and in transgenic animals to mimic and understand Parkinson's disease. A decade of transgenesis has lead to overexpression of wild type and mutated alpha-synuclein, but without faithful reproduction of human neuropathology and movement disorder. In particular, widespread regional neuronal cell death in the substantia nigra associated with human disease has not been described. The intraneuronal protein aggregates (inclusions) in all of the human chronic neurodegenerative diseases contain ubiquitylated proteins. There could be several reasons for the accumulation of ubiquitylated proteins, including malfunction of the ubiquitin proteasome system (UPS). This hypothesis has been genetically tested in mice by conditional deletion of a proteasomal regulatory ATPase gene. The consequences of gene ablation in the forebrain include extensive neuronal death and the production of Lewy-like bodies containing ubiquitylated proteins as in dementia with Lewy bodies. Gene deletion in catecholaminergic neurons, including in the substantia nigra, recapitulates the neuropathology of Parkinson's disease.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismo , Animales , Humanos , Cuerpos de Lewy , Enfermedades Neurodegenerativas/genética , alfa-Sinucleína/genéticaRESUMEN
The airway epithelium is altered in respiratory disease and is thought to contribute to disease etiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of this study was to extensively characterize the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI-1 or hTERT using viral vector systems. Cells were characterized at passage (p) early (p5), mid (p10), and late (p15) stage for: BMI-1, p16, and CK14 protein expression, viability and the ability to differentiate at air-liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), scanning electron microscopy (SEM), MUC5AC and beta tubulin (BTUB) staining. BMI-1-expressing cells maintained elevated levels of the BMI-1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI-1-expressing cells had a viability advantage, differentiated at ALI, and had a normal karyotype. In contrast, hTERT-expressing cells had a reduced viability, showed limited differentiation, and had an abnormal karyotype. We therefore provide extensive characterization of the plasticity of BMI-1 expressing cells in the context of the ALI model. These cells retain properties of wild-type cells and may be useful to characterize respiratory disease mechanisms in vitro over sustained periods.
Asunto(s)
Bronquios/citología , Plasticidad de la Célula/genética , Células Epiteliales/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Adulto , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Senescencia Celular/genética , Genes p16 , Ingeniería Genética/métodos , Humanos , Técnicas In Vitro , Cariotipo , Lentivirus/genética , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Retroviridae/genética , Telomerasa/metabolismoRESUMEN
The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1(+/-)) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1(+/-) MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1(+/-) MEFs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Encéfalo/metabolismo , Fibroblastos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/genética , Animales , Encéfalo/patología , Células Cultivadas , Fibroblastos/citología , Puntos de Control de la Fase G2 del Ciclo Celular , Heterocigoto , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Ratones Noqueados , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Modelos Biológicos , Poliubiquitina/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Interferencia de ARN , Transfección , Técnicas del Sistema de Dos Híbridos , Ubiquitinación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
We have generated a human feeder cell line from early second trimester Placental Stromal Fibroblasts (ihPSF) stably over-expressing the polycomb protein BMI-1. These feeder cells retain the ability to maintain human Embryonic Stem cells (hESc) over long-term culture whereas hTERT or BMI-1/hTERT immortalised feeder cell lines do not. ihPSFs were able to support the derivation of a new hESc line in near xenofree (free of non-human animal components) conditions and support continued culture of newly derived hESc and human induced Pluripotent Stem (hiPS) cell lines in complete xenofree conditions necessary for clinical use.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Diferenciación Celular , Proliferación Celular , Citometría de Flujo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ/métodosRESUMEN
In the past twenty years, evidence has accumulated to show that ubiquitinated proteins are a consistent feature of the intraneuronal protein aggregates (inclusions) that characterize chronic neurodegenerative disease. These findings may indicate that age-related dysfunction of the 26S proteasome may be central to disease pathogenesis. The aggregate-prone proteins can also be eliminated by autophagy. We have used the Cre-recombinase/loxP genetic approach to ablate the proteasomal Psmc1 ATPase gene and deplete 26S proteasomes in neurons in different regions of the brain to mimic neurodegeneration. Deletion of the gene in dopaminergic neurons in the substantia nigra generates a new model of Parkinson disease. Ablation of the gene in the forebrain creates the first model of dementia with Lewy bodies. In both neuroanatomical regions, gene ablation causes the formation of Lewy-like inclusions together with extensive neurodegeneration. There is some evidence for neuronal autophagy in areas adjacent to inclusions. The models indicate that neuronal loss in neurodegenerative diseases can be attributed to proteasomal malfunction accompanied by Lewy-like inclusions as seen in dementia with Lewy bodies and Parkinson disease.
Asunto(s)
Autofagia , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Enfermedad Crónica , Humanos , Ratones , Pliegue de ProteínaRESUMEN
Gankyrin is an oncoprotein overexpressed in hepatocarcinoma cells that binds to the cell-cycle regulator CDK4 and the S6b ATPase subunit of the regulatory component of the proteasome. It belongs to the family of ankyrin-repeat proteins that appear to mediate protein-protein interactions in diverse biochemical processes. Gankyrin has been crystallized from polyethylene glycol solutions and diffraction data have been obtained from these crystals that extend to 2.1 A spacing.
Asunto(s)
Proteínas Oncogénicas/química , Cristalización/métodos , Humanos , Polietilenglicoles , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas , Difracción de Rayos X/métodosRESUMEN
A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.